USOO7704694B2

(12) United States Patent (10) Patent No.: US 7.704,694 B2 Hirao et al. (45) Date of Patent: Apr. 27, 2010

(54) METHOD FOR DETECTING TARGET PLANT Singhet al.; “Distribution of rDNA Loci in the Genus Glycine willd. GENUS Theor. App. Genet. (2001) 103:212-218. Buck, G.A., et al., “Design Strategies and Performance of Custom DNA Sequencing Primers'. BioTechniques (Sep. 1999), vol.27, No. (75) Inventors: Takashi Hirao, Osaka-Fu (JP); 3, pp. 528-536. Masayuki Hiramoto, Osaka-Fu (JP) Hartmann, Stefanie, et al., “Extensive Ribosomal DNA Genic Varia tion in the Columnar Cactus Lophocereus”. J. Mol. Evol. (2001), vol. (73) Assignee: House Foods Corporation, Osaka-Fu 53, pp. 124-134. (JP) Holzhauser, T., et al., “Polymerase chain reaction (PCR) for detection of potentially allergenic hazelnut residues in complex food (*) Notice: Subject to any disclaimer, the term of this matrixes'. Eur, Food Res. Technol. (2000), vol. 211, pp. 360-365. patent is extended or adjusted under 35 Lavin, Matt, et al., “The Dalbergioid Legumes (Fabaceae): Delimi U.S.C. 154(b) by 100 days. tation of a Pantropical Monophyletic Clade'. American Journal of Botany (2001), vol. 88(3), pp. 503-533. (21) Appl. No.: 12/139,701 Lott, Timothy J., et al., “Nucleotide Sequence Analysis of the 5.8S rDNA and Adjacent ITS2 Region of Candida albicans and Related (22) Filed: Jun. 16, 2008 Species”. Yeast (1993), vol. 9, pp. 1199-1206. Proft, Jana, et al., “Identification of six sibling species of the Anoph (65) Prior Publication Data eles macilipennis complex (Diptera: Culicidae) by a polymerase chain reaction assay”. Parasitol Res. (1999), vol. 85, pp. 837-843. US 2008/O28O3O4 A1 Nov. 13, 2008 Shin, Jong Hee, et al., “Rapid Identification of up to Three Candida Species in a Single Reaction Tube by a 5' Exonuclease Assay using Related U.S. Application Data Fluorescent DNA Probes'. Journal of Clinical Microbiology (Jan. 1999), vol. 37, No. 1, pp. 165-170. (62) Division of application No. 1 1/581,872, filed on Oct. Allmann, Michael, et al., “Polymerase chain reaction (PCR): a pos 17, 2006, now Pat. No. 7,402,391, which is a division sible alternative to immunochemical methods asuring safety and of application No. 10/285,061, filed on Oct. 31, 2002, quality of food'. Z. Lebensm Unters Forsch (1993), vol. 196, pp. now Pat. No. 7,144,702. 248-251. Primary Examiner Kenneth R. Horlick (30) Foreign Application Priority Data Assistant Examiner David C Thomas

Nov. 1, 2001 (JP) . ... 2001-336571 (74) Attorney, Agent, or Firm Hoffman & Baron, LLP Sep. 27, 2002 (JP) ...... 2002-284222 (57) ABSTRACT (51) Int. Cl. CI2O I/68 (2006.01) A method for detecting species in a target plant genus com CI2P 19/34 (2006.01) prises the steps of conducting PCR using at least one member selected from the group consisting of primers (A) and (B), (52) U.S. Cl...... 435/6: 435/91.1; 435/91.2 which can hybridize under Stringent conditions to a nucleic (58) Field of Classification Search ...... None acid molecule having a common nucleotide sequence for all See application file for complete search history. species in the target plant genus in 45S rRNA precursor gene (56) References Cited sequence thereof, wherein 3' end of primer (A) can comple mentarily bind to a base in ITS-1 sequence of the target plant U.S. PATENT DOCUMENTS genus when the primer hybridizes to the nucleic acid mol 5,876,977 A 3/1999 Wang et al. ecule while 3' end of primer (B) can complementarily bind to 5,962,665 A 10/1999 Kroeger et al. a base in ITS-2 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule, and identify OTHER PUBLICATIONS ing the presence of the resulting amplification product from PCR containing at least a part of ITS-1 or ITS-2 sequence of Hirao et al., “Method for Detecting Soybean with High Sensitivity the target plant genus Using PCR Which Gives Characteristic Ladder Product': Annual Meeting of Japan Society for Bioscience, Biotechnology, and The method for detecting species in a target plant genus, Agrochemistry, 2001 Lecture Abstracts (Mar. 2001), p. 45 (1M6p3). Yasui et al. “Phylogenetic Relationships Among Fagopyrum Species particularly an allergenic plant genus Such as the genus Revealed by the Nucleotide Sequences of the ITS Region of the Fagopyrum, can make it possible to detect with high sensi Nuclear rRNA Gene”, Genes Genet. Syst. (1998) 73 p. 201-210. tivity, for example, about 1 ppm of the plant(s) in cases where Yasui et al. “Phylogenetic Relationships of Fagopyrum Revealed by the plant(s) is contained in a food ingredient or food product. Comparative rRNA Gene Sequences'; Japanese Journal of Breeding, 46 Supp. 2 (1996) p. 318. 16 Claims, 10 Drawing Sheets U.S. Patent Apr. 27, 2010 Sheet 1 of 10 US 7.704,694 B2

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US 7,704,694 B2 1. 2 METHOD FOR DETECTING TARGET PLANT Thus, the conventional methods mentioned above are to GENUS detect one specific species in a mixture and to identify a bio specimen exclusively derived from a single species of the CROSS-REFFERENCE TO RELATED genus, and therefore, the methods do not relate to a method APPLICATIONS for detecting the target genus broadly in cases where even one kind of the target genus is contained in a mixture. In addition, This application is a divisional application of U.S. patent the primer sequences common to several species are located application Ser. No. 1 1/581,872, filed Oct. 17, 2006, which is on SSU rRNA, 5.8S rRNA and LSU rRNA gene sequence, a divisional application of U.S. patent application Ser. No. and therefore, primer sequences common to several species 10/285,061, filed Oct. 31, 2002, now U.S. Pat. No. 7,144,702, 10 are not found in ITS-1 or ITS-2 sequence. which claims priority to Japanese Patent Application No. On the other hand, regarding detection of allergenic plants 2002-284222 filed on Sep. 27, 2002 and Japanese Patent in food, a method for detecting whether some wheat is con Application No. 2001-336571, filed Nov. 1, 2001. tained in a food sample of interest is disclosed by Allmann M. et al. (Z Lebensm Unters Forsch, 196: 248-251 (1993)). The BACKGROUND OF THE INVENTION 15 method uses primers which specifically hybridize to a IGS sequence between 25S rRNA (LSU rRNA) and 18S rRNA The 45S rRNA precursor gene sequence (Small Subunit (SSU rRNA) gene sequences of wheat. However, it is hard to ribosomal RNA (SSU rRNA) gene-Internal Transcribed evaluate the specificity of the primers by simulation and the Spacer-1 (ITS-1)-5.8S ribosomal RNA (5.8S rRNA) like because the primers have to be designed based on little gene-Internal Transcribed Spacer-2 (ITS-2)-Large Subunit information about the IGS sequence in the method. There ribosomal RNA (LSU rRNA) gene) has been used for the fore, it would be difficult to judge the reliability of the analy classification of species. For example, according to the S1S. method developed by Shin J. H. et al. (J. Clin. Microbiol. 37: 165-170 (1999)), 5 candida species (fungi) of the genus Can SUMMARY OF THE INVENTION dida can be detected and identified using two primers hybrid 25 ized to the 5.8S rRNA and 28S rRNA (LSU rRNA) gene An object of the present inventions is to provide a method sequences common to fungi and 5 separate probes each of for detecting species (a plant or plants) in a target plant genus, which can specifically hybridize to the ITS-2 sequence of its particularly an allergenic plant genus Such as the genus corresponding species. The method is different from the Fagopyrum, which makes it possible to detect with high sen present invention as described below. Firstly, the method is 30 sitivity, for example, about 1 ppm of the plant(s) in cases aimed at fungi, specifically candida (fungi). Secondly, the where the plant(s) is contained in a food ingredient or food method does not use the primers, which hybridize to ITS-1 or product. ITS-2 sequence. Consequently, these primer pairs do not Since a trace of allergenic food ingredients, particularly assure the specificity to the genus Candida, whereas each of plants in an allergenic plant genus may be unintentionally five probes can independently recognize its corresponding 35 contaminated in the food ingredient or product at the stages of candida species (fungi) of the genus Candida. In other words, production, distribution and fabrication, it is important that only one species of the genus Candida can be detected and providers of the food ingredient or product conduct quality identified when a single set of the primer pair and a probe is control to detect whether these plants have contaminated the used. Thirdly, the above publication does not describe about food ingredient or product. the sensitivity of the detection, which is very important for the 40 For example, regarding buckwheat, though it is reported detection methods of allergenic plants in food. Lastly, the that patients are affected with anaphylaxis by pillows made of method needs expensive reagents and instruments due to the buckwheat chaffand die due to anaphylactic shock and traces use of probes. of buckwheat may effect a severe symptom in allergic According to the method developed by ProftJ, et al. (Para patients for buckwheat, there is no method for detecting buck sitol. Res., 85: 837-843 (1999)), a certainanopheles mosquito 45 wheat in the food ingredient or product in the world. For can be classified into one of 6 species of the genus Anopheles example, it is considered that contamination of buckwheat using 6 primer pairs. The method uses a primer that can into the food ingredient or product occurs in a case where hybridize to the 5.8 rRNA gene sequence common to the six buckwheatgrown near afield cultivated with plants other than anopheles mosquito species of the genus Anopheles in com buckwheat is contaminated in the food ingredient harvest bination with 6 primer pairs each of which can specifically 50 time. Therefore, in order to find the contamination of trace of hybridize to the ITS-2 sequence of its corresponding anoph unintended buckwheat, it is desirable that a method for eles mosquito species of the genus Anopheles. Based on the detecting buckwheat be built up, wherein the method can size of the amplification product obtained by PCR method, detect as sensitive as possible, for example, even 1 ppm of the the anopheles mosquito of interest can be classified into one buckwheat in a food ingredient and product. Furthermore, as of the 6 species of the genus Anopheles. The method is dif 55 for grain allergies, it is said that Some cross-reaction occurs ferent from the present invention as described below. Firstly, among taxonomically related plants, and therefore, it is desir the method is aimed at mosquitoes, specifically the anopheles able that the method be able to detect a wide range of any mosquitoes. Secondly, due to the properties of designed plants in the genus Fagopyrum without limiting the detectable primer pairs, only one species of the genus Anopheles can be plants to eatable buckwheat. detectable when a single primer pair is used. Thirdly, an 60 Regarding a method for detecting peanuts, an ELISA kit, object of the method is to identify a specimen exclusively which can detect about 2.5 ppm of peanuts using specific derived from a single species of mosquitoes. Consequently, antibodies for proteins inherent to peanuts, have been sold the object of the method is not to analyze anopheles mosqui and used in the world. When positive finding in ELISA, toes in a mixture. Lastly, the above publication does not whether it is false positive or truly positive can be confirmed describe about the sensitivity of the detection, which is very 65 in detail by Western Blot etc., but it confirms only the size of important for the detection methods of allergenic plants in protein involving antigen-antibody reaction. A method for food. detecting a DNA inherent to peanuts has not been reported. In US 7,704,694 B2 3 4 order to detect peanuts in a food ingredient and product for plants in the genus can be chosen. Moreover, as the through a variety of processing steps, it is desirable that there sequence of 45S rRNA precursor gene is present in most is built up a method for detecting target DNA sequences, plants, it can be advantageously applied on a variety of plants. which will have a relatively high resistivity against the pro Based on this knowledge, the present inventions have been cessing rather than proteins Furthermore, as it is the same as completed. In this connection, the following method for in buckwheat, it is desirable that the method be able to detect detecting plants can be applied not only to the allergenic plant a wide range of plants in the genus Arachis. genus but also to other plant genus. Thus, it is important to detect a plant(s) in the allergenic Accordingly, the present invention provides a method for plant genus with high sensitivity in cases where even only one detecting species (a plant(s)) in a target plant genus, which kind of the plants is contained in the food ingredient, product 10 comprises the steps of conducting PCR using at least one and the like. member selected from the group consisting of primers (A) In cases of genetically modified products and the like, and (B), which can hybridize under stringent conditions to a DNA sequences to be detected are limited to recombinant nucleic acid molecule having a nucleotide sequence common DNA sequences. On the other hand, in cases of plants which to all species in the target plant genus in 45S rRNA precursor originally exist in nature, there has not been clear knowledge 15 gene sequence thereof, wherein 3' end of primer (A) can how to choose a target DNA sequence from a large number of complementarily bind to a base in a ITS-1 sequence of the DNA sequences, and whether the thus chosen DNA sequence target plant genus when the primer hybridizes to the nucleic is useful and universal for a variety of plants. It has been acid molecule while 3' end of primer (B) can complementa conducted to choose a specific protein to a target plant, and to rily bind to a base in a ITS-2 sequence of the target plant genus detect a DNA sequence coding for the protein, but it is nec when the primer hybridizes to the nucleic acid molecule, and essary to choose a separate specific protein to each plant. identifying the presence of the resulting amplification prod Furthermore, even if such a specific protein can be chosen, uct from PCR containing at least a part of the ITS-1 or ITS-2 when the copy number of a DNA sequence coding for the sequence of the target plant genus. protein is Small, there are some cases where the method may Herein, the phrase “hybridize under stringent conditions” not have a sufficient sensitivity and therefore it will be disad 25 means that two DNA fragments hybridize to each other under Vantage for the detection of traces of a contaminating plant. the standard hybridization condition described by Sambrook Under Such circumstances, in order to develop a method for J. et al. (Expression of Cloned Genes in E. coli (Molecular detecting a plant(s) in an allergenic plant genus and the like in Cloning: A laboratory Manual (1989)) Cold Spring Harbor cases where even only one kind of the plants is contained in a Laboratory Press, New York, USA, 9.47-9.62 and 11.45 food ingredient and product, the present inventors have 30 11.61). More specifically, for example, it means that a hybrid focused their attention on the gene sequences of a target plant ization and washing (for example, about 2.0xSSC, 50° C.) are genus to vigorously conduct the research. In order to detect conducted on the basis of Tm value obtained by the following whether one specific plant has contaminated a food ingredient equation. or product, it may be conducted to detect a specific gene Tim=81.5+16.6(logoNa)+0.41 (fraction G+C)- sequence of the plant in the food ingredient and product. 35 However, in order to detect a case where even only one kind (600/N) of the plants is contained in a genus in a food ingredient and In addition, the term genus as used in the present specifi product, such method is very complicated and inefficient cation means a group including all species in the genus or because it is necessary to repeat the same operation for Some species chosen from the species in the genus. respective plants in a specific genus. 40 In order to solve this problem, the inventors have con BRIEF DESCRIPTION OF THE DRAWINGS ducted further research, collected some information on gene sequences of plants in the genus Fagopyrum (21 sequences FIG. 1 is an electrophoretogram showing the results elec registered in GenBank) and in other genus and studied on a trophoresed on a 2% agarose gel in Example 1. variety of viewpoint, and thereby, the inventors have found 45 FIG. 2 is an electrophoretogram showing the results elec that a specific common sequence for plants in the genus trophoresed on a 2% agarose gel in Example 1. Fagopyrum, which differs from a sequence of plants in other FIG. 3 is an electrophoretogram showing the results elec genus, is present in gene sequences of the plant in the genus trophoresed on a 2% agarose gel in Example 1. Fagopyrum (21 sequences registered in GenBank). As the FIG. 4 is nucleotide sequences obtained by a sequence result of an investigation conducted based on this knowledge 50 analysis of the target amplification product from buckwheat for other plant genus Such as the genus Arachis, the inventors chaff. have also found that there is similar tendency among them. FIG. 5 is an electrophoretogram showing the results elec Based on this knowledge, it has been found that a method trophoresed on a 2% agarose gel in Example 3. for detecting each allergenic plant genus using a sequence of FIG. 6 is an electrophoretogram showing the results elec 45S rRNA precursor gene, as a sequence which exists as a 55 sequence having a large copy number in plant DNA and is trophoresed on a 2% agarose gel in Example 3. specific to eachallergenic plant genus, can be useful in attain FIG. 7 is nucleotide sequences obtained by a sequence ing the object. When positive indication appears in PCR, analysis of the target amplification product from Shirahana differently from ELISA, as an amplification product can be soba. analyzed not only in the size thereof but also in detail 60 FIG. 8 is an electrophoretogram showing the results elec sequence thereof by sequencing the amplification product, it trophoresed on a 2% agarose gel in Example 5. can be confirmed more precisely whether it is false positive or FIG. 9 is nucleotide sequences obtained by a sequence truly positive. Furthermore, it has been found that, by choos analysis of the target amplification product from peanut. ing a region including ITS-1 or ITS-2 sequence as a target FIG. 10 is an electrophoretogram showing the results elec sequence, the method is useful in detecting trace of plants in 65 trophoresed on a 2% agarose gel in Example 6. the target plant genus in a mixture because the specific FIG. 11 is an electrophoretogram showing the results elec sequence can be obtained and common region of sequences trophoresed on a 2% agarose gel in Example 6. US 7,704,694 B2 5 6 FIG. 12 is nucleotide sequences obtained by a sequence genus, the studied subject is not contaminated by a plant(s) in analysis of the target amplification product from peanut. the target plant genus. Furthermore, the method of the present FIG. 13 is 45S rRNA precursor gene structure. invention can detect with high sensitivity, for example, about 1 ppm level of a contamination. DESCRIPTION OF THE PREFERRED For example, at least 2 primers may be used in the method EMBODIMENTS of the present invention. In cases where at least 2 kinds of the target plant genus are detected at the same time, at least 3 Although a target plant genus to be detected by the method primers may be used provided that it is important to use at of the present invention may be any plant genus, because the least one member selected from the group consisting of prim method can detect a presence of trace of a plant(s) in the target 10 ers (A) and (B), which can hybridize under stringent condi plant genus in a food ingredient or product, the method is tions to a nucleic acid molecule having a nucleotide sequence particularly useful in detecting whether plants in the aller common to all species in the target plant genus in 45S rRNA genic plant genus Such as the genus Fagopyrum, genus Ara precursor gene sequence thereof, wherein 3' end primer (A) chis, genus Triticum and genus Glycine are contaminated in can complementarily bind to a base in ITS-1 sequence of the the food ingredient or product. 15 target plant genus when the primer hybridizes to the nucleic The method of the present inventions uses at least one acid molecule while 3' end of primer (13) can complementa member selected from the group consisting of primers (A) rily bind to a base in ITS-2 sequence of the target plant genus and (S.), which can hybridize under stringent conditions to a when the primer hybridizes to the nucleic acid molecule. In nucleic acid molecule having a nucleotide sequence common this connection, examples of the primer (A) include primers, to all species in the target plant genus in a 45S rRNA precur which can hybridize to a nucleic acid molecule having a sor gene sequence thereof, wherein 3' end of primer (A) can boundary between a ITS-1 sequence and a 5.8S rRNA gene complementarily bind to a base in a ITS-1 sequence of the sequence or which can hybridize to a nucleic acid molecule target plant genus when the primer hybridizes to the nucleic having a boundary between a ITS-1 sequence and a SSU acid molecule, while 3' end of primer (B) can complementa rRNA gene sequence. Likewise, examples of the primer (1B) rily bind to a base ina ITS-2 sequence of the target plant genus 25 include primers, which can hybridize to a nucleic acid mol when the primer hybridizes to the nucleic acid molecule to ecule having aboundary betweena ITS-2 sequence and a 5.8S conduct PCR amplification for DNA isolated from a subject rRNA gene sequence or which can hybridize to a nucleic acid to which the method is to be applied. In the PCR amplifica molecule having a boundary between a ITS-2 sequence and a tion, based on a conventional procedure described in publi LSU rRNA gene sequence. Preferably the primers (A) and cations, for example, Saiki R K, et al., Science, 230: 1350 30 (B) have at least 15 bases, more preferably 15 to 30 bases. 1354 (1985) and Shyokubutsu no PCR Zikken Protocol Since the ITS-1 sequence and the ITS-2 sequence contain Idenshi no Tanri Hatsugen Kara Genome Kaiseki Made— many specific sequences for species, the primer (A) or (13), (Saiboukougaku Bessatsu Saiboukougaku Series 2), General which has a specificity common to the target plant genus, can Editors Shimamoto, K. and Sasaki, T., Shujunsha Co., Ltd., be obtained by choosing a Suitable nucleic acid molecule Tokyo, 1995 and the like, optimal conditions are chosen from 35 having a specific nucleotide sequence common to the target appropriate modification oftemperature and time of each step plant genus in the ITS-1 and ITS-2 sequences, as a nucleic of denaturation, annealing and extension, a kind and concen acid molecule having a nucleotide sequence common to the tration of enzyme (DNA polymerase), concentrations of target plant genus in the 45S rRNA precursor gene sequence. dNTP primer and magnesium chloride, an amount of tem One or two or more member(s) selected from the group con plate DNA and the like. 40 sisting of the primer (A) and the primer (B) may also be used, In addition, PCR amplification may be conducted at an but if at least two members are used, the method of the present annealing temperature of the primer and the template DNA invention can become more highly sensitive to the target plant higher than Tm value of the primer, preferably the Tm value genus, particularly genus Fagopyrum. plus 10 to 3° C., and Subsequently at an annealing tempera In another embodiment of the method for detection of the ture near the Tm value, preferably the Tm value plus 7 to 0° 45 present invention, primer (A) is used together with a primer C., wherein the Tm value is determined by commercially (C) which can hybridize under Stringent conditions to a available software such as HYB SimulatorTM version 4.0 nucleic acid molecule having a part of a nucleotide sequence (Advanced Gene Computing Technologies, Inc.) and Primer continuously bonded ITS-1, 5.8S rRNA gene, ITS-2 and LSU ExpressTM version 1.5 (PE Applied Biosystems). rRNA gene of the target plant genus. Alternatively, primer (A) After the PCR amplification of DNA isolated from a sub 50 is used together with a primer (E) which can hybridize under ject to be studied Such as a food ingredient or product, the stringent conditions to a nucleic acid molecule having a part resulting reaction solution is analyzed by for example, elec of a nucleotide sequence continuously bonded SSU rRNA trophoresis to determine whether the target plant genus is gene and ITS-1 of the target plant genus. In a further embodi present in the subject. The determination is based on whether ment of the method for detection of the present invention, the any PCR amplification products having target size are present 55 primer (B) is used together with a primer (D) which can in the reaction solution after the PCR amplification, and if the hybridize under Stringent conditions to a nucleic acid mol PCR amplification products are present in the reaction solu ecule having a part of a nucleotide sequence continuously tion, whether at least a part of the ITS-1 or ITS-2 sequence of bonded SSU rRNA gene, ITS-1, 5.8S rRNA gene and ITS-2 the target plant genus is present in the sequence of the PCR of the target plant genus. Alternatively, primer (B) is used amplification products. That is, if the PCR amplification 60 together with a primer (F) which can hybridize under strin products, which have the target size and contain at least a part gent conditions to a nucleic acid molecule having a part of a of the ITS-1 or ITS-2 sequence of the target plant genus, are nucleotide sequence continuously bonded ITS-2 and LSU present in the reaction solution, the studied Subject is con rRNA gene of the target plant genus. In this connection, 5.8S taminated by a plant(s) in the target plant genus. On the other rRNA gene is highly preservative and contains many hand, if the PCR amplification products are not present in the 65 sequences common to a great majority of plants. Therefore, as reaction Solution or even though it exists, unless it contains at a primer (C), by appropriately choosing a primer, which can least a part of ITS-1 or ITS-2 sequence of the target plant hybridize under Stringent conditions to a nucleic acid mol US 7,704,694 B2 7 8 ecule having a part of a nucleotide sequence of 5.8S rRNA genus from GenBank, conducting an alignment and search gene, wherein 3' end thereof can complementarily bond to a ing a region having a high specificity common to the target nucleotide sequence in 5.8S rRNA gene sequence when the plant genus. In addition, among the regions thus identified, a primer hybridizes to the nucleic acid molecule, or as sprimer base, which can assure that the base is specific to the target (D), by appropriately choosing a primer, which can hybridize plant genus and not to plants thought to be related species under stringent conditions to a nucleic acid molecule having thereof, can be determined as 3' end of the primers to select a a part of a nucleotide sequence of 5.8S rRNA gene, wherein primer sequence. 3' end thereof can complementarily bond to a nucleotide When the target plant genus is the genus Fagopyrum, sequence in 5.8S rRNA gene sequence when the primer examples of a commonly specific nucleotide sequences in the hybridizes to the nucleic acid molecule, the resulting primer 10 ITS-1 sequence thereof include a nucleotide sequence indi can be commonly used for a variety of plants. If said primer is cated by any of SEQ NO:8, 9 or 10, or a complementary fixed and a common specific primer is chosen for the species nucleotide sequence thereof. Preferably, they include a nucle in the target plant genus from the ITS-1 or ITS-2 region otide sequence of positions 11 to 61 of the nucleotide thereof, then the primers can be easily designed to detect with sequence of SEQ NO:8 or a complementary nucleotide high sensitivity the contaminated plants in the target plant 15 sequence thereof, or a nucleotide sequence of positions 11 to genus. Preferably, the primers (C) to (F) have at least 15 67 of the nucleotide sequence of SEQNO:9 or a complemen bases, more preferably 15 to 30 bases. tary nucleotide sequence thereof. In addition, SEQ NO:10 is When these primers are designed, it will be sufficient to particularly useful as a region for selecting primers for detect design them based on, for example, PCR Hou Saizensen ing specifically F. esculentum (common buckwheat), F. Kisogizyutsu Kara Ouyou Made tataricum (Tartarian buckwheat), F homotropicum and/or F. (Tanpakushitsu Kakusan Kouso Rinzizoukan), ed. Sekiya, T. cymosum, which are members of the genus Fagopyrum. and Fujinaga, K., Kyoritsu Shuppan Co. Ltd., Tokyo, 1997, Preferably, the primer (A) is an oligonucleotide indicated Baio Zikken Illustrated 3 Hontouni Hueru PCR (Saiboukou by any of SEQ NOs: 11 to 16 wherein the oligonucleotide gaku Besshi Me de Miru Zikken Note Series), Nakayama, H., indicated by any of SEQ NOs: 11 to 14 hybridizes to a Shujunsha Co., Ltd., Tokyo, 1996 or PCR Technology: Prin 25 complementary strand of SEQ NO:8 and the oligonucleotide ciples and Applications of DNA Amplification, ed. Erlich, H. indicated by any of SEQ NOs: 15 and 16 hybridizes to a A., Stockton Press, Inc., NY, 1989. However, since there is a nucleic acid molecule of SEQNO:9. The primer (A) may also low possibility that the target DNA is decomposed when the be an oligonucleotide indicated by any nucleotide sequence DNA is detected in un-processed materials, the primers may of SEQ NOs: 11 to 16, wherein one or several base(s) thereof be those which can induce an amplification product within 30 are deleted or substituted, or one or several base(s) are added 700 bases, and since there is a possibility that the target DNA thereto. Furthermore, examples of the common specific is decomposed to become short when the DNA is detected in nucleotide sequence in ITS-2 include a nucleotide sequence processed foods, the primers, which can induce an amplifi indicated by any of SEQ NO:21 or 22, or a complementary cation product within 200 bases, are preferable in view of that nucleotide sequence thereof. These nucleotide sequences are the primers provide high sensitivity. 35 particularly useful as a region for selecting primers for detect In view of the above, it is preferable that the primer (C) or ing specifically F. esculentum (common buckwheat), F. (D) be able to hybridize under stringent conditions to a tataricum (Tartarian buckwheat), F homotropicum and/or F. nucleic acid molecule having a nucleotide sequence indicated cymosum, which are members of the genus Fagopyrum. In by SEQ NO:1 or a complementary nucleotide sequence addition, it is preferable to use a combination of the primer of thereof. Said primer is preferable because the region indi 40 any of SEQ NOs: 11 to 14 and the primer of any of SEQ cated by SEQ NO:1 has an especially high homology, a NOS:15, 16 or 2 to 4. primer which hybridize to any region of 5.8S rRNA gene When the target plant genus is the genus Arachis, examples sequence may be used because the sequences of species in the of a common specific nucleotide sequences in the ITS-1 allergenic plant genus have a high homology over almost the sequence thereof include a nucleotide sequence indicated by whole region of 5.8S rRNA gene sequence. More preferably, 45 SEQ NO:17, or a complementary nucleotide sequence it is a primer, which can hybridize under Stringent conditions thereof. Preferably, they include a nucleotide sequence of to a nucleic acid molecule having positions 11 to 63 of the positions 1 to 60 of the nucleotide sequence of SEQNO:17 or nucleotide sequence of SEQNO:1 or a complementary nucle a complementary nucleotide sequence thereof, or a nucle otide sequence thereof. Preferably, primer (C) is an oligo otide sequence of positions 43 to 99 of the nucleotide nucleotide indicated by any of SEQ NO:2, 3 or 4, which 50 sequence of SEQ NO:17 or a complementary nucleotide hybridizes to the nucleic acid molecule of SEQ NO:1. Pref sequence thereof. More preferably, they include a nucleotide erably, primer (D) is also an oligonucleotide indicated by any sequence of positions 11 to 50 of the nucleotide sequence of of SEQ NO:5, 6 or 7, which hybridizes to a complementary SEQ NO:17 or a complementary nucleotide sequence strand of SEQ NO:1. Said primers have to hybridize under thereof, or a nucleotide sequence of positions 53 to 89 of the stringent conditions specific to the target nucleic acid mol 55 nucleotide sequence of SEQ NO:17 or a complementary ecule and 3' end thereof have to be a complementary base to nucleotide sequence thereof. the target part of DNA sequence so that the hybridized prim Preferably, the primer (A) is an oligonucleotide indicated ers can function as one primer and an extension reaction by any of SEQ NOs: 18 to 20, which hybridizes to a comple occurs. Therefore, as long as the primers meet the above mentary strand of SEQNO:17. The primer (A) may also be an requirement, the primers may be an oligonucleotide indicated 60 oligonucleotide indicated by any nucleotide sequence of SEQ by any nucleotide sequence of SEQ NOs:2 to 7, wherein one NOs: 18 to 20, wherein one or several base(s) thereof are or several base(s) thereofare deleted or substituted, or one or deleted or substituted, or one or several base(s) are added several base(s) are added thereto. thereto. Furthermore, examples of the common specific The specific nucleotide sequence common to the target nucleotide sequence in ITS-2 sequence of the genus Arachis plant genus in ITS-1 or ITS-2 sequence can be identified by 65 include a nucleotide sequence of SEQ NO:23 or a comple obtaining the ITS-1-5.8S rRNA gene-ITS-2 sequence of a mentary nucleotide sequence thereof. Preferably, it is a nucle plant(s) in the target plant genus to be detected and other plant otide sequence of positions 11 to 47 of the nucleotide US 7,704,694 B2 9 10 sequence of SEQ NO:23 or a complementary nucleotide For example, in order to design the primer for detecting the sequence thereof. Moreover, it is preferable that the primer genus Fagopyrum, a common region having a high specificity (B) be an oligonucleotide indicated by SEQ NO:24, which for all of the 21 DNA sequences of plants in genus Fagopyrum hybridizes to a nucleic acid molecule of SEQ NO:23. The including eatable buckwheat (common buckwheat and Tar primer (13) may also be an oligonucleotide indicated by any tarian buckwheat) is selected from the region of ITS-1-5.8S nucleotide sequence of SEQ NO:24, wherein one or several rRNA gene-ITS-2 sequence, and further, a base, which can base(s) thereof are deleted or substituted, or one or several assure the specificity to other plants, is selected as 3' end of the base(s) are added thereto. In addition, it is preferable to use a primer to determine the primer sequence. However, the spe combination of the primer of any of SEQ NOs: 18 to 20 and cies in the genus Fagopyrum have the ITS-1-5.8S rRNA the primer of any of SEQ NOs: 2 to 4, a combination of the 10 primer of any of SEQ NOs: 18 to 20 and the primer of SEQ gene-ITS-2 sequence from which apart thereof is deleted and NO:24 or a combination of the primer of SEQ NO:24 and the from which a number of bases are deleted, which differ from primer of any of SEQ NOS:5 to 7, and more preferably, a each other, and therefore, it is necessary to conduct further combination of the primer of any of SEQ NOs: 18 to 20 and selection in order to obtain a same size of amplification prod the primer of any of SEQ NOS:2 to 4. 15 uct for the 21 plants in the genus Fagopyrum. If the same size When the target plant genus is genus Triticum, examples of of amplification product can be obtained for the 21 plants in common specific nucleotide sequences in ITS-2 sequence the genus Fagopyrum, the presence of the genus Fagopyrum thereof include a nucleotide sequence indicated by any of can be easily detected. In the genus Fagopyrum, particularly SEQ NO:25, 26 or 27, or a complementary nucleotide by selecting the primer (A) and the primer (C) or two primers sequence thereof. Preferably, it is a nucleotide sequence of (A), the simulation has confirmed that the same size of ampli positions 11 to 50 of the nucleotide sequence of SEQ NO:25 fication product would be obtained for all of 21 plants in the or a complementary nucleotide sequence thereof, a nucle genus Fagopyrum. There can be designed primers by which otide sequence of positions 11 to 47 of the nucleotide nonspecific products can be easily identified in light of the sequence of SEQ NO:26 or a complementary nucleotide size of the products. sequence thereof, or a nucleotide sequence of positions 11 to 25 As mentioned above, regarding the designed primer, it was 47 of the nucleotide sequence of SEQ NO:27 or a comple mentary nucleotide sequence thereof. confirmed by PCR simulation whether or not the target ampli Preferably, the primer (B) is an oligonucleotide indicated fication product could be obtained and the results were almost by any of SEQ NOs: 28 to 30 wherein the oligonucleotide of the same as the results of actual PCR, and therefore, the SEQNO:28 hybridizes to the complementary strand of SEQ 30 simulation results possess high reliability In this connection, NO:25, the oligonucleotide of SEQ NO:29 hybridizes to the the above described PCR simulation software, Amplify 1.0 nucleic acid molecule of SEQNO:26 and the oligonucleotide (Bill Engels) and the like can be used in the PCR simulation. of SEQ NO:30 hybridizes to the nucleic acid molecule of An amplification of DNA sequence using the primers SEQ NO:27. The primer (B) may also be an oligonucleotide described above can be conducted by PCR methods (Poly indicated by any nucleotide sequence of SEQ NOs:28 to 30, 35 merase Chain Reaction: for example, Saiki R K, et al., Sci wherein one or several base(s) thereof are deleted or substi ence, 230: 1350-1354 (1985)), as well as LAMP (Loop-Me tuted, or one or several base(s) are added thereto. In addition, diated Isothermal Amplification: Notomi T, et al., Nucleic it is preferable to use a combination of the primer of SEQ Acids Res., 28 e 63 (2000)) or by other appropriate methods. NO:28 and at least one primer selected from the group con In addition, though the amplification product is generally sisting of SEQ NOS:29 and 30. 40 detected by electrophoresis, other methods can be used. When the target plant genus is genus Glycine, examples of commonly specific nucleotide sequences in ITS-2 sequence EXAMPLES thereof include a nucleotide sequence indicated by any of SEQ NO:31, 32 or 33, or a complementary nucleotide The present invention will be described more specifically sequence thereof. Preferably, it is a nucleotide sequence of 45 with reference to the following Examples. positions 11 to 48 of the nucleotide sequence of SEQ NO:31 or a complementary nucleotide sequence thereof, a nucle Example 1 otide sequence of positions 11 to 55 of the nucleotide sequence of SEQ NO:32 or a complementary nucleotide sequence thereof, or a nucleotide sequence of positions 11 to 50 A. Design of Oligonucleotide Primers for Detection of DNA 52 of the nucleotide sequence of SEQ NO:33 or a comple from Buckwheat mentary nucleotide sequence thereof. (1) DNA Sequences of the Genus Fagopyrum Preferably, the primer (B) is an oligonucleotide indicated Regarding the genus Fagopyrum, 5.8S rRNA gene, ITS-1 by any of SEQ NOS:34 to 41 wherein the oligonucleotide of and ITS-2 sequences in the following 21 DNA sequences SEQ NO:34 hybridizes to a complementary strand of SEQ 55 registered in GenBank were examined to select suitable NO:31, the oligonucleotide of any of SEQ NOS:35 to 40 regions for the primers. hybridizes to a nucleic acid molecule of SEQ NO:32 and the oligonucleotide of SEQ NO:41 hybridizes to a nucleic acid 1. Fagopyrum urophyllum (AB000342) molecule of SEQ NO:33. The primer (B) may also be an 2. Fagopyrum urophyllum (AB000341) oligonucleotide indicated by any nucleotide sequence of SEQ 60 3: Tartarian buckwheat: Fagopyrum tataricum (Sub Species: NOs:34 to 41, wherein one or several base(s) thereof are potamini) (AB000340) deleted or substituted, or one or several base(s) are added 4: Tartarian buckwheat: Fagopyrum tataricum (AB000339) thereto. It is preferable to use a combination of the primer of 5. Fagopyrum statice (AB000338) SEQ NO:34 and at least one primer selected from the group consisting of SEQ NOS:35 to 41. 65 6. Fagopyrum statice (AB000337) In order to design these primers and to evaluate the 7. Fagopyrum pleioramosum (AB000336) designed primers, a PCR simulation may be used. 8. Fagopyrum lineare (AB000335) US 7,704,694 B2 11 12 9. Fagopyrum leptopodum (AB000334) 10: Fagopyrum homotropicum (AB000333) Sense primer 11. Fagopyrum gracilipes (AB000332) 5'-GGA CCA CGA ACA GAA. GCG CGT CCC G-3 '' (SEQ NO:11) 12: Common buckwheat: Fagopyrum esculentum ancestralis 5 From among 5.8S rRNA gene sequences of the aforemen (AB000331) tioned 21 DNA sequences of the genus Fagopyrum and 8 13: Common buckwheat: Fagopyrum esculentum DNA sequences of other common allergenic plants, there was (AB000330) determined a nucleotide sequence which would hybridize to 14. Fagopyrum cymosum (AB000329) 10 all of these sequences through the study of the sequences. The 15. Fagopyrum cymosum (AB000328) thus determined nucleotide sequence is indicated as SEQ 16. Fagopyrum cymosum (AB000327) NO:3. Subsequently, the oligonucleotide primer with the 17. Fagopyrum cymosum (AB000326) SEQ NO:3 was synthesized. 18. Fagopyrum cymosum (AB000325) 15 19. Fagopyrum cymosum (AB000324) Antisense primer (SEQ NO:3) 2O. Fagopyrum capillatum (AB000323) s" - ATC GCA TTT CGC TAC GTT CTT CAT CG-3 21. Fagopyrum callianthum (AB000322) Regarding the sense and antisense primer pair, the simula (2) DNA Sequences of Other Common Allergenic Plants tion was conducted with PCR simulation software, Amplify As sequences of peanut, wheat, soybean, walnut, mat 1.0 (Bill Engels). As a result, it was predicted that target 140 Sutake mushroom, peach, apple and orange, 5.8S rRNA gene, bp amplification products would be obtained from the afore ITS-1 and ITS-2 sequences in the following DNA sequences mentioned 21 DNA sequences of the genus Fagopyrum. In registered in GenBank were selected. 25 contrast, no 140 bp amplification product was predicted from 1: peanut: Arachis hypogaea (AF156675) the aforementioned 8 DNA sequences of common allergenic 2: wheat: Triticum aestivum (AJ301799) plants other than genus Fagopyrum (peanut, wheat, soybean, 3: soybean: Glycine max (U60551) walnut, matsutake mushroom, peach, apple and orange) and 4: walnut: Juglans regia (AF303809) 30 the 4 DNA sequences of plants widely used for a food ingre 5. matsutake mushroom. 7 Ticholoma matsutake (U62964) dient (corn, brown rice, pepper and mustard). However, the 6: peach: Prunus persica (AF185621) results of the simulation indicated Some possibility that non 7: apple: Malusxdomestica (AF186484) specific amplification products, which were different from 8: Valencia orange: Citrus sp. (E08821) 35 the target one in size, would be obtained from soybean, apple and orange in light of weak amplified signals. On the other (3) DNA Sequences of Plants Widely Used for a Food Ingre hand, no amplification product was predicted from the 5 DNA dient sequences of other common allergenic plants (peanut, wheat, As sequences of corn, brown rice, pepper and mustard, 40 walnut, matsutake mushroom and peach) and the 4 DNA 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following sequences of plants widely used for a food ingredient (corn, DNA sequences registered in GenBank were selected. brown rice, pepper and mustard). The simulation results are 1: corn: Zea mays (U46648) shown in Tables 1A and 1B. The meanings of symbols and 2: brown rice: Oryza sativa (AF169230) 45 numerical values in Tables 1A and 1B are explained below. 3: pepper: Piper nigrum (AF275197) k: An obtained amplification product whose size almost 4: mustard: Sinapis alba (X15915) matched to the target product size 140 bp (+10 bp), which would be obtained. (4) Oligonucleotide Primer Synthesis and Evaluation W 2-6: Probability of obtaining amplification products Among ITS-1 sequences of the aforementioned 21 DNA 50 sequences of the genus Fagopyrum, there was determined a High Probability W6>W5>W4>W3>W2 Low Prob nucleotide sequence which would specifically hybridize to all ability of the 21 DNA sequences of the genus Fagopyrum through Numerical values followed by bp: the study of the ITS-1 sequences. The thus determined nucle 55 Each value was obtained by subtracting 2 from the value otide sequence is indicated as SEQ NO:11. Subsequently, the obtained in the simulation. oligonucleotide primer with SEQ NO:11 was synthesized. (-) No amplification product was predicted. TABLE 1A SEQ NO. 11 & SEQ NO. 3 primer: Amplification products

GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Genus Fagopyrim frFagopyrum urophyllum AB000342 140 bp – kFagopyrum urophyllum AB000341 140 bp – US 7,704,694 B2 13 14 TABLE 1 A-continued SEQ NO. 11 & SEQ NO. 3 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 kFagopyrum tataricum ABOOO340 140 bp 64 bp (Tartarian buckwheat) kFagopyrum tataricum ABOOO339 140 bp 64 bp (Tartarian buckwheat) st Fagopyri in Statice ABOOO338 40 bp — st Fagopyri in Statice ABOOO337 40 bp — kFagopyrum pieioramosum AB000336 140 bp — kFagopyrum lineare ABOOO335 40 bp — st Fagopyrim lepiopodiin ABOOO334 140 bp kFagopyrum homotropicum AB000333 40 bp — 326 bp st Fagopyrim gracilipes ABOOO332 140 bp st Fagopyrim escientiin ABOOO331 40 bp — 326 bp (Common buckwheat) st Fagopyrim escientiin AB000330 140 bp – 325 bp (Common buckwheat) st Fagopyri in cymost in ABOOO329 140 bp 333 bp – st Fagopyri in cymost in ABOOO328. 140 bp 321 bp — st Fagopyri in cymost in ABOOO327 140 bp 321 bp — st Fagopyri in cymost in ABOOO326 40 bp — st Fagopyri in cymost in ABOOO325 40 bp — 321 bp — st Fagopyri in cymost in ABOOO324 140 bp 333 bp – st Fagopyrim capillatin ABOOO323 40 bp — kFagopyrum callianthum ABOOO322 140 bp

TABLE 1B SEQ NO. 11 & SEQ NO. 3 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Other Common Allergenic Arachis hypogaea (Peanut) AF1566.75 Plants Triticum aestivum (Wheat) AJ301799 Glycine max (Soybean) U60551 227 bp — Jugians regia (Walnut) AF303809 - Tichoioma matsutake U62964 (Matsutake mushroom) Prunus persica (Peach) AF185621 Maius X domestica (Apple) AF186484 - – 275 bp Citrus sp. (Valencia orange) E08821 — 312 bp 215 bp Plants Widely Used for a Food Zea mays (Corn) U46648 Ingredient Oryza sativa (Brown rice) AF169230 - Piper nigrum (Pepper) AF275197 Sinapis alba (Mustard) X15915

B. Preparation of Template DNA for PCR Preparation of Black Pepper Powder Containing Buckwheat 50 Chaff (1) Samples Used for DNA Isolation 0.1 g of ground buckwheat chaff was added to 0.9 g of Buckwheat: ground black pepper to obtain black pepper powder contain Commercially available seeds of Shirahana soba (common ing 10% buckwheat chaff. buckwheat) and Dattan soba (Tartarian buckwheat) were (2) Isolation of DNA from Buckwheat, Buckwheat Chaff. used. 55 Black Pepper, White Pepper and Black Pepper Powder Con taining Buckwheat Chaff Buckwheat Chaff: DNA isolation was conducted by using the QIAGEN Commercially available buckwheat chaffused for pillows Genomic-tip according to the procedures described in the was used. Genomic DNA Handbook with a few modifications thereto as 60 stated below. Pepper: Into a 15 ml-tube was transferred 1 g of a ground sample, Commercially available seeds of black pepper and white added 4 ml of Carlson Lysis Buffer (0.1 M Tris-HCl(pH 9.5), pepper were used. 2% CTAB, 1.4 M Polyethylene Glycol # 6000, 20 mM EDTA), 8 ul of RNase A (100 mg/ml), 10ul of 2-mercapto Soybean, Wheat, Corn and Mustard: 65 ethanol and 80 ul of proteinase K (20 mg/ml) thereto and Leaves that germinated from commercially available seeds mixed, and the resulting mixture was incubated for 20 min. at of soybean, wheat, corn and mustard were used. 740 C. US 7,704,694 B2 15 16 After cooling down to room temperature, to the mixture ppb. 10 ppb, and 1 ppb of buckwheat seed DNA. The DNA was added 5 ml of phenol/chloroform/isoamyl alcohol (25/ preparation from buckwheat seeds was obtained according to 24/1) at room temperature and mixed well by inverting the the procedures described in (2). The DNA preparation from tube. After centrifuging them, a resulting upper water layer, wheat leaves was obtained according to the procedures was collected. The water layer was mixed well with the same described in (3). volume of chloroform/isoamyl alcohol (24/1) and after cen C. PCR trifuging, a resulting upper water layer was collected. The PCR was conducted using the QIAGEN HotStarTaq Mas water layer was mixed well with chloroform/isoamyl alcohol ter Mix Kit according to the procedures described in the (24/1), and after centrifuging, a resulting water layer was HotStarTaq PCR Handbook as stated below. collected again and used in the next step. 10 PCR was carried out using final volumes of 25 ul of a Half of the volume of the water layer obtained above was solution containing 12.5 ul of 2x HotStarTaq Master Mix subjected to isopropanol precipitation to collect crude DNA. (HotStarTaq DNA Polymerase, PCR Buffer with 3 mM The collected crude DNA was dissolved in 500 ul of Buffer MgCl, 400 uM each dNTP), 0.2 uM of each primer (SEQ QBT and the resulting solution was applied to the Genomic NO:11 and SEQ NO:3), the template DNA and sterilized tip 20/G column equilibrated with 1 ml of Buffer QBT to 15 ultrapure water in 0.2-ml microcentrifuge tubes. Amplifica adsorb DNA. Subsequently, the column was washed with 5 tion was performed using a GeneAmp PCR System 9600 ml of Buffer QBT and then with 2 ml of Buffer QC. Finally, (Applied Biosystems) according to the following PCR pro DNA was eluted with 1.7 ml of Buffer QF, and the resulting gram: pre-incubation at 95°C. for 15 min.: 45 cycles consist eluate was subjected to isopropanol precipitation to collect ing of denaturation at 95°C. for 1 min. annealing at 68°C. for DNA, which was then dissolved in 40 ul of sterilized ultra 2 min. and extension at 72°C. for 1 min.; followed by a final pure water. After the concentration of the resulting DNA extension at 72°C. for 4 min. The PCR reaction mixture was preparation was determined, the DNA preparation was used electrophoresed on a 2% agarose gel containing ethidium for a PCR template. bromide. After the electrophoresis, the gel was analyzed (3) Isolation of DNA from Leaves of Wheat, Soybean, Corn 25 using a Fluorimager 595 (Amersham Pharmacia Biotech). and Mustard The results are shown in FIGS. 1-3. The meanings of numeri DNA isolation was conducted by using the QIAGEN cal values, abbreviations and symbols used in FIGS. 1-3 areas DNeasy Plant Mini Kitaccording to the procedures described follows. in the DNeasy Plant Mini Kit Handbook mentioned below. M: 100 bp DNA Ladder Marker 0.5g of a ground sample was transferred to a 15 ml-tube, 30 (-): Negative Control (no DNA) added 3 ml of Buffer AP1 and 30 ul of RNase A (100 mg/ml), Numerical values above sample names: the amounts of the and mixed well with them. Then the resulting mixture was template DNA incubated for 15 min. at 65° C. 975 ul of Buffer AP2 was Arrow: indicates the target amplification product (140 bp) added to the mixture. The resulting mixture was incubated for The quality of each of the template DNA used here was 10 min. on ice and then centrifuged to obtain a Supernatant. 35 sufficient enough to be used for PCR based on the result The supernatant was applied to a QIAshredder Spin Column of a separate PCR, in which target products were and a flow-through fraction was obtained by centrifuging the obtained using a primer pair to amplify a part of a plant column. To the flow-through fraction was added 0.5 volume chloroplast DNA. of Buffer AP3 and 1 volume of ethanol, and mixed. The D. PCR Results resulting mixture was divided into halves to be applied to two 40 PCR described above was conducted using primers separate DNeasy Spin Columns. 650 ul of the mixture was designed in the present invention. The results are shown in applied to a DNeasy Spin Column and the column was cen FIGS. 1-3. As shown in FIG. 1, target 140 bp amplification trifuged for 1 min. at 6,000xg to adsorb DNA. This step was products, predicted from the simulation results of the 21 DNA repeated with the remaining mixture. In order to wash the sequences of the genus Fagopyrum, were obtained from column, to the column was added 500 ul of Buffer AW and 45 Shirahana soba (common buckwheat) and Dattan soba (Tar was centrifuged for 1 min. at 6,000xg. To the column was tarian buckwheat). On the other hand, no 140 bp amplification added 500 uL of Buffer AW again and was centrifuged for 1 product was obtained from wheat, mustard, soybean, corn, min. at a maximum speed to flush out the remaining Buffer and white pepper. However, nonspecific amplification prod AW. Finally, to the column was added 120 ul of preheated ucts whose sizes were different from the target product were (65°C.) Buffer AE and was centrifuged for 1 min. at 6,000xg 50 obtained from soybean (approximately 230 bp) and wheat to obtain a DNA eluate. After the concentration thereof was (approximately 2,300 bp). Because these results almost determined, the DNA eluate was used for a PCR template. matched the simulation results shown in Table 1, the simula (4) Preparation of DNA Solutions for Evaluation of Sensitiv tion results were thought to be reliable. Consequently, it was ity (Buckwheat in Black Pepper Powder) confirmed that a wide range of plants in the genus Fagopyrum A DNA preparation derived from black pepper powder 55 including common buckwheat and Tartarian buckwheat were containing 10% buckwheat chaff was diluted stepwise with a detectable using the present invention. DNA preparation from black pepper to obtain black pepper As shown in FIG. 2, target 140 bp amplification products, DNA solutions containing 1%, 0.1%, 100 ppm, 10 ppm, 1 predicted from the simulation results of the 21 DNA ppm, 100 ppb, and 10 ppb of buckwheat chaff DNA. Both sequences of the genus Fagopyrum, were obtained from black DNA preparations used above were obtained according to the 60 pepper powder samples containing 10 to 1 ppm of buckwheat procedures described in (2). chaff. This result showed that buckwheat chaff present in an amount of more than 1 ppm in black pepper is detectable. (5) Preparation of DNA Solutions for Evaluation of Sensitiv As shown in FIG. 3, the target 140 bp amplification prod ity (Buckwheat in Wheat) ucts, predicted from the simulation results of the 21 DNA A DNA preparation derived from buckwheat seeds was 65 sequences of the genus Fagopyrum, were obtained from the diluted stepwise with a DNA preparation from wheat leaves wheat DNA sample containing 10 to 1 ppm of buckwheat to obtain wheat leaf DNA solutions containing 1 ppm, 100 DNA. US 7,704,694 B2 17 18 The nonspecific amplification product that was obviously sequence, lines underneath this sequence and other symbols different from the target product in size did not interfere in the used in FIG. 4 are described below. detection of 1 ppm of buckwheat DNA. This result showed Number Symbols Nucleotide numbers of amplification prod that buckwheat DNA present in an amount of more than 1 lucts ppm in wheat DNA is detectable. Asterisks (*): Identical nucleotides between two nucleotide E. Preparation of a Sequencing Sample Sequences Dash (-): Unidentifiable nucleotide (1) Purification of the Amplification Product from Buckwheat S: Mixed nucleotide with C and G Chaff Single underline: The sense primer region Purification of the amplification product from buckwheat 10 Double underline: The antisense primer region chaff obtained in section D was conducted by using the QIAGEN QIAquick PCR Purification Kit according to the G. The result of Sequence Analysis procedures described in the QIAquick Spin Handbook as As shown in FIG. 4, the amplification product from buck stated below. wheat chaff obtained by PCR using the primers designed in To 1 volume of PCR reaction mixture was added 5 volumes 15 the present invention almost matched the sequence of com of Buffer PB and mixed well. After being spun down by mon buckwheat, Fagopyrum esculentum (AB000330). 87 out centrifugation, to a QIAquick Spin Column was applied the of89 bases between two primer regions were determined in a mixture and centrifuged for 1 min. at 10,000xg to adsorb sequence of the product and 86 of these bases matched the DNA. Then, to the column was added 750 ul of buffer PE to sequence of common buckwheat If the base 44, determined as wash and centrifuged for 1 min. at 10,000xg. In addition, the a mixture of two bases, is considered to match the sequence of column was centrifuged for 1 min. at 10,000xg to remove common buckwheat, all 87 of these bases matched to the Buffer PE completely. Finally, to the column was added 50 ul sequence of common buckwheat. This result indicates the of Buffer EB, let stand for 1 min., and then centrifuged for 1 target ITS-1-5.8S rRNA gene sequence of plants in the genus min. at 10,000xg. The resulting eluted DNA was used for a Fagopyrum can be detected without fail. Furthermore, the sequencing sample. 25 source of the buckwheat chaff, used in the aforementioned experiments, was traced back to common buckwheat (Fa (2) Sequencing PCR Reaction and Removal of Excess Dye gopyrum esculentum). Labeled Dideoxynucleotides PCR for sequencing of the purified amplification product Example 2 from buckwheat chaff obtained in (1) was conducted by using 30 the Applied Biosystems BigDye Terminator Cycle Sequenc A. Design of Oligonucleotide Primers for Detection of DNA ing FS Ready Reaction Kit according to the procedures from Buckwheat described in the manufacturer's manual as stated below. The PCR for sequencing was carried out using final vol (1) DNA Sequences of the Genus Fagopyrum. Other Com umes of 20 ul of a solution containing 8 ul of BigDye Termi mon Allergenic Plants and Plants Widely Used for a Food nator RRMix, 3.2 umol of primer (SEQNO:3), 2ng template 35 Ingredient DNA and sterilized ultrapure water in 0.2-ml microcentrifuge The DNA sequences described in Example 1 (1) “DNA tubes. Amplification was performed using a GeneAmp PCR Sequences of the Genus Fagopyrum', (2) “DNA Sequences System 9600 (Applied Biosystems) according to the follow of Other Common Allergenic Plants” and (3) “DNA ing PCR program: pre-incubation at 96° C. for 1 min.; 25 Sequences of Plants Widely Used for a Food Ingredient' were cycles consisting of denaturation at 96° C. for 10 secs. and 40 examined to select Suitable regions for the primers. annealing and extension at 60° C. for 1 min. (2) DNA Sequence of Related Species of the Genus Fagopy Subsequently, removal of the excess dye-labeled dideoxy FiFi. nucleotides from the sequencing reaction mixture was con As representatives of the DNA sequences of related species ducted by using the Amersham Pharmacia Biotech AutoSeq 45 of the genus Fagopyrum, 5.8S rRNA gene, 1TS-1 and ITS-2 G-50 according to the procedures described in the manufac sequences in the following 27 DNA sequences registered in turer's manual as stated below. GenBank were selected. In this connection, the 27 DNA The AutoSeqG-50 column was uncapped and 100 ul of 10 sequences were selected as representatives of the DNA mM EDTA was added to the resin in the column. The column sequences of related species of the genus Fagopyrum, each of was then capped and the resin inside was suspended thor 50 which had the highest score in the corresponding genus other oughly by Vortexing. The cap was then loosened and the than genus Fagopyrum and a score of 60 bits or more among bottom closure of the column was snapped off. The column sequences of species belonging to the corresponding genus was then uncapped and placed in a 2-ml microcentrifuge tube for support and centrifuged for 1 min. at 2,000xg. The column selected from sequences registrated in GenBank through a was then placed in a new 2-ml microcentrifuge tube and the BLAST homology search using the ITS-1 sequence of buck 55 wheat (Fagopyrum esculentum AB000330). sample was applied to the resin in the column. After being 1: Aconogonum sp. Won 152 (AF189731) capped, the column was centrifuged for 1 min. at 2,000xg, 2: Fallopia scandens (AF040069) and the flow-through fraction obtained was used in the next 3: Polygonum virginianum (U51274) step. 4. Rumex acetosella (AF189730) F. Sequence Analysis 60 5. Talinum paraguayense (L78056) The sample for sequencing from buckwheat chaff obtained 6. Bruinsmia styracoides (AF396438) in E (2) was analyzed using an ABI PRISM 310 Genetic 7. Talinella pachypoda (L78054) Analyzer (Applied Biosystems). The obtained nucleotide 8. Rehderodendron kwangtungense (AF396448) sequence of the amplification product was compared with the 9. Pterostyrax corymbosus (AF396445) sequence of common buckwheat, Fagopyrum esculentum 65 10: Anredera cordifolia (L78086) (AB000330), registered in GenBank. The result is shown in 11: Cistanthe quadripetala (L78062) FIG. 4. The meanings of number symbols above a nucleotide 12. Xenia vulcanensis (L78060) US 7,704,694 B2 19 20 13: Talinopsis frutescens (L78058) obtained from the aforementioned 8 DNA sequences of other 14: Talinaria palmeri (L78052) common allergenic plants (peanut, wheat, soybean, walnut, 15: Portulaca sp. (L78049) matsutake mushroom, peach, apple and orange), the 4 DNA 16. Phemeranthus confertiflorus (L78039) sequences of plants widely used as a food ingredient (corn, 17: Montiopsis umbellata (L78033) brown Lice, pepper and mustard) and the 2 DNA sequences 18. Grahamia bracteata (L78028) among related species of the genus Fagopyrum belonging to 19. Hermiaria glabra (AJ310965) Polygonaceae and the 23 DNA sequences of related species 20: Alluaudia duwosa (L78011) of the genus Fagopyrum not belonging to Polygonaceae. In 21. Sinojackia xylocarpa (AF396451) this connection, the results of simulation indicated Some pos 22. Halesia macgregori (AF396442) 10 sibility that an amplification product, whose size almost 23. Changiostyrax dolichocarpa (AF396439) matched the target product size of 146 bp, would be obtained 24. Alectryon subdentatus (AF314765) from the sequences of Aconogonum sp. Won 152 and Fallopia 25. Anacampseros recurvata (L78014) scandens in the related species of the genus Fagopyrum 26. Weinmannia racemosa (AF485597) belonging to Polygonaceae. However, by sequence analysis 27: Bursera tecomaca (AF08.0029) 15 of the amplification products, it is possible to identify either (3) Oligonucleotide Primer Synthesis and Evaluation the genus Fagopyrum or the related species thereof. The Among ITS-1 sequences of the aforementioned 21 DNA simulation results are shown in Tables 2A to 2C. The mean sequences of the genus Fagopyrum, there was determined a ings of symbols and numerical values in Tables 2A to 2C are nucleotide sequence which would specifically hybridize to all explained below. of the 21 DNA sequences of the genus Fagopyrum and would k: An obtained amplification product whose size almost not induce nonspecific amplification products from soybean matched the target product size 146 bp (+10 bp). through the study of the ITS-1 sequences. The thus deter W 2-6: Probability of obtaining amplification products mined nucleotide sequence is indicated as SEQ NO:14. Sub High Probability W6>W5>W4->W3>W Low Prob sequently, the oligonucleotide primer with SEQ NO:14 was 25 ability synthesized. Numerical values followed by bp: Each value was obtained by subtracting 2 from the value Sense primer obtained in the simulation. 5 - CGC CAA. GGA, CCA CGA ACA GAA. G-3 (SEQ NO:14) (-): No amplification product was predicted. 30 The oligonucleotide primer with SEQ NO:3 was also used Related Species of the genus Fagopyrum. as an antisense primer, the same as in Example 1. Sequences similar to the ITS-1 sequence of Fagopyrum Regarding the sense and antisense primer pair, the simula esculentum (AB000330) were searched by means of a tion was conducted with PCR simulation software, Amplify BLAST homology search and the sequences having a 1.0 (Bill Engels), which is the same as in Example 1. As a score of 60 bits or more were selected from among them. result, it was predicted that target 146 bp amplification prod 35 Each sequence having the highest score in each genus ucts would be obtained from the aforementioned 21 DNA and having a score of 60 bits or more is shown in the sequences of the genus Fagopyrum. In contrast, obtaining of following Table 2C as the representative of the DNA any 146 bp amplification product was not predicted to be sequences of related species of the genus Fagopyrum.

TABLE 2A Buckwheat, SEQ NO: 14 & SEQ NO. 3 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Genus Fagopyrim kFagopyrum urophyllum ABOOO342 1.46 bp 439 bp — kFagopyrum urophyllum ABOOO341 46 bp kFagopyrum tataricum ABOOO34O 146 bp (Tartarian buckwheat) kFagopyrum tataricum ABOOO339 146 bp (Tartarian buckwheat) st Fagopyrim Statice ABOOO338 146 bp st Fagopyrim Statice ABOOO337 146 by st Fagopyrim pieioranostin ABOOO336 146 bp kFagopyrum lineare ABOOO335 146 bp st Fagopyrim lepiopodium ABOOO334 146 bp kFagopyrum homotropicum ABOOO333 146 bp st Fagopyrim gracipes ABOOO332 1.46 bp st Fagopyrim escientiin ABOOO331 46 bp (Common buckwheat) st Fagopyrim escientiin ABOOO33O 146 bp (Common buckwheat) st Fagopyri in cynostin ABOOO329 146 bp st Fagopyri in cynostin ABOOO328. 146 bp st Fagopyri in cynostin ABOOO327 146 by st Fagopyri in cynostin ABOOO326 146 bp st Fagopyri in cynostin ABOOO325 146 bp st Fagopyri in cynostin ABOOO324 146 bp US 7,704,694 B2

TABLE 2A-continued

Buckwheat, SEQNO: 14 & SEQNO:3 primer: Amplification products

GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2

st Fagopyrim capillatin AB000323 146 bp kFagopyrum callianthum AB000322 146 bp 439 bp —

TABLE 2B Buckwheat, SEQNO: 14 & SEQNO:3 primer: Amplification products

GenBank Scientific Name Accession (Common Name) No. W6 WS WA. W3 W2 Other Common Allergenic Arachis hypogaea (Peanut) AF1566.75 Plants Triticum aestivum (Wheat) AJ301799 Glycine max (Soybean) U60551 Jugians regia (Walnut) AF303809 - Tichoioma matsutake U62964 (Matsutake mushroom) Prunus persica (Peach) AF185621 Maius X domestica (Apple) AF186484 - Citrus sp. (Valencia orange) E08821 Plants Widely Used for a Food Zea mays (Corn) U46648 Ingredient Oryza sativa (Brown rice) AF169230 - Piper nigrum (Pepper) AF275197 Sinapis alba (Mustard) X15915

TABLE 2C Buckwheat, SEQ NO: 14 & SEQ NO. 3 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Related Species of the Genus kAconogonum sp. Won 152 AF189731 — 149bp — Fagopyrim Belonging to kFallopia scandens AF040069 – 145 bp – Plygonaceae Polygontin virginianum US 1274 Rumex acetoseiia AF18973O Related Species of the Genus Fagopyrim Taintin paraguayense L78056 Not Belonging to Polygonaceae Bruinsmia styracoides AF396438 - Taiinelia pachypoda L78054 Rehderodendron kwangtungense AF396448 — Pterostyrax corymbosus AF3964.45 Anredera cordifolia L78086 Cistanthe quadripetalia L78062 Xenia vulcanensis L78060 Tainopsis finitesCens L78058 Tainaria palmeri L78052 Portulaca sp. L78049 Phemeranthus confertifiorus L78039 Montiopsis umbellata L78O33 Grahamia bracteata L78028 Henniaria glabra A31096S Alluaudia dimosa L78O11 Sinojackia xylocarpa AF3964S1 Halesia macgregori AF396442 - Changiostyrax doichocarpa AF396439 - Alecinyon subdentatus AF314765 Anacampseros recurvata L78O14 Weinmannia racemosa AF485597 Birsera teconaca AFO80O29 US 7,704,694 B2 23 24 B. Preparation of Template DNA for PCR E. Preparation of a Sequencing Sample The DNA samples isolated from buckwheat, pepper, (1) Purification of the Amplification Product from Shirahana wheat, soybean, corn and mustard in Examples 1B (2) and (3) Soba (Common Buckwheat) and the DNA solutions for evaluation of sensitivity prepared Purification of the amplification product from Shirahana in Example 1B (5) were used. Soba (common buckwheat) obtained in D was conducted in C. PCR the same way as in Example 1E PCR was conducted in the same way as in Example 1C, (1) “Purification of the Amplification Product from Buck except for the use of the following primer and PCR program. wheat Chaff. 10 (2) Sequencing PCR Reaction and Removal of Excess Dye Primer: Labeled Dideoxynucleotides Each primer of SEQNO:14 and SEQNO:3 was used at 0.2 PCR for sequencing of the purified amplification product uM of final concentration. from Shirahana soba (common buckwheat) obtained in (1) was conducted in the same way as in Example 1E (2) PCR Program: “Sequencing PCR Reaction and Removal of Excess Dye PCR was conducted according to the following PCR pro 15 Labeled Dideoxynucleotides”, except for the use of primers gram. of SEQ NO:14 and SEQ NO:3). Pre-incubation at 95°C. for 15 min...: thereafter 45 cycles F. Sequence Analysis consisting of denaturation at 95°C. for 1 min., annealing at The sample for sequencing from Shirahana Soba (common 66°C. for 2 min. and extension at 72°C. for 1 min.; followed buckwheat) obtained in E (2) was analyzed using an ABI by a final extension at 72°C. for 4 mm. PRISM 310 Genetic Analyzer (Applied Biosystems). The The results are shown in FIGS. 5 and 6. The meanings of obtained nucleotide sequence of the amplification product numerical values, abbreviations and symbols used in FIGS. 5 was compared with the sequence of common buckwheat, and 6 are as follows. Fagopyrum esculentum (AB000331) and F homotropicum M: 100 bp DNA Ladder Marker 25 (AB000340) in GenBank. The result is shown in FIG. 7. The (-): Negative control (no DNA) meanings of number symbols above a nucleotide sequence, Numerical values above sample names: the amounts of the lines underneath this sequence and other symbols used in template DNA FIG. 7 are described below. Arrow: indicating the target amplification product (146 bp) Number Symbols: Nucleotide numbers of amplification 30 products The quality of each of the template DNAs used here was sufficient enough to be used for PCR based on the result Asterisks (*): Nucleotides of Shirahana soba (common buck of a separate PCR, in which target products were wheat), which are identical with those of both nucleotide obtained using a primer pair to amplify a part of plant sequences of F esculentum (AB000331) and F homotro chloroplast DNA. picum (AB000340) 35 *: Nucleotides of Shirahana soba (common buckwheat), D. PCR Results which are identical with those of only one of the nucleotide PCR described above was conducted using primers sequences of F esculentum (AB000331) and F homotro designed in the present invention. The results are shown in picum (AB000340) FIGS.5 and 6. As shown in FIG. 5, target 146 bp amplification Single underline The sense primer region products, predicted from the simulation results of the 21 DNA 40 Double underline The antisense primer region sequences of the genus Fagopyrum, were obtained from G. The result of Sequence Analysis Shirahana soba (common buckwheat) and Dattan soba (Tar As shown in FIG. 7, all 89 bases between two primer tarian buckwheat). On the other hand, no 146 bp amplification regions in the amplification product derived from Shirahana product was obtained from wheat, mustard, soybean, corn, 45 soba (common buckwheat) by PCR using the primers and white pepper (nonspecific amplification obtained from designed in the present invention are completely identical Soybean in Example 1 was not obtained either). Likewise, no with one of sequences of common buckwheat, Fesculentum 146 bp amplification product was obtained from brown rice (AB000331) or F homotropicum (AB000340). This result (not shown in FIG. 5). However, nonspecific amplification indicates the target ITS-1-5.8S rRNA gene sequence of products whose sizes were different from that of the target 50 plants in the genus Fagopyrum can be detected without fail. product were obtained from wheat (approximately 2,300 bp). Furthermore, the source of the Shirahana soba (common Because these results almost matched the simulation results buckwheat), used in the aforementioned experiments, was shown in Table 2, the simulation results were thought to be traced back to Fagopyrum esculentum or F homotropicum. reliable. Consequently, it was confirmed that a wide range of plants in the genus Fagopyrum including shirahana Soba Example 3 (common buckwheat) and Tartarian buckwheat were detect 55 able using the present invention. As shown in FIG. 6, the target 140 bp amplification prod A. Design of Oligonucleotide Primers for Detection of DNA uct, predicted from the simulation results of the 21 DNA from Peanut sequences of the genus Fagopyrum, was obtained from the 60 (1) DNA Sequences of the Genus Arachis wheat DNA sample containing 10 to 1 ppm of buckwheat Regarding the genus Arachis, 5.8S rRNA gene, ITS-1 and DNA. ITS-2 sequences in the following 11 DNA sequences regis The nonspecific amplification product that was obviously tered in GenBank were examined to select suitable regions for different from the target in size did not interfere in the detec the primers. tion of 1 ppm of buckwheat DNA. This result showed that 65 1: Arachis batizocoi (AF203553) buckwheat DNA present in an amount of more than 1 ppm in 2: Arachis correntina (AF203554) wheat DNA is detectable. 3. Arachis hermannii (AF203556) US 7,704,694 B2 25 26 4. Arachis hoehnei (AJ320395) 17. Stylosanthes hispida (AJ320328) 5: Arachis hypogaea (AF156675) 18. Stylosanthes humilis (AJ320323) 6. Arachis magna (AF203555) 19. Stylosanthes ingrata (AJ320329) 7: Arachis major (AF203552) 2O. Stylosanthes leiocarpa (AJ320332) 8. Arachis palustris (AF203557) 21. Stylosanthes linearifolia (AJ320367) 9. Arachis pintoi (AF203551) 22. Stylosanthes macrocarpa (AJ320369) 10: Arachis triseminata (AF204233) 23. Stylosanthes macrocephala (AJ320371) 11: Arachis villosa (AF203558) 24. Stylosanthes macrosoma (AJ320333) (2) DNA Sequences of Other Common Allergenic Plants 25. Stylosanthes mexicana (AJ320374) The DNA sequences described in Example 1A (2) “DNA 10 26. Stylosanthes montevidensis (AJ320336) Sequences of Other Common Allergenic Plants' were 27. Stylosanthes pilosa (AJ320377) selected. Regarding buckwheat, 5.8S rRNA gene, ITS-1 and 28. Stylosanthes scabra (AJ320382) ITS-2 sequences in the following DNA sequence registered in 29. Stylosanthes seabrana (AJ320384) GenBank were also selected. 15 30. Stylosanthes sericeiceps (AJ320386) 1: buckwheat: Fagopyrum esculentum (AB000330) 31. Stylosanthes subsericea (AJ320387) (3) DNA Sequences of Plants Widely Used for a Food Ingre 32. Stylosanthes Sundaica (AJ320389) dient 33. Stylosanthes sympodialis (AJ320391) The DNA sequences described in Example 1A (3) “DNA 34. Stylosanthes tomentosa (AJ320337) Sequences of Plants Widely Used for a Food Ingredient' were 35. Stylosanthes tuberculata (AJ320392) selected. 36. Stylosanthes viscosa (AJ320340) (4) DNA Sequences of Leguminous Plants Widely Used for a 37. Ormocarpum bernierianum (AF189036) Food Ingredient 38. Ormocarpum coeruleum (AF189037) Regarding French bean, lima bean, lentil, chickpea, mung 39. Ormocarpum drakei (AF189039) bean and adzuki bean, 5.8S rRNA gene. ITS-1 and ITS-2 25 40. Ormocarpum flavum (AF189041) sequences in the following DNA sequences registered in 41. Ormocarpum keniense (AF068.155) GenBank were selected. In the case of adzuki bean, only the 42. Ormocarpum kirkii (AF068152) ITS-1 sequence of Vigna angularis var. nipponensis 43. Ormocarpum klainei (AF189044) (AB05.9747) was selected because the 5.8S rRNA gene 30 44. Ormocarpum megalophyllum (AF068.154) sequence was not registered in GenBank. 45. Ormocarpum muricatum (AF068156) 1: French bean: Phaseolus vulgaris (AF115169) 2: lima bean: Phaseolus lunatus (AF115175) 46. Ormocarpum orientale (AF068159) 3: lentil: Lens culinaris subsp. culinaris (AF228066) 47. Ormocarpum pubescens (AF189045) 4: chickpea: Cicer arietinum (AJ237698) 48. Ormocarpum rectangulare (AF189046) 5: mung bean: Vigna radiata (X14337) 35 49. Ormocarpum schliebenii (AfE189047) 6: adzuki bean: Vigna angularis var. nipponensis 50. Ormocarpum sennoides (AF068153) (AB05.9747) 51 Ormocarpum Somalense (AF 189048) (5) DNA Sequences of Plants in Related Species of the Genus 52. Ormocarpum trachycarpum (AF189049) Arachis 40 53. Ormocarpum trichocarpum (AF068.158) As representatives of the DNA sequences of related species 54. Ormocarpum verrucosum (AF189050) of the genus Arachis, 5.8S rRNA gene, ITS-1 and ITS-2 55. Chapmannia floridana (AF203543) sequences in the following 69 DNA sequences registered in 56. Chapmannia prismatica (AJ320400) GenBank were selected. In this connection, the 69 DNA 57. Chapmannia Somalensis (AF203544) sequences were selected as representatives of the DNA 45 58. Ormocarpopsis aspera (AF068148) sequence of related species of the genus Arachis, each of 59. Ormocarpopsis calcicola (AF068145) which had the highest score in the corresponding genus other 60. Ormocarpopsis itremoensis (AF068149) than genus Arachis and a score of 60 bits or more among sequences of species belonging to the corresponding genus 61. Ormocarpopsis mandrarensis (AF068.147) 62. Ormocarpopsis parvifolia (AF068144) selected from ITS-1 sequence of buckwheat through BLAST 50 homology search. (Arachis hypogaea AF 156675) 63. Ormocarpopsis tulearensis (AF068146) 1. Stylosanthes acuminata (AJ320282) 64. Diphysa humilis (AF068162) : Stylosanthes angustifolia (AJ320284) 65. Diphysa macrophylla (AF189029) * Stylosanthes aurea (AJ320285) 66. Diphysa suberosa (AF189034) Stylosanthes biflora (AJ320289) 55 67. Spigelia coelostylioides (AF177992) * Stylosanthes bracteata (AJ320346) 68. Spigelia hedyotidea (AF 178005) * Stylosanthes calcicola (AJ320348) 69. Spigelia marilandica (AF177991) * Stylosanthes campestris (AJ320291) * Stylosanthes capitata (AJ320350) (6) Oligonucleotide Primer Synthesis and Evaluation 9.* Stylosanthes cavennensis (AJ320292) 60 Among ITS-1 sequences of the aforementioned 11 DNA 10: Stylosanthes erects (AJ320352) sequences of the genus Arachis, there was determined three : Stylosanthes fruticosa (AJ320356) nucleotide sequences which would specifically hybridize to 12. Stylosanthes gracilis (AJ320296) all of the 11 DNA sequences of the genus Arachis through the 13. Stylosanthes grandifolia (AJ320299) study of the ITS-1 sequences. The thus determined nucleotide 14. Stylosanthes guianensis subsp. dissitiflora (AJ320301) 65 sequences are indicated as SEQ NOs: 18, 19 and 20. Subse 15. Stylosanthes hamata (AJ320365) quently, the oligonucleotide primers with SEQ NOs: 18, 19 16. Stylosanthes hippocampoides (AJ320317) and 20 were synthesized. US 7,704,694 B2 27 28 obtained from 7 DNA sequences of common allergenic plants other than peanut (buckwheat, wheat, soybean, walnut, mat Sense primers Sutake mushroom, peach and orange), the 4 DNA sequences 5 " - GCG GAA AGC GCC AAG GAA. GC-3' (SEQ NO:18) of plants widely used as a food ingredient (corn, brown rice, 5 - CGG CTT CCG GAG ACG GCA-3' (SEQ NO:19) 5 pepper and mustard) and the 6 DNA sequences of leguminous plants widely used for a food ingredient (French bean, lima 5 - CGG CTC CGG AGA CGG CA-3' (SEQ NO: 2O) bean, lentil, chickpea, mung bean and adzuki bean). In this As an antisense primer, the oligonucleotide primer with connection, the results of the simulation indicated Some pos SEQ NO:3 was also used, the same as in Example 1. sibility that amplification products having almost 156 bp Regarding the sense and antisense primer pairs, a simula 10 would be obtained from apple from among the other common tion was conducted with PCR simulation software, Amplify allergenic plants. However, by a sequence analysis of the 1.0 (Bill Engels), in the same manner as in Example 1. As a amplification products, it is possible to identify either peanut result, it was predicted that 156 to 157 bp (a combination of or apple. The simulation results are shown in Tables 3A and the primers with SEQ NOs: 18 and 3), 114 to 116 bp (a 3B. The meanings of symbols and numerical values in Tables combination of the primers with SEQNOS:19 and 3) and 113 15 3A and 3B are explained below. to 115 bp (a combination of the primers with SEQ NOs:20 k: An obtained amplification product whose size almost and 3) of target amplification products would be obtained matched the target product size 156 bp (+10 bp). from the aforementioned 11 DNA sequences of the genus W 2-6: Probability of obtaining amplification products Arachis. High Probability W6>W5>W4>W3>W2 Low Prob Furthermore, regarding the sense and antisense primer ability pairs, it was predicted whether amplification products would Numerical values followed by bp: be obtained from the aforementioned 8 DNA sequences of Each value was obtained by subtracting 2 from the value common allergenic plants other than peanut (buckwheat, obtained in the simulation. wheat, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used as a 25 (-): No amplification product was predicted. food ingredient (corn, rise, pepper and mustard) and the 6 (-): No annealing site of the primer (SEQ NO: 18) was DNA sequences of leguminous plants widely used for a food predicted within the ITS-1 sequence of Vigna angularis ingredient (French bean, lima bean, lentil, chickpea, mung var. nipponensis (adzuki bean). bean and adzuki bean). In the case of adzuki bean, only the ITS-1 sequence was Regarding the combination of the primers with SEQ NOs: 30 selected because the 5.8S rRNA gene sequence of Vigna 18 and 3, the result of a simulation indicated that desired angularis var. nipponensis (AB059747) was not regis amplification products having almost 156 bp would not be tered in GenBank.

TABLE 3A Peanut, SEQ NO: 18 & SEQ NO. 3 primer: Amplification products

Scientific Name GenBank (Common Name) Accession No. W6 W5 W4 W3 W2 Genus Arachis Arachis fatizocoi AF2O3SS3 156 bp Arachis correntina AF2O3SS4 156 bp Arachis hermannii AF2O3SS6 156 bp Arachis hoehnei AJ32O395 156 bp y Arachis hypogaea AF1566.75 156 bp (Peanut) kArachis magna AF2O3555 156 bp kArachis major AF2O3SS2 156 bp kArachis palustris AF2O3557 156 bp kArachis pintoi AF2O3SS1 157 bp Arachis iriseminata AF2O4233 156 bp Arachis viliosa AF2O3SS8 156 bp Other Common Allergenic Plants Fagopyrum esculentum AB000330 (Common buckwheat) Trictim aestivitin AJ301799 (Wheat) Glycine max U60551 (Soybean) Jugians regia AF303809 (Walnut) Tricholoma matsutake U62964 (Matsuttake mushroom) Prunus persica AF185621 (Peach) Maius X domestica AF1864.84 155 bp — 424 bp — (Apple) 467 bp Citrus sp. E08821 (Valencia orange) US 7,704,694 B2 29 30

TABLE 3B Peanut, SEQ No. 18 & SEQ No. 3 primer: Amplification products Scientific Name GenBank (Common Name) Accession No. W6 WS WA. W3 W2 Plants Widely Used Zea mayS U46648 for a Food Ingredient (Corn) Oryza sativa AF169230 (Brown rice) Piper nigrum AF275197 (Pepper) Sinapis alba X15915 (Mustard) Leguminous Plants Widely Used for a Phaseolus vulgaris AF115169 Food Ingredient (French bean) Phaseolus iunatus AF115175 (Lima bean) Lens culinaris Subsp. AF228.066 cuinaris (Lentil) Cicer arrieintin A237698 (Chickpea) Vigna radiata X14337 (Mung bean) Vigna angularis var. ABOS9747 nipponensis (Adzuki bean)*

Regarding the combination of the primers with SEQ NOs: simulation results are shown in Tables 4A to 4E. The mean 20 and 3, no amplification product having almost 114 bp was ings of symbols and numerical values in Tables 4A to 4E are predicted from 6 DNA 5 sequences of common allergenic 30 explained below. As to the simulation in which SEQNO: 19 and SEQNO:3 plants other than peanut (wheat, walnut, matsutake mush were used, the result is not shown here because it was later room, peach, apple and orange), the 4 DNA sequences of found that this primer pair was not suitable for PCR analysis plants widely used as a food ingredient (corn, brown lice, to detect the genus Arachis. pepper and mustard) and the 5 out of 6 DNA sequences of 35 k: An obtained amplification product whose size almost leguminous plants widely used for a food ingredient (French matched the target 114 bp (+10 bp). bean, lima bean, lentil, chickpea and mung bean). In this W 2-6: Probability of Obtaining Amplification Products connection, the results of the simulation indicated Some pos High Probability W6>W5>W4>W3>W2 Low Prob sibility that nonspecific amplification products having almost ability 40 Numerical values followed by bp: 114 bp would be obtained from buckwheat and from soybean Each value was obtained by subtracting 2 from the value from among the other common allergenic plants and from obtained in the simulation. adzuki bean in the leguminous plants widely used for a food (-): No amplification product was predicted. ingredient in even weak amplified signals. Here, regarding Related Species of the genus Arachis: the DNA sequence of adzuki bean (Vigna angulans var. nip 45 Sequences similar to the ITS-1 sequence of Arachis hypogaea (AF156675) were searched by means of a ponensis AB05.9747), as the ITS-1 sequence was registered in BLAST homology search and the sequences having a GenBank, but the 5.8S rRNA gene sequence was not regis score of 60 bits or more were selected among them. Each tered therein, the amplification product having almost 100 bp sequence having highest score in each genus and having was estimated based on the predicted annealing site of SEQ a score of 60 bits or more is shown in the following NO: 20 to the ITS-1 sequence and the assumption that Vigna Tables 4D-4E as the representatives of the DNA angulans var. nipponensis (adzuki bean) had the same 5.8S sequences of related species of the genus Arachis. rRNA gene sequence as Arachis hypogaea (peanut) and the (+): An annealing site of the primer (SEQ NO: 20) was primer with SEQ NO:3 had an annealing site within the 5.8S predicted within the ITS-1 of Vigna angularis var. nippon rRNA gene sequence. However, despite the prediction of 55 ensis (adzuki bean). amplification, the probability of obtaining these amplification In the case of adzuki bean, only the ITS-1 sequence regis tered in GenBank was used because the 5.8S rRNA gene products compared with obtaining the target amplification sequence of Vigna angularis var. nipponensis product of the genus Arachis were lower than the probability (AB05.9747) was not registered in GenBank. Further of obtaining amplification for peanut, and by a sequence more, the size of the amplification product (approxi analysis of the amplification products, it is also possible to 60 mately 100 bp) was estimated based on the predicted identify whether they are peanut or not. annealing site of the SEQ NO: 20 within the ITS-1 sequence and the assumption described below. It was In addition, regarding the combination of the primers with assumed that Vigna angularis var. nipponensis (adzuki SEQNOS:20 and 3, no amplification products having almost bean) had the same 5.8S rRNA gene sequence as Arachis 100 bp were predicted from the 69 DNA sequences of related 65 hypogaea (peanut) and the primer with the SEQ NO: 3 species of both the genus Arachis belonging to leguminous had an annealing site within the 5.8S rRNA gene plants and those not belonging to leguminous plants. The Sequence. US 7,704,694 B2 31 32

TABLE 4A

Peanut, SEQNO:20 & SEQNO:3 primer: Amplification products

GenBank Scientific Name Accession (Common Name) No.

Genus Arachis Arachis batizocoi 114 bp — 281 b 52 bp Arachis correntina 114 bp — 282 b Arachis hermannii 114 bp — 281 b Arachis hoehnei 114 bp — 284 b y Arachis hypogaea F156675 – 113 bp 286 b (Peanut) kArachis magna 114 bp — 282 b kArachis major 114 bp — 281 b kArachis palustris 114 bp — 282 b kArachis pintoi 115 bp – 283 b Arachis iriseminata 114 bp — 281 b Arachis viliosa 114 bp – 283 b Other Common Allergenic Plants st Fagopyrim escientiin 109 b (Common buckwheat) Trictim aestivum A. 301799 247 b (Wheat) 154 b Glycine max U60551 (Soybean) Jugians regia AF303809 (Walnut) Trichoioma matsutake U62964 (Matsutake mushroom) Prunus persica AF185621 (Peach) Maius X domestica AF1864.84 (Apple) Citrus sp. E08821 (Valencia orange)

TABLE 4B Peanut, SEQ NO. 20 & SEQ NO. 3 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 WS W4 W3 W2 Plants Widely Used Zea mayS U46648 for a Food Ingredient (Corn) Oryza sativa AF169230 (Brown rice) Piper nigrum AF275197 (Pepper) Sinapis alba X15915 (Mustard) Leguminous Plants Widely Used Phaseolus vulgaris AF115169 - for a Food Ingredient (French bean) Phaseolus lunatus AF115175 (Lima bean) Lens culinaris Subsp. AF228.066 — Culinaris (Lentil) Cicerarietintin A237698 (Chickpea) Vigna radiata X14337 (Mung bean) k Vigna angularis var. AB05.9747 +* (approximately 100 bp) nipponensis (Adzuki bean)* US 7,704,694 B2 33 34

TABLE 4C Peanut, SEQ NO. 20 & SEQ NO. 3 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 Related Species of the Genus Arachis Stylosanthes actiminata 32O282 Belonging to Leguminous Plants Stylosanthes angustifolia 32O284 Stylosanthes at trea 32O28S Stylosanthes biflora 32O289 Stylosanthes bracteata 32O346 Stylosanthes calcicola 32O348 Stylosanthes Campestris 32O291 Stylosanthes Capitata 32O350 217 bp 351 bp 384 bp Stylosanthes Cayennensis Stylosanthes erecta Stylosanthes finiticosa Stylosanthes gracilis Stylosanthes grandifolia Stylosanthes guianensis subsp. dissitiflora Stylosanthes hanaia 32O36S Stylosanthes hippocampoides 32O317 Stylosanthes hispida 32O328 Stylosanthes humilis 32O323 Stylosanthes ingrata 32O329 Stylosanthes leiocarpa 32O332

TABLE Peanut, SEQ NO. 20 & SEQ NO. 3 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W3 Related Species of the Genus Arachis Stylosanthes linearifolia 32O367 Belonging to Leguminous Plants Stylosanthes macrocarpa 32O369 Stylosanthes macrocephala 32O371 217 bp 384 bp Stylosanthes macrosoma 32O333 Stylosanthes mexicana 32O374 Stylosanthes montevidensis 32O336 Stylosanthes pilosa 32O377 Stylosanthes scabra 32O382 Stylosanthes Seabrana 32O384 Stylosanthes sericeiceps 32O386 Stylosanthes subsericea 32O387 Stylosanthes Sundaica 32O389 Stylosanthes Sympodialis 32O391 Stylosanthesiomentosa 32O337 Stylosanthes tuberculata 32O392 Stylosanthes viscosa 32O340 Ormocarpum bernierianum F189036 Ormocarpum coeruleum F189037 Ormocarpum drakei F189039 Ormocarpum flavum F189041 Ormocarpum keniense FO68155 Ormocarpum kirkii FO68152 Ormocarpum kiainei F189044

TABLE 4E Peanut, SEQ NO: 18 & SEQ NO. 3 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W3 Related Species of the Genus Arachis Ormocarpum megalophyllum AFO68154 Belonging to Leguminous Plants Ormocarpum muricatum AFO681S6 260 bp Ormocarpum orientale AFO68159 Ormocarpin pubescens AF189045 215 bp Ormocarpum rectangulare AF189046 US 7,704,694 B2 35 36

TABLE 4E-continued Peanut, SEQ NO: 18 & SEQ NO. 3 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. Ormocarpum Schliebenii F189047 Ormocarpum Sennoides FO68153 Ormocarpiim Somaliense F189048 Ormocarpum trachycarpum F189049 Ormocarpum trichocarpum FO681.58 Ormocarpum verrucosum F1890SO Chapmannia floridana F2O3S43 Chapmannia prismatica 3204OO Chapmannia Somaliensis F2O3S44 Ormocarpopsis aspera FO68148 Ormocarpopsis calcicola FO681.45 Ormocarpopsis it remoensis FO681.49 Ormocarpopsis mandrarensis FO68147 Ormocarpopsis parvifolia FO68144 Ormocarpopsis titlearensis FO68146 Diphysa humilis FO68162 Diphysa macrophylla F189029 Diphysa Suberosa F189034 Related Species of the Genus Spigeia coeliostylioides F177992 Arachis Not Belonging to Spigeia heavoidea F178005 Leguminous Plants Spigeia marilandica F177991

B. Preparation of Template DNA for PCR concentration of the resulting DNA preparation was deter (1) Samples Used for DNA Extraction mined, the DNA preparation was used for a PCR template. 30 Peanut: (3) Isolation of DNA from Leaves of Shirahana Soba (Com 6 commercially available peanuts were used. mon Buckwheat), Wheat, Soybean, Adzuki Bean and Corn Buckwheat, Wheat, Soybean, Adzuki Bean and Corn: DNA isolation was conducted by using the QIAGEN Leaves that germinated from commercially available seeds DNeasy Plant Mini Kit according to the procedures described of Shirahana Soba (common buckwheat), wheat, 2 soybeans, 35 in the DNeasy Plant Mini Kit Handbook as shown below. 2 adzuki beans and corn were used. Into a 1.5 ml-tube 50 mg of a ground sample was trans (2) Isolation of DNA from Peanut ferred, added 600 ul of Buffer AP1 and 6 ul of RNase A (100 DNA isolation was conducted by using the QIAGEN mg/ml), and they were mixed well. Then the resulting mixture Genomic-tip and the resulting isolate was purified by MACH 40 was incubated for 1 hour at 65° C. 2001c1 of Buffer AP2 was EREYNAGEL NucleoSpin as stated below. then added to the mixture. The resulting mixture was incu Into a 15 ml-tube 1 g of a ground sample was transferred, bated for 10 min. on ice and then centrifuged to obtain a added 10 ml of Buffer G2, 100 ul of Proteinase K (20 mg/ml) Supernatant. The resulting Supernatant was applied to a and 10ul of RNase A (100 mg/ml), and they were mixed. The QIAshredder Spin Column and a flow-through fraction was resulting mixture was incubated for 1 hour at 50° C. Then the 45 obtained by centrifuging the column To the flow-through resulting mixture was centrifuged for 10 min. at 3,000xg to obtain a Supernatant. The resulting Supernatant was applied to fraction was added 0.5 volume of Buffer AP3 and 1 volume of the Genomic-tip 20/G column equilibrated with 1 ml of ethanol, and mixed with them. The resulting mixture was Buffer QBT to adsorb DNA to the column. Subsequently, the divided into two halves to be applied to two DNeasy Spin column was washed with 4 ml of Buffer QC and DNA was 50 Columns. 650 ul of the mixture was applied to a DNeasy Spin eluted with 1 ml of preheated (50° C.) Buffer QF. To the eluate Column and the column was centrifuged for 1 min. at 6,000xg was added 4 volume of Buffer NT2, mixed with it, and then to adsorb DNA to the column. This step was repeated with the the resulting mixture was divided into two halves to be remaining mixture. In order to wash the column, to the col applied to two separate NucleoSpin Extract Columns. 650 ul umn was added 500 ul of Buffer AW and was centrifuged for of the mixture was applied to a NucleoSpin Extract Column 55 1 min. at 6,000xg. To the column was added 500 ul of Buffer and then the column was centrifuged for 1 min. at 6,000xg to AW again and was centrifuged for 1 min. at a maximum speed adsorb DNA to the column. This step was repeated with the to flush out the Buffer AW remaining in the column. Finally, remaining mixture. In order to wash the column, to the col umn was added 600 ul of Buffer NT3 and was centrifuged for to the column was added 100 ul of preheated (65°C.) Buffer 1 min. at 6,000xg. To the column was added 600 ul of Buffer 60 AE and was centrifuged for 1 min. at a maximum speed, and NT3 again and was centrifuged for 1 min. at a maximum added another 100 ul of preheated (65°C.) BufferAE and was speed to flush out the Buffer NT3 remaining in the column. centrifuged for 1 min. at a maximum speed to obtain a DNA Finally, to the column was added 100 ul of Buffer NE and was eluate from the column, and the resulting eluate was Sub centrifuged for 1 min. at a maximum speed to obtain a DNA jected to isopropanol precipitation to collect DNA, which eluate from the column and the resulting eluate was subjected 65 were then dissolved in 50 ul of sterilized ultrapure water. to isopropanol precipitation to collect DNA, which were then After the concentration of the resulting DNA preparation was dissolved in 40 ul of sterilized ultrapure water. After the determined, the DNA eluate was used for a PCR template. US 7,704,694 B2 37 38 (4) Preparation of DNA Solutions for Evaluation of Sensitiv F. Sequence Analysis (Part 1: a combination of primers with ity (Peanut in Wheat) SEO NOs: 18 and 3) A DNA preparation derived from peanut seeds was diluted The sample for sequencing from peanut obtained in E (2) stepwise with a DNA preparation from wheat leaves to obtain was analyzed using an ABI PRISM 310 Genetic Analyzer wheat leaf DNA solutions containing 10 ppm and 1 ppm of 5 (Applied Biosystems). The obtained nucleotide sequence of peanut seed DNA. The DNA preparation from peanut seeds the amplification product was compared with the sequence of was obtained according to the procedures described in (2). the genus Arachis, Arachis hypogaea (AF156675), A. corren The DNA preparation from wheat leaves was obtained tina (AF203554) and A. villosa (AF203558) in GenBank. The according to the procedures described in (3). result is shown in FIG. 9. The meanings of number symbols 10 above a nucleotide sequence, lines underneath a sequence and C. PCR (Part 1: a combination of primers with SEO NOs: 18 other symbols used in FIG. 9 are described below. Number Symbols: Nucleotide numbers of amplification and 3) products PCR was conducted using the Applied Biosystems Ampli Asterisks (*): Nucleotides of the peanuts, which are identical Taq Gold(R) & 10xPCR Buffer II & MgCl, Solution with 15 with those of all of the nucleotide sequences of Arachis dNTP as Stated below. hypogaea (AF156675), A. correntina (AF203554) and A. PCR was carried out using final volumes of 25 ul of a villosa (AF203558) in GenBank solution containing 2.5ul of 10xPCR Buffer II, 0.125 ul of Single underline: The sense primer region AmpliTaq Gold (5 U?ul), 2.5ul of dNTPs Mix (2 mM each), Double underline: The antisense primer region 1.5 ul of MgCl, Solution (25 mM), 0.5 LM of each primer (SEQ NO:18 and SEQ NO:3), the template DNA and steril G. The result of Sequence Analysis (Part 1: a combination of ized ultrapure water in 0.2-ml microcentrifuge tubes. Ampli primers with SEO NOs: 18 and 3) fication was performed using a GeneAmp PCR System 2400 As shown in FIG. 9, the amplification products derived (Applied Biosystems) according to the following PCR pro from all of the 6 commercially available peanuts obtained by gram: pre-incubation at 95°C. for 15 min.: 45 cycles consist 25 PCR using the primers designed in the present invention ing of denaturation at 95°C. for 1 min. annealing at 66°C. for completely (100%) matched from one another, and both the 2 min. and extension at 72°C. for 1 min.; followed by a final sequence of A. correntina (AF203554) and that of A. villosa extension at 72°C. for 4 min. The PCR reaction mixture was (AF156675) completely matched 114 bases between two electrophoresed on a 2% agarose gel containing ethidium primer regions. This result indicates the target ITS-1-5.8S bromide. After the electrophoresis, the gel was analyzed 30 rRNA gene sequence of the genus Arachis can be detected using a Fluorimager 595 (Amersham Pharmacia Biotech). without fail. In this connection, the target amplification prod The results are shown in FIG.8. The meanings of numerical uct differed from A. hypogaea (AF156675) at 5 positions, values, abbreviations and symbols used in FIG. 8 are as fol particularly the 48" base “T” thereof was not present in the lows. genus Arachis except for A. hypogaea (AF156675) in Gen 35 Bank and it is not present in the commercially available M: 100 bp DNA Ladder Marker peanuts experimented on here. It was found that, in order to (-): Negative control (no DNA) design widely detectable primers to the genus Arachis, which Numerical values above sample names: the amounts of the includes commercially available peanuts, a DNA sequence of template DNA commercially available peanuts, A. correntina (AF203554) Arrow: indicates the target amplification product (156 bp) 40 or A. villosa (AF203558), was preferably used in place of a DNA sequence of A. hypogaea (AF156675) in GenBank, and D. PCR Results (Part 1: a combination of primers with SEO therefore, that the sense primer with SEQ NO:20 designed NOs: 18 and 3) from the sequence of commercially available peanuts, A. PCR described above was conducted using primers correntina (AF203554) or A. villosa (AF203558) was used designed in the present invention. The results are shown in 45 rather than the sense primer with SEQ NO:19 designed from FIG. 8. As shown in FIG. 8, target 156 bp amplification the sequence of A. hypogaea (AF156675). H. PCR (Part 2: a products, predicted from the simulation results of ITS-1-5.8S combination of primers with SEO NOS:20 and 3) rRNA gene sequences of the genus Arachis, were obtained PCR was conducted using the QIAGEN HotStarTaq Mas from 6 commercially available peanuts. Because this result ter Mix Kit according to the procedures described in the almost matched the simulation results shown in Table 3, the 50 HotStarTaq PCR Handbook as stated below. simulation results were thought to be reliable. PCR was carried out using final volumes of 25 ul of a solution containing 12.5 ul of 2x HotStarTaq Master Mix E. Preparation of a Sequencing Sample (Part 1: a combination (HotStarTaq DNA Polymerase, PCR Buffer with 3 mM of primers with SEQ NOs: 18 and 3) MgCl, 400 uM each dNTP), 0.5 M of each primer (SEQ (1) Purification of the Amplification Product from Peanut 55 NO:20 and SEQ NO:3), the template DNA and sterilized A purification of the amplification product from peanut ultrapure water in 0.2-ml microcentrifuge tubes. Amplifica obtained in D was conducted in the same way as in Example tion was performed using a Sequence Detection System ABI 1E (1) “Purification of the Amplification Product from Buck PRISM 7700 (Applied Biosystems) according to the follow wheat Chaff. ing PCR program: pre-incubation at 95°C. for 15 min.; 25 60 cycles consisting of denaturation at 95° C. for 30 secs. (2) Sequencing PCR Reaction and Removal of Excess Dye annealing and extension at 75° C. for 30 secs. respectively Labeled Dideoxynucleotides and 30 cycles consisting of denaturation at 95°C. for 30 secs. PCR for sequencing of the purified amplification product annealing and extension at 72° C. for 30 secs. respectively; from peanut obtained in (1) was conducted in the same way as followed by a final extension at 72°C. for 5 min. The resulting in Example 1E (2) “Sequencing PCR Reaction and Removal 65 PCR reaction mixture was electrophoresed on a 2% agarose of Excess Dye-Labeled Dideoxynucleotides”, except for use gel containing ethidium bromide. After the electrophoresis, of primers with SEQ NOs: 18 and 3. the gel was analyzed using a Fluorimager 595 (Amersham US 7,704,694 B2 39 40 Pharmacia Biotech). The results are shown in FIGS. 10 and K. Sequence Analysis (Part 2: A Combination of Primers with 11. The meanings of numerical values, abbreviations and SEO NOs:20 and 3) symbols used in FIGS. 10 and 11 are as follows. The sample for sequencing from peanut obtained in J (2) was analyzed using an ABI PRISM 310 Genetic Analyzer M: 100 bp DNA Ladder Marker (Applied Biosystems). The obtained nucleotide sequence of (-): Negative control (no DNA) the amplification product was compared with the sequence of Numerical values above sample names: the amounts of the Arachis hypogaea (AF156675), A. correntina (AF203554), template DNA A. villosa (AF203558), A. major (AF203552) and A. herman Arrow: indicates the target amplification product (114 bp) nii (AF203556) in the genus Arachis and the nucleotide The quality of each of the template DNA used here was 10 sequence of the commercially available peanut obtained in G. sufficient enough to be used for PCR based on the result of a The result is shown in FIG. 12. The meanings of number separate PCR, in which target products were obtained using a symbols above a nucleotide sequence, lines underneath this primer pair to amplify a part of plant chloroplast DNA. sequence and other symbols used in FIG. 12 are described below. I. PCR Results (Part 2: a combination of primers with SEO 15 Number Symbols: Nucleotide numbers of amplification NOs:20 and 3) products PCR described above was conducted using primers Commercially available peanuts: A part of the nucleotide designed in the present invention. The results are shown in sequence of the commercially available peanuts obtained FIGS. 10 and 11. As shown in FIG. 10, target 114 bp ampli in G fication products, predicted from the simulation results of the Asterisks (*): Nucleotides of the peanuts, which are identical ITS-1-5.8S rRNA gene sequence of the genus Arachis, were with those of all of the sequences of Arachis hypogaea obtained from peanuts. On the other hand, no 114 bp ampli (AF156675), A. correntina (AF203554), A. villosa fication product was obtained from buckwheat, wheat, soy (AF203558), A. major (AF203552) and A. hermannii bean, adzuki bean and corn. However, nonspecific amplifica (AF203556) in the genus Arachis and the sequence of the tion products, which were different from the target product in 25 commercially available peanut obtained in G. size, were obtained from wheat (approximately 250 bp). Single underline: The sense primer region Because these results almost matched the simulation results Double underline: The antisense primer region shown in Table 4, the simulation results were thought to be reliable. Consequently, it was confirmed that a wide range of L. The Result of Sequence Analysis (Part 2: a Combination of plants in the genus Arachis including commercially available 30 Primers with SEO NOs:20 and 3) peanuts were detectable using the present invention. As shown in FIG. 12, a part of the nucleotide sequence of As shown in FIG. 11, the target 114 bp amplification prod an amplification product derived from commercially avail uct, predicted from the simulation results of the ITS-1-5.8S able peanuts obtained by PCR using the primers designed in rRNA gene sequence of the genus Arachis, was obtained from the present invention completely (100%) matched 75 bases 35 between two primer regions of respective sequences of Ara the wheat DNA sample containing 1 ppm of peanut DNA. chis correntina (AF203554), A. villosa (AF203558Y. A. This result showed that even where 10 to 1 ppm of peanut Major (AF203552) and A. hermannii (AF203556). In this DNA is present in wheat DNA, the peanut can be detectable. connection, the target amplification product differed from Furthermore, the nonspecific amplification product from Arachis hypogaea (AF156675) at 4 positions, but completely wheat that was obviously different from the target product in 40 (100%) matched the nucleotide sequence of the commer size did not interfere in the detection of 1 ppm of peanut DNA. cially available peanuts obtained in G. This result indicates In this connection, as shown in Examples 1, 2 and 3, where the target ITS-1-5.8S rRNA gene sequence of the genus a W value, which shows a possibility of obtaining amplifica Arachis can be detected without fail. tion products in a simulation by Amplify, is not more than W4. it has been found that the amplification products, which differ 45 Example 4 from the target one, are obtained in Some cases and not obtained in other cases in actual PCR. Please note that Tables 1 to 4 show the data of the simulation results of Amplify A. Design of Oligonucleotide Primers for Detection of DNA which are of W2 value or higher but do not show those of a from Buckwheat lower Wvalue such as W1 and W0 wherein the possibility of 50 (1) DNA Sequences of the Genus Fagopyrum. Other Com obtaining the amplification products is considered to be low. mon Allergenic Plants and Plants Widely Used for a Food Ingredient J. Preparation of a Sequencing Sample (Part 2: a Combina The DNA sequences described in Example 1A (1) “DNA tion of Primers with SEO NOs:20 and 3) sequences of the Genus Fagopyrum', (2) “DNA Sequences of (1) Purification of the Amplification Product from Peanut 55 other common allergenic plants' and (3) “DNA Sequences of The purification of the amplification product from peanut Plants Widely Used for a Food Ingredient' were examined to obtained in I was conducted in the same way as in Example 1E select suitable regions for the primers. (1) “Purification of the Amplification Product from Buck (2) DNA Sequence of Plants in Related Species of the Genus wheat Chaff. Fagopyrum 60 (2) Sequencing PCR Reaction and Removal of Excess Dye The DNA sequences described in Example 2A (2) “DNA Labeled Dideoxynucleotides Sequence of Plants in Related Species of the Genus Fagopy PCR for sequencing of the purified amplification product rum' were selected. from peanut obtained in (1) was conducted in the same way as (3) Oligonucleotide Primer Synthesis in Example 1E (2) “Sequencing PCR Reaction and Removal 65 Among ITS-1 sequences of the aforementioned 21 DNA of Excess Dye-Labeled Dideoxynucleotides”, except for use sequences of the genus Fagopyrum, there was determined a of primers with SEQ NOs:20 and 3. nucleotide sequence which would specifically hybridize to all US 7,704,694 B2 41 42 of the 21 DNA sequences of the genus Fagopyrum through C. Preparation of Template DNA for PCR the study of the ITS-1 sequences. The thus determined nucle The DNA samples isolated from Shirahana soba (common otide sequence is indicated as SEQ NO:15. Subsequently, the buckwheat) and Dattan soba (Tartalian buckwheat) in oligonucleotide primer with SEQ NO:15 was synthesized. Example 1B (2) were diluted stepwise with sterilized ultra pure water to use them. The DNA samples isolated from white pepper in Example 1B (2), mustard in Example 1B (3), Sense primer peanut in Example 3B (2) and wheat, soybean, and corn in s" - CGT TGC CGA GAG TCG TTC TGT TT-3' (SEQ NO:15) Example 3B (3) were also used. In addition, the DNA samples isolated from brown lice seeds and Fallopia convolvulus in The oligonucleotide primer with SEQ NO:14 was also 10 the same way as in Example 1B (3) were used. The DNA used as an antisense primer, the same as in Example 2. samples from Fallopia convolvulus were diluted stepwise B. PCR Simulation with sterilized ultrapure water to use. Regarding the sense and antisense primer pair, the simula D. PCR tion was conducted with PCR simulation software, Amplify 15 PCR was conducted in essentially the same way as in 1.0 (Bill Engels), which is the same as in Example 1, to Example 1C, except for use of the following primers and PCR examine whether a target size of amplification products program. would be obtained from the 21 DNA sequences of the genus Primers: Fagopyrum, the 8 DNA sequences of common allergenic Each primer of SEQ NO:14 and SEQ NO:15 was used at plants other than buckwheat (peanut, wheat, soybean, walnut, 0.5uM of a final concentration. matsutake mushroom, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, PCR Program: brown rice, pepper and mustard) and the 27 sequences of PCR was conducted according to the following PCR pro related species of the genus Fagopyrum. gram. 25 The simulation results are shown in Tables 5A and 5C. The Pre-incubation at 95°C. for 15 min...: thereafter 45 cycles meanings of symbols and numerical values in Tables 5A and consisting of denaturation at 95°C. for 1 min., annealing at 5C are explained below. 66° C. for 2 min. and extension at 72°C. for 1 min.; followed k: An obtained amplification product whose size almost by a final extension at 72°C. for 4 min. matched the target 140 bp (+10 bp). 30 E. Results W 2-6: Probability of Obtaining Amplification Products Regarding the primers of the present invention, the simu lation was conducted with PCR simulation software, Amplify High Probability W6>W5>W4>W3>W2 Low Prob 1.0 (Bill Engels) to examine the specificity to the ITS-1-5.8S ability rRNA gene sequence of each plant. As a result, as shown in Numerical values followed by bp: 35 Tables 5A to 5C, it was predicted that target 101 bp amplifi Each value was obtained by subtracting 2 from the value cation products would be obtained from the aforementioned 21 DNA sequences of the genus Fagopyrum. On the other obtained in the simulation. hand, it was predicted that no target 101 bp amplification (-): No amplification product was predicted. product would be obtained from the 8 DNA sequences of Related Species of the genus Fagopyrum. 40 other common allergenic plants (peanut, wheat, soybean, Sequences similar to the ITS-1 sequence of Fagopyrum walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient esculentum (AB000330) were searched by means of a (corn, brown rice, pepper and mustard) and the 27 DNA BLAST homology search and the sequences having a score of sequences of related species of the genus Fagopyrum both 60 bits or more were selected from among them. Each 45 belonging to Polygonaceae and not belonging to Polygo sequence having the highest score in each genus and having a naceae. Also, no nonspecific amplification product could be score of 60 bits or more is shown in the following Tables predicted. As a result, it was confirmed that a wide range of 5B-5C as the representative of the DNA sequences of related plants in the genus Fagopyrum would be specifically detect species of the genus Fagopyrum. able using the present invention. TABLE 5A Buckwheat, SEQ No. 14 & SEQ No. 15 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Genus Fagopyrim Fagopyri in tirophyllum AB000342 101 bp – 439 bp – kFagopyrum urophyllum AB000341 101 bp – kFagopyrum tataricum AB000340 101 bp – (Tartarian buckwheat) kFagopyrum tataricum AB000339 101 bp – (Tartarian buckwheat) st Fagopyrim Statice AB000338 101 bp – st Fagopyrim Statice AB000337 101 bp – kFagopyrum pieioramosum AB000336 101 bp — Fagopyrim lineare AB000335 101 bp – st Fagopyrim lepiopodium AB000334 101 bp – kFagopyrum homotropicum AB000333 101 bp — Fagopyrim gracipes AB000332 101 bp – US 7,704,694 B2 43 44

TABLE 5A-continued Buckwheat, SEQ No. 14 & SEQ No. 15 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 st Fagopyrim escientiin AB000331 101 bp – (Common buckwheat) st Fagopyrim escientiin AB000330 101 bp – (Common buckwheat) st Fagopyri in cymost in AB000329 101 bp – st Fagopyri in cymost in AB000328 101 bp – st Fagopyri in cymost in AB000327 101 bp – st Fagopyri in cymost in AB000326 101 bp – st Fagopyri in cymost in AB000325 101 bp – st Fagopyri in cymost in AB000324 101 bp – st Fagopyrim capillatin AB000323 101 bp – kFagopyrum callianthum AB000322 101 bp – 440 bp

TABLE 5B Buckwheat, SEQ No. 14 & SEQ No. 15 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 Other Common Allergenic Plants Arachis hypogaea AF1566.75 (Peanut) Trictim aestivum AJ301799 (Wheat) Glycine max U60551 (Soybean) Jugians regia AF303809 - (Walnut) Trichoioma matsutake U62964 (Matsutake mushroom) Prunus persica AF185621 (Peach) Maius X domestica AF186484 - (Apple) Citrus sp. E08821 (Valencia orange) Plants Widely Used Zea mayS U46648 for a Food Ingredient (Corn) Oryza sativa AF169230 (Brown rice) Piper nigrum AF275197 (Pepper) Sinapis alba X15915 (Mustard) Related Species of the Genus Aconogonum sp. Won 152 AF189731 Fagopyrim Belonging to Fallopia Scandens AFO40069 - Polygonaceae Polygonim virginianum U51274 Rumex acetoseiia AF18973O

TABLE SC Buckwheat, SEQ No. 14 & SEQ No. 15 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. Related Species of the Genus Fagopyrum Taintin paraguayense L78056 Not Belonging to Polygo Bruinsmia styracoides AF396438 Taiinelia pachypoda L78OS4 Rehderodendron AF3964.48 kwangtungense Pterostyrax corymbosus AF3964.45 Anredera cordifolia L78O86 Cistanthe quadripetalia L78062 Xenia vulcanensis L78060 Tainopsis finitesCens L78058 US 7,704,694 B2 45 46

TABLE 5C-continued Buckwheat, SEQ No. 14 & SEQ No. 15 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 WS W4 W3 W2 Tainaria palmeri Portulaca sp. Pheneranthus confertifiorus Montiopsis umbellata L78O33 Grahamia bracteata L78028 Henniaria glabra A31096S Alluaudia dimosa L78O11 Sinojackia xylocarpa AF3964S1 Halesia macgregori AF3964.42 Changiostyrax doichocarpa AF396439 Alecinyon subdentatus AF314765 Anacampseros recurvata L78O14 Weinmannia racemosa AF485597 Birsera teconaca AFO80O29

PCR described above was conducted using primers Sequences of Other Common Allergenic Plants’. Example designed in the present invention. In this case, target 101 bp 3A (3) “DNA Sequences of Plants Widely Used for a Food amplification products, predicted from the simulation results 25 Ingredient' and Example 3A (4) “DNA Sequences of Legu of the ITS-1-5.8S rRNA gene sequences of the genus minous Plants Widely Used for a Food Ingredient were Fagopyrum, were obtained from 500 to 50 fg of Shirahana examined to select Suitable regions for the primers. In addi soba (common buckwheat) DNA and Dattan soba (Tartarian tion, as a DNA sequence of adzuki bean, 5.8S rRNA gene, buckwheat) DNA. As a result, it was found that even where ITS-2 sequences in the following DNA sequences registered 500 to 50 fg ofbuckwheat DNA is present, the buckwheat can 30 in GenBank were selected. be detected. In this connection, Such sensitivity corresponds 1: adzuki bean: Vigna angularis Vars nipponensis to a sensitivity wherein there can be detected 10 to 1 ppm of (AB060088) buckwheat DNA contained in the sample DNA when PCR (2) DNA Sequences of Plants in Related Species of the Genus was conducted with, as a template, 50 ng of DNA isolated Arachis from Some samples. On the other hand, no amplification 35 As representatives of the DNA sequences of related species product having 101 bp or nonspecific amplification products of the genus Arachis, respective 5.8S rRNA gene, ITS-1 and was obtained from wheat, peanut, soybean, corn, mustard, ITS-2 sequences in the following 45 DNA sequences regis pepper and brown rice. Furthermore, regarding Fallopia con tered in GenBank were examined to select suitable regions for volvulus, when an amount of template DNA is 50 to 5 ng, a the primers. In this connection, the 45 DNA sequences were target size of an amplification product was obtained with a 40 selected as representations, each of which was of highest very weak signal, but when an amount of template DNA is Score in the species which were other than peanut (Arachis 500 pg or less, no amplification product in a target size or hypogaea AF 156675) and were of Score of 60 bits or more nonspecific amplification product was obtained at all. In this among sequences of said species hit to ITS-2 sequence of connection, regarding Fallopia convolvulus, when PCR was peanut through BLAST homology search. conducted with, as a template, 50 ng of DNA isolated from 45 1. Chapmannia floridana (AF203543) Some samples, even if 1% of Fallopia convolvulus was 2: Chapmannia gracilis (AF203546) present in sample DNA, the level of Fallopia convolvulus : Chapmannia prismatica (AJ320400) DNA corresponds to a non-detected levelas false positive. By : Chapmannia reghidensis (AF204232) modification of PCR program, there is a possibility that : Chapmannia sericea (AF203548) amplification products in a target size will not be obtained 50 : Chapmannia Somalensis (AF203544) from 50 to 5 ng of DNA from Fallopia convolvulus. : Chapmannia tinireana (AF203547) Consequently, in conjunction with the results of specificity 8: hebrigiella gracilis (AF203561) studied by PCR simulation and of sensitivity and specificity 9. Fissicalyx fendleri (AF1890.61) studied by PCR, it was confirmed that a wide range of the 10. Stylosanthes acuminata (AJ320282) genus Fagopyrum including common buckwheat and Tartar 55 11. Stylosanthes angustifolia (AJ320284) ian buckwheat were detectable using the present invention. 12. Stylosanthes aurea (AJ320285) Example 5 13. Stylosanthes biflora (AJ320289) 14. Stylosanthes bracteata (AJ320346) 15. Stylosanthes calcicola (AJ320348) A. Design of Oligonucleotide Primers for Detection of DNA 60 16. Stylosanthes campestris (AJ320291) from Peanut 17: Stylosanthes capitata (AJ320350) (1) DNA Sequences of the Genus Arachis, Other Common 18. Stylosanthes cayennensis (AJ320292) Allergenic Plants, Plants Widely Used for a Food Ingredient 19: Stylosanthes erecta (AJ320352) and Leguminous Plants Widely Used for a Food Ingredient 65 20: Stylosanthes fruticosa (AJ320356) The DNA sequences described in Example 3A (1) “DNA 21. Stylosanthes gracilis (AJ320296) Sequences of the Genus Arachis’. Example 3A (2) “DNA 22. Stylosanthes grandifolia (AJ320299) US 7,704,694 B2 47 48 23. Stylosanthes guianensis subsp. dissitiflora (AJ320301) peanut (buckwheat, wheat, soybean, walnut, matsutake 24. Stylosanthes hamata (AJ320365) mushroom, peach, apple and orange), the 4 DNA sequences 25. Stylosanthes hippocampoides (AJ320316) of plants widely used for a food ingredient (corn, brown rice, 26. Stylosanthes hispida (AJ320328) pepper and mustard), the 6 DNA sequence of leguminous 27. Stylosanthes humilis (AJ320327) plants widely used for a food ingredient (French bean, lima 28. Stylosanthes ingrata (AJ320329) bean, lentil, chickpea, mung bean and adzuki bean) and the 29. Stylosanthes leiocarpa (AJ320332) DNA sequences of plants in related species of the genus 3O. Stylosanthes linearifola (AJ320367) Arachis. 31. Stylosanthes macrocarpa (AJ320369) 32. Stylosanthes macrocephala (AJ320371) 10 (a) The simulation results using a combination of primers of 33. Stylosanthes macrosoma (AJ320333) SEQ NOs:7 and 24 are shown in Tables 6A to 6D. The 34. Stylosanthes mexicana (AJ320373) meanings of symbols and numerical values in Tables 6A to 35. Stylosanthes montevidensis (AJ320336) 6D are explained below. 36. Stylosanthes pilosa (AJ320377) k: An obtained amplification product whose size almost 37. Stylosanthes scabra (AJ320382) 15 matched the target 140 bp (+10 bp). 38. Stylosanthes seabrana (AJ320384) 39. Stylosanthes sericeiceps (AJ320386) W 2-6. Probability to obtain amplification products 40. Stylosanthes subsericea (AJ320387) High Probability W6>W5>W4>W3>W2 Low Prob 41. Stylosanthes Sundaica (AJ320389) ability 42. Stylosanthes sympodialis (AJ320391) Numerical values followed by bp: 43. Stylosanthes tomentosa (AJ320337) Each value was obtained by subtracting 2 from the value 44. Stylosanthes tuberculata (AJ320392) obtained in the simulation. 45. Stylosanthes viscosa (AJ320340) (-): No amplification product was predicted. In addition, if PCR simulation is conducted by selecting a 25 Related Species of the genus Arachis: primer hybridized to ITS-1 sequence, the DNA sequences Sequences similar to the ITS-2 sequence of Arachis described in Example 3A (5) “DNA Sequences of Plants in hypogaea (AF156675) were searched by means of a Related Species to the Genus Arachis' were also selected. BLAST homology search and the sequences having a (3) Oligonucleotide Primer Synthesis score of 60 bits or one were selected among them. Each (a) Among 5.8S rRNA gene sequences of the aforementioned 30 sequence having the highest score in each genus and 11 DNA sequences of the genus Arachis and the aforemen having a score of 60 bits or more is shown in the follow tioned 8 DNA sequences of other common allergenic plants, ing Tables GB-6D as the representative of the DNA there was determined nucleotide sequences which would sequences of related species of the genus Arachis. hybridize to all DNA sequences of these plants through the (-): No annealing site of the primer (SEQ NO: 24) was study of the sequences. The thus determined nucleotide 35 predicted within the ITS-2 sequence of Vigna angularis sequence is indicated as SEQNO:7. Subsequently, the oligo var. nipponensis (adzuki bean). nucleotide primer with SEQ NO:7 was synthesized. In the case of adzuki bean, only the ITS-2 sequence was selected because the 5.8S rRNA gene sequence of Vigna angularis var. nipponensis (AB059747) was not regis Sense primer 40 tered in GenBank. (SEO NO:7) 5 " - GAT GAA GAA CGT AGC GAA. ATG CGA TAC T-3' (b) The simulation results using a combination of primers of Among ITS-2 sequences of the aforementioned 11 DNA SEQ NOs: 18 and 24 are shown in Tables 7A to 7E. The sequences of the genus Arachis, there was determined nucle meanings of symbols and numerical values in Tables 7A to 7E otide sequences which would specifically hybridize to all of 45 are explained below. the 11 DNA sequences of the genus Arachis through the study k: An obtained amplification product whose size almost of the sequences. The thus determined nucleotide sequence is matched the target 140 bp (+10 bp). indicated as SEQ NO:24. Subsequently, the oligonucleotide W 2-6. Probability to obtain amplification products primer with SEQ NO:24 was synthesized. 50 High Probability W6>W5>W4>W3>W2 Low Prob ability Antisense primer Numerical values followed by bp: s' - CCA TCT, GCC GCG. GTG CC-3' (SEQ NO:24) Each value was obtained by subtracting 2 from the value obtained in the simulation. (b) Furthermore, in another combination of primers, the 55 (-): No amplification product was predicted. primer of SEQNO:18 described in Example 3 was selected as Related Species of the genus Arachis: sense primer on the ITS-1 sequence and the primer of SEQ Sequences Similar to the ITS-1 or ITS-2 sequence of Ara NO:24 was selected as antisense primer on the ITS-2 chis hypogaea (AF156675) were searched by means of a Sequence. 60 BLAST homology search and the sequences having a B. PCR Simulation score of 60 bits or more were selected among them. Each Regarding the sense and antisense primer pairs, the simu sequence having the highest score in each genus and lation was conducted with PCR simulation software, Amplify having a score of 60 bits or more is shown in the follow 1.0 (Bill Engels), which is the same as in Example 1 to ing Tables 7B-7E as the representative of the DNA examine whether target size of amplification products are 65 sequences of related species of the genus Arachis. obtained from the 11 DNA sequences of the genus Arachis, (-): No annealing site of the primer (SEQ NO:18) was the 8 DNA sequences of common allergenic plants other than predicted within the ITS-1 sequence of Vigna angularis US 7,704,694 B2 49 50 var. nipponensis (adzuki bean) and no annealing site of the E. Results primer (SEQ NO:24) was predicted within the ITS-2 (a) Results of the Combination of the Primers of SEQ NOs:7 sequence of Vigna angularis var. nipponensis (adzuki and 24 bean). Regarding the primers of the present invention (the com In the case of adzuki bean, only either the ITS-1 or ITS-2 bination of the primers of SEQNOs:7 and 24), the simulation sequence was selected respectively because full length of the was conducted with PCR simulation software, Amplify 1.0 ITS-1-5.8S rRNA gene-ITS-2 sequence of Vigna angularis (Bill Engels) to examine the specificity to 5.8S rRNA var. nipponensis (AB05.9747) was not registered in GenBank. gene-ITS-2 sequence of each plants. As a result, as shown in C. Preparation of Template DNA for PCR Tables GA to GD, it was predicted that target 253 to 259 bp A DNA preparation from peanut isolated in Example 3B 10 amplification products would be obtained from the aforemen (2) was diluted stepwise with sterilized ultrapure water to use. tioned 11 DNA sequences of the genus Arachis. On the other hand, it was predicted that no target 253 to 259bp amplifica D. PCR tion products would be obtained from the 8 DNA sequences PCR was conducted in substantially the same way as in of other common allergenic plants (buckwheat, wheat, soy Example 1C, except for use of following primers and PCR 15 bean, walnut, matsutake mushroom, peach, apple and program. orange), the 4 DNA sequences of plants widely used for a Primer: food ingredient (corn, brown rice, pepper and mustard), the 6 (a) Each primer of SEQNO:7 and SEQNO:24 was used at 0.5 DNA sequence of leguminous plants widely used for a food uM of final concentration. ingredient (French bean, lima bean, lentil, chickpea, mung (b) Each primer of SEQ NO:18 and SEQ NO:24 was used at bean and adzuki bean) and the 41 DNA sequences of plants in 0.5, M of final concentration. related species of the genus Arachis belonging to leguminous plants. In this connection, among the plants in related species PCR Program: of the genus Arachis belonging to leguminous plants, it was PCR was conducted according to the following PCR pro predicted by simulation that amplification products having gram. 25 almost 253 to 259 bp would be obtained from the DNA Pre-incubation at 95°C. for 15 min...: thereafter 45 cycles sequences of Stylosanthes Cayennensis, Stylosanthes hispida, consisting of denaturation at 95°C. for 1 min., annealing at Stylosanthes viscosa and Fissicalyx fendleri, but these ampli 68°C. for 1 min. and extension at 72°C. for 1 min.; followed fication products can be identified by sequence analysis. by a final extension at 72°C. for 4 min. Optionally, whether these amplification products are peanut The quality of each of the template DNA used here was 30 may be also identified by PCR showing in Example 3 and the sufficient enough to be used for PCR based on the result of a like. As a result, it was confirmed that wide ranges of plants in separate PCR, in which target products were obtained using a the genus Arachis would be specifically detectable using the primer pair to amplify a part of plant chloroplast DNA. present invention.

TABLE 6A Peanut, SEQ NO: 7 & SEQ NO. 24 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Genus Arachis Arachis batizocoi AF203553 – 253 bp 318 bp 214 bp – 49bp Arachis correntina AF203554 – 254 bp 323 bp 219 bp – SOb Arachis hermannii AF203556 – 253 bp 320 bp 216 bp – 49bp Arachis hoehnei AJ32O395 – 256 bp 52 by y Arachis hypogaea AF156675 – 259 bp 968 bp 222 bp 69 bp (Peanut) 941 bp. 328 by 7S by 53 by kArachis magna AF203555 – 254 bp 322 bp 218bp – SOb kArachis major AF203552 – 253 bp 320 bp 216 bp – 49bp kArachis palustris AF203557 – 254 bp 323 bp 219 bp – SOb kArachis pintoi AF2O3SS1 – 254 bp 323 bp 219 bp – SOb Arachis iriseminata AF204233 – 253 bp 49bp Arachis viliosa AF203558 – 255 bp 324 bp 220 bp – S1 by Other Common Allergenic Plants Fagopyrim escientiin ABOOO330 - 160 bp — (Common buckwheat) Triticum aestivum (Wheat) AJ301799 Glycine max (Soybean) U60551 Jugians regia (Walnut) AF3O3809 - Tricholoma matsutake U62964 (Matsuttake mushroom) US 7,704,694 B2 51 52

TABLE 6A-continued Peanut, SEQNO: 7 & SEQNO: 24 primer: Amplification products

GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Prunus persica (Peach) AF185621 Maius X domestica AF186484 - (Apple) Citrus sp. E08821 (Valencia orange)

TABLE 6B Peanut, SEQ NO. 7 & SEQ NO. 24 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Plants Widely Used Zea mayS U46648 82 bp for a Food (Corn) Ingredient Oryza sativa AF169230 - 99 bp (Brown rice) 92 bp Piper nigrum AF275197 (Pepper) Sinapis alba X15915 (Mustard) Leguminous Plants Widely Used Phaseolus vulgaris AF115169 - for a Food Ingredient (French bean) Phaseolus iunatus AF11517S (Lima bean) Lens clinaris Subsp. AF228.066 - 98 bp culinaris (Lentil) Cicer arietintin A237698 (Chickpea) Vigna radiata X14337 (Mung bean) Vigna angularis var. ABO60O88 * nipponensis (Adzuki bean)* Related Species of the Genus Chapmannia floridana AF2O3543 - – 330 bp 35 bp 226 bp Arachis Belonging to leguminous Chapmannia gracilis AF2O3546 - Plants Chapmannia prismatica AJ320400 Chapmannia reghidensis AF204232 — Chapmannia sericea AF2O3548 - Chapmannia Somaliensis AF203544 — Chapmannia tinireana AF2O3S47 Fiebrigiella gracilis AF203561 kFissicalyx fendieri AF189061 — 252 bp — 349 bp 170 bp 340 bp 258 bp 148 bp 66 bp

TABLE 6C Peanut, SEQ NO. 7 & SEQ NO. 24 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Related Species of the Genus Arachis Stylosanthes acuminata AJ32O282 Belonging to Leguminous Plants Stylosanthes angustifolia AJ32O284 Stylosanthes at trea AJ32O285 Stylosanthes biflora AJ32O289 Stylosanthes bracteata AJ32O346 Stylosanthes calcicola AJ32O348 Stylosanthes Campestris AJ32O291 Stylosanthes Capitata AJ32O3SO k Stylosanthes cayennensis AJ320292 – 253 bp 455 bp - 372 bp 170 bp US 7,704,694 B2 53

TABLE 6C-continued Peanut, SEQ NO. 7 & SEQ NO. 24 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. Stylosanthes erecta AJ32O352 Stylosanthes finiticosa AJ32O356 Stylosanthes gracilis AJ32O296 Stylosanthes grandifolia AJ32O299 Stylosanthes guianensis AJ32O3O1 Subsp. dissitiflora Stylosanthes hanaia AJ32O365 Stylosanthes AJ32O316 hippocampoides k Stylosanthes hispida AJ32O328 – 253 bp 455 bp - 372 bp 170 bp Stylosanthes humilis AJ32O327 170 bp – 66 bp

TABLE 6D Peanut, SEQ NO. 7 & SEQ NO. 24 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. Related Species of the Genus Arachis Stylosanthes ingrata AJ32O329 Belonging to Leguminous Plants Stylosanthes leiocarpa AJ32O332 175 bp – 71 bp Stylosanthes linearifolia AJ32O367 Stylosanthes macrocarpa AJ32O369 Stylosanthes macrocephala AJ320371 Stylosanthes macrosoma AJ32O333 Stylosanthes mexicana AJ320373 Stylosanthes AJ32O336 montevidensis Stylosanthes pilosa AJ32O377 Stylosanthes scabra AJ32O382 Stylosanthes Seabrana AJ32O384 Stylosanthes sericeiceps AJ32O386 Stylosanthes subsericea AJ32O387 Stylosanthes Sundaica AJ32O389 Stylosanthes Sympodialis AJ32O391 Stylosanthesiomentosa AJ32O337 Stylosanthes tuberculata AJ32O392 k Stylosanthes viscosa AJ32O340 436 bp 149 bp 253 bp

PCR described above was conducted using primers 45 rRNA gene-ITS-2 sequence of each plant. As a result, as designed in the present invention. In this case, target 253 to shown in Tables 7A to 7E, it was predicted that target 384 to 259bp amplification products, expected from the simulation 390 bp amplification products would be obtained from the results of the 5.8S rRNA gene-ITS-2 sequences of the genus aforementioned 11 DNA sequences of the genus Arachis. On Arachis, were obtained from 500 to 50 fg of peanut DNA. It 50 the other hand, it was predicted that no target 384 to 390 bp is found from the results that even where 500 to 50 fg of amplification products would be obtained from the 8 DNA peanut DNA is present, the peanut can be detected. In this sequences of other common allergenic plants (buckwheat, connection, this sensitivity correspond to a sensitivity wheat, soybean, walnut, matsutake mushroom, peach, apple wherein there can be detected 10 to 1 ppm of peanut DNA and orange), the 4 DNA sequences of plants widely used for contained in the sample DNA when PCR was conducted with, as a template, 50 ng of DNA isolated from some samples. 55 a food ingredient (corn, brown lice, pepper and mustard), the Consequently, in conjunction with the results of specificity 7 DNA sequence of leguminous plants widely used for a food studied by PCR simulation, and of sensitivity and specificity ingredient (French bean, lima bean, lentil, chickpea, mung studied by PCR, it was confirmed that a wide range of the bean and adzuki bean), the 71 DNA sequences of plants in genus Arachis including peanut were detectable using the related species of the genus Arachis belonging to leguminous 60 plants and the 3 DNA sequences of plants in related species of present invention. the genus Arachis not belonging to leguminous plants. (b) Results of the Combination of the Primers of SEQNOs: 18 Among the plants in related species of the genus Arachis and 24 belonging to leguminous plants, it was predicted by simula Regarding the primers of the present invention (the com tion that amplification products, which were almost matched bination of the primers of SEQ NOs: 18 and 24), the simula 65 384 to 390 bp of the target one in size, would be obtained from tion was conducted with PCR simulation software, Amplify the DNA sequences of Stylosanthes cavennensis, Stylosan 1.0 (Bill Engels) to examine the specificity to ITS-1-5.8S thes hispida, Stylosanthes viscosa and Fissicalyx fendleri, but US 7,704,694 B2 55 56 these amplification products can be identified by sequence Example 3 and the like. As a result, it was confirmed that wide analysis. Optionally, whether these amplification products ranges of plants in the genus Arachis would be specifically are peanut may be also identified by PCR showing in detectable using the present invention.

TABLE 7A Peanut, SEQ NO: 18 & SEO NO. 24 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2

Genus Arachis Arachis fatizocoi AF2O3SS3 - 384 bp 49 bp 214 by 449 by 72 bp Arachis correntina AF2O3SS4 385 by SObb 219 by 454 bp Arachis hermannii AF2O3SS6 - 384 bp 49 bp 216 bp 451 bp. Arachis hoehnei AJ32O395 - 387 by S2 bb 60 bp y Arachis hypogaea AF1566.75 - 390 bp 306 bp 1099 bp 69 bp (Peanut) 459 by 53 bp 1072 bp 222 bp kArachis magna AF2O3555 - 385 by SObb 218 by 453 by 72 bp kArachis major AF2O3552 – 384 bp 149 by 216 bp 451 bp. kArachis palustris AF2O3SS7 385 by SObb 219 by 454 bp 72 bp kArachis pintoi AF2O3SS1 - 386 by SObb 219 by 45Sb Arachis iriseminata AF2O4233 - 384 bp 49 bp Arachis viliosa AF2O3SS8 - 386 by S1 bp. 220 bp 45Sb Other Common Allergenic Plants Fagopyrum esculentum AB000330 — 160 by (Common buckwheat) Trictim aestivitin AJ301799 (Wheat) Glycine max (Soybean) U60551 Jugians regia (Walnut) AF303809 Tricholoma matsutake U62964 (Matsutake mushroom) Prunus persica (Peach). AF185621 Maius X domestica AF186484 - 467 bp (Apple) 424 bp Citrus sp. E08821 103 bp 280 bp (Valencia orange)

TABLE 7B Peanut, SEQ NO: 18 & SEQ NO. 24 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Plants Widely Used Zea mays U46648 82 bp for a Food (Corn) Ingredient Oryza sativa AF169230 - 99 bp (Brown rice) 92 bp Piper nigrum AF275197 (Pepper) Sinapis alba X15915 (Mustard) Leguminous Plants Widely Used Phaseolus vulgaris AF115169 - for a Food Ingredient (French bean) Phaseolus itinati is AF115175 (Lima bean) Lens citiinaris Subsp. AF228.066 - 98 bp culinaris (Lentil) Cicer arietintin A237698 95 bp (Chickpea) Vigna radiata (Mung bean) X14337 Vigna angularis var. ABOS9747 * nipponensis (Adzuki bean)* US 7,704,694 B2 57 58

TABLE 7B-continued

Peanut, SEQ NO: 18 & SEQ NO: 24 primer: Amplification products

GenBank Scientific Name Accession (Common Name) No. W6 W5 W3

Vigna angularis var. ABO60O88 nipponensis (Adzuki bean)* Related Species of the Genus Arachis Stylosanthes actiminata AJ32O282 Belonging to Leguminous Stylosanthes angustifolia AJ32O284 Stylosanthes at trea AJ32O285 Stylosanthes biflora AJ32O289 Stylosanthes bracteata AJ32O346 Stylosanthes calcicola AJ32O348 Stylosanthes Campestris AJ32O291 Stylosanthes Capitata AJ32O3SO k Stylosanthes cayennensis AJ320292 384 bp 455 bp 301 bp 372 bp Stylosanthes erecta AJ32O352 Stylosanthes finiticosa AJ32O356 Stylosanthes gracilis AJ32O296 Stylosanthes grandifolia AJ32O299

TABLE 7C Peanut, SEQ NO: 18 & SEQ NO. 24 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W3 Related Species of the Genus Arachis Stylosanthes guianensis Belonging to Leguminous Subsp. dissitiflora Stylosanthes hanaia 32O365 Stylosanthes 32O317 hippocampoides k Stylosanthes hispida 32O328 384 bp 455 bp 301 bp 372 bp Stylosanthes humilis 32O323 Stylosanthes ingrata 32O329 Stylosanthes leiocarpa 32O332 306 bp 71 bp Stylosanthes linearifolia 32O367 Stylosanthes macrocarpa 32O369 Stylosanthes macrocephala 32O371 Stylosanthes macrosoma 32O333 Stylosanthes mexicana 32O374 Stylosanthes 32O336 montevidensis Stylosanthes pilosa 32O377 Stylosanthes scabra 32O382 Stylosanthes Seabrana 32O384 Stylosanthes sericeiceps 32O386 Stylosanthes subsericea 32O387 Stylosanthes Sundaica 32O389 Stylosanthes Sympodialis 32O391 Stylosanthesiomentosa 32O337 Stylosanthes tuberculata 32O392 k Stylosanthes viscosa 32O340 385bp 149 bp 436 bp Stylosanthes 32O316 hippocampoides Stylosanthes humilis 320327 301 bp 66 bp Stylosanthes mexicana 32O373 Ormocarpum bernierianum F189036 Ormocarpum coeruleum F189037 Ormocarpum drakei F189039 90 bp US 7,704,694 B2 59 60

TABLE

Peanut, SEQ NO: 18 & SEQ NO: 24 primer: Amplification products

GenBank Scientific Name Accession (Common Name) No. W6 W2

Related Species of the Genus Arachis Ormocarpum flavum F189041 Belonging to Leguminous Ormocarpum keniense FO68155 Ormocarpum kirkii FO68152 Ormocarpum kiainei F189044 Ormocarpum FO68154 megalophyllum Ormocarpum muricatum FO681S6 Ormocarpum orientale FO68159 Ormocarpin pubescens F189045 Ormocarpum rectangulare F189046 Ormocarpum Schliebenii F189047 Ormocarpum Sennoides FO68153 Ormocarpiim Somaliense F189048 Ormocarpum trachycarpum F189049 Ormocarpum trichocarpum FO681.58 Ormocarpum ecoStil F1890SO 91 bp Chapmannia floridana F2O3S43 462 bp 35 bp 226 bp Chapmannia prismatica 320400 Chapmannia Somaliensis F2O3S44 Chapmannia gracilis F203546 Chapmannia reghiaiensis F2O4232 Chapmannia sericea F2O3S48 Chapmannia tinireana F203547 Ormocarpopsis aspera FO68148 Ormocarpopsis calcicola FO681.45 Ormocarpopsis FO681.49 69 bp itremoensis Ormocarpopsis A. FO68147 69 bp mandrafensis Ormocarpopsis parvifolia A. FO68144 Ormocarpopsis A. FO68146 69 bp titlearensis Diphysa humilis AFO 68162 90 bp Diphysa macrophylla AF189029

TABLE 7E Peanut, SEQ NO: 18 & SEQ NO. 24 primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 Related Species of the Diphysa Suberosa AF189034 - 90 bp Genus Arachis Belonging Fiebrigiella gracilis AF203561 to leguminous kFissicalyx fendieri AF189061 — 384 bp 302 bp

Related Species of the Spigeia coeliostylioides AF177992 — Genus Arachis Not Spigeia hedyotidea AF1780OS Belonging to Spigeia marilandica AF177991 Leguminous US 7,704,694 B2 61 62 PCR described above was conducted using primers ITS-1 and ITS-2 sequences in the following DNA sequences designed in the present invention. In this case, target 253 to registered in GenBank were selected. 259 bp amplification products, 5 expected from the simula 1: Buckwheat: Fagopyrum esculentum (AB000330) tion results of the ITS-1-5.8S rRNA gene-ITS-2 sequences (3) DNA Sequences of Rye, Barley and Oats of the genus Arachis, were obtained from 500 to 50 fg of 5 Regarding rye, barley and oats, 5.8S rRNA gene, ITS-1 and peanut DNA. It is found from the results that even where 500 ITS-2 sequences in the following DNA sequences registered to 50 fg of peanut DNA is present, the peanut can be detected. in GenBank were selected. In this connection, this sensitivity corresponds to a sensitivity 1: Rye: Secale cereale (L36504) wherein there can be detected 10 to 1 ppm of peanut DNA 2: Barley: Hordeum vulgare (AF440678) contained in the sample DNA when PCR was conducted with, 10 as a template, 50 ng of DNA isolated from some samples. 3: Oat: Avena sativa (Z96893) Consequently, in conjunction with the results of specificity (4) DNA Sequences of Related Species of the genus Triticum studied by PCR simulation, and the results of sensitivity and As representatives of the DNA sequences of related species specificity studied by PCR, it was confirmed that a wide range of the genus Triticum, 5.8S rRNA gene. ITS-1 and ITS-2 of plants in the genus Arachis including peanut were detect 15 sequences in the following 70 DNA sequences registered in able using the present invention. GenBank were selected. In this connection, the 70 DNA sequences were selected as representatives of the DNA Example 6 sequences of related species of the genus Triticum, each of which had the highest score in the corresponding genus other than genus Fagopyrum and a score of 60 bits or more among A. Design of Oligonucleotide Primers for Detection of DNA sequences of species belonging to the corresponding genus from Wheat selected from sequences registrated in GenBank through a (1) DNASequences of the Genus Triticum BLAST homology search using the ITS-2 sequence of wheat Regarding the genus Triticum, 5.8S rRNA gene, ITS-1 and (Triticum aestivum Z11761). ITS-2 sequences in the following 29 DNA sequences regis 25 1: Ancestral species of wheat: Aegilops Sharonensis tered in GenBank were examined to select suitable regions for (AF149195) the primer. : Taeniatherum caput-medusae (L36505) Triticum aestivum (AF440679) : Agropyron puberulum (L36482) : Triticum aestivum (AF440676) : Thinopyrum intermedium (AF507809) 30 ... Lophopyrum elongatum (L36495) Triticum aestivum (AF4381.91) : Pseudoroegneria spicata (L36502) Triticum aestivum (AF438.188) : Peridictyon sanctum (L36497) Triticum aestivum (AF438187) . Australopyrum pectinatum (L36484) : Triticum aestivum (AF438186) 9. Amblyopyrum muticum (AF149202) Triticum baeoticum (AJ238901) 35 10. Henrardia persica (L36491) Triticum urartu (AJ301803) 11: Eremopyrum bonaepartis (L36490) : Triticum turgidium subsp. dicoccum (AJ301801) 12: Crithopsis delileana (L36487) : Triticum monococcum (AJ301800) 13: Psathyrostachys fragilis (L36498) : Triticum aestivum (AJ301799) 14. Heteranthelium piliferum (L36492) 40 15. Critesion violaceum (L36488) : Triticum monococcum (AJ245404) 16: Secale Sylvestre (AJ409210) : Triticum turgidium (AJ238919) 17: Haynaldia villosa (L36489) : Triticum turgidium (AJ238918) 18. Bromus tectorum (L36485) : Triticum turgidium (AJ238917) 19: Helictotrichon gervaisii (AJ389134) : Triticum turgidium (AJ238915) 45 20: Festuca lasto (AF303418) 21: Lagurus ovatus (AJ389166) : Triticum turgidium (AJ238913) 22. Poa pratensis (AF171183) : Triticum turgidium (AJ238912) 23. Pseudarrhenatherum longifolium (AJ389 162) : Triticum turgidium (AJ238911) 24. Alopecurus vaginatus (Z96921) : Triticum timopheevi (AJ238924) 50 25. Calamagrostis epigeios (AJ306448) : Triticum timopheevi (AJ238923) 26. Thisetum spicatum (AJ389168) : Triticum timopheevi (AJ238922) 27. Koeleria pyramidata (Z96911) : Triticum timopheevi (AJ238921) 28. Beckmannia eruciformis (AJ389164) : Triticum timopheevi (AJ238920) 29: Lolium persicum (AF171157) 55 30: Diarrhena americana (AF019798) : Triticum turgidium (AJ238916) 31: Arrhenatherum elatius (AF019795) : Triticum turgidium (AJ238914) 32. Deschampsia christophersenii (AF486267) : Triticum urartu (AJ238902) 33. Piptochaetium fimbriatum (L36523) : Triticum aestivum (Z11761) 34. Vulpia fasciculata (AF303402) 29. Triticum monococcum (L11581) 60 35: Phalaris truncata (L36522) 36. Holcus lanatus (Z96919) (2) DNA Sequences of Other Common Allergenic Plants and 37: Merxmuellera stricta (AF019871) Plants Widely Used for a Food Ingredient 38. Brachypodium mexicanum (AFO 19805) The DNA sequences described in Example 1A (2) “DNA 39. Austrostipa nodosa (AFO19804) Sequences of Other Common Allergenic Plants’ and (3) 65 40. Ampelodesmos mauritanica (AFO 19799) “DNA Sequences of Plants Widely Used for a Food Ingredi 41. Nassella viridula (L36521) ent” were selected. Regarding buckwheat, 5.8S rRNA gene, 42. Melica imperfecta (L36519) US 7,704,694 B2 63 64 43. Achnatherum hymenoides (L36507) k: An obtained amplification product whose size almost 44. Austrodanthonia auriculata (AF367604) matched the target 140 bp (+10 bp). 45. Notodanthonia laevis (AF019875) W 2-6: Probability to obtain amplification products 46. Oryzopsis exigua (AFO19801) High Probability W6>W5>W4>W3>W2 Low Prob 47. ability Chionochloa rigida (AF367597) Numerical values followed by bp: 48. Thysanolaena maxima (AFO 19854) Each value was obtained by subtracting 2 from the value 49. Monachather paradoxus (A-FO19852) obtained in the simulation. 50. Stipagrostis zeyheri (A-FO19845) (-): No amplification product was predicted. 51 Arundo donax (AF019809) 10 Related Species of the genus Triticum. 52. Zingeria biebersteiniana (AJ428836) Sequences similar to the ITS-2 sequence of Triticum aes 53. Centothecalappacea (AFO19814) tivum (Z11761) were searched by means of a BLAST 54. Briza minor (L36510) homology search and the sequences having a score of 60 55. Thibolium hispidum (AF367602) bits or more were selected from among them. Each 56. Rytidosperma pumilum (AFO 19878) 15 sequence having the highest score in each genus and having a score of 60 bits or more is shown in the follow 57. Karroochloa purpurea (AFO19874) ing Tables 8C-8F as the representative of the DNA 58. Centropodia glauca (AFO19861) sequences of related species of the genus Triticum. 59. Cortaderia archboldii (AF367620) 60. Lamprothyrsus peruvianus (AF367605) C. Preparation of Template DNA for PCR 61. Imperata cylindrica (A1F345653) (1) Samples Used for DNA Extraction 62. Zizania latifolia (A-F 169234) Wheat: 63. Prionanthium ecklonii (AF019866) Commercially available seeds of wheat were used. 64. Pentaschistis aspera (AFO 19865) 65. 25 (2) DNA. Isolation from Wheat Pentameris macrocalycina (A-FO19864) A DNA was isolated from wheat in the same way as in 66. Molinia caerulea (AF019857) Example 1B (3). The isolated DNA preparation of wheat was 67. Dregeochloa pumilla (AFO 19853) diluted stepwise with sterilized ultrapure water to use as 68. Diplopogon setaceus (AFO 19851) template DNA for PCR. 69. Amphipogon amphopogonoides (AFO 19850) 30 D. PCR 70. Aristida purpurea (AF019807) PCR was conducted in the substantially same way as (5) Oligonucleotide Primer Synthesis and Evaluation Example 1C, except for use of the following primers and PCR Among ITS-2 sequences of the aforementioned 29 DNA program. sequences of the genus Triticum, there was determined nucle 35 Primer: otide sequences which would specifically hybridize to all of The primer of SEQ NO:28 was used at 0.5 M of final the 29 DNA sequences of the genus Triticum through the concentration and each primer of SEQ NOs:29 and 30 was study of the sequences. The thus determined nucleotide used at 0.25uM of final concentration. sequences are indicated as SEQ NOs:28, 29 and 30. Subse PCR Program: quently, the oligonucleotide primers with SEQ NOS:28, 29 40 PCR was conducted according to the following PCR pro and 30 were synthesized. gram. Pre-incubation at 95°C. for 15 min...: thereafter 45 cycles Sense primer consisting of denaturation at 95°C. for 1 min., annealing at s" - CGG CAT CTG GTC CCT CGT CT-3' (SEQ NO: 28) 66° C. for 1 min. and extension at 72°C. for 1 min.; followed 45 by a final extension at 72°C. for 4 min. Antisense primer The quality of each of the template DNA used here was 5 - GCG. AGG ACG. CCC ACC AT-3' (SEQ NO: 29) sufficient enough to be used for PCR based on the result of a 5 GCA AAG ACG CCC ACC AT-3' (SEQ NO:30) separate PCR, in which target products were obtained using a primer pair to amplify a part of plant chloroplast DNA. 50 B. PCR Simulation E. Results Regarding the sense and antisense primer pairs, the simu Regarding the primers of the present invention, the simu lation was conducted with PCR simulation software, Amplify lation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels), which is the same as in Example 1 to 1.0 (Bill Engels) to examine the specificity to ITS-2 sequence examine whether target size of amplification products are 55 of each plant. As a result, as shown in Tables 8A to 8F, it was obtained from the 29 DNA sequences of the genus Triticum, predicted that target 93 to 95bp amplification products would the 8 DNA sequences of common allergenic plants other than be obtained from the aforementioned 29 DNA sequences of wheat (buckwheat, peanut, soybean, walnut, matsutake the genus Triticum. On the other hand, it was predicted that no mushroom, peach, apple and orange), the 4 DNA sequences target 93 to 95bp amplification product would be obtained of plants widely used for a food ingredient (corn, brown rice, 60 from the 8 DNA sequences of common allergenic plants other pepper and mustard), the DNA sequences of rye, barley and than wheat (buckwheat, peanut, soybean, walnut, matsutake oat and the DNA sequences of Aegilops termed ancestral mushroom, peach, apple and orange), the 4 DNA sequences species of wheat and plants in related species of the genus of plants widely used for a food ingredient (corn, brown lice, Triticum used for breed improvement of wheat belonging to pepper and mustard), the DNA sequences of rye, barley and the tribe Triticeae. The simulation results are shown in Tables 65 oat, the 2 DNA sequences of related species of the genus 8A to 8F. The meanings of symbols and numerical values in Triticum belonging to the tribe Triticeae and the 51 DNA Tables 8A to 8F are explained below. sequences of related species of the genus Triticum not belong US 7,704,694 B2 65 66 ing to the tribe Triticeae. In this connection, it was predicted species of the genus Triticum used for breed improvement of by simulation that amplification products having almost 93 to wheat belonging to the tribe Triticeae and Some plants in 95 bp would be obtained from the DNA sequences of related species of the genus Triticum not belonging to tribe Aegilops termed ancestral species of wheat, plants in related Triticeae.

TABLE 8A Wheat, SEQ No. 28 & SEQ NOS: 29 and 30 (two types) primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Genus Triticum Triticum aestivum AF440679 94 bp – 335 bp – 287 by 218 by kTriticum aestivum AF440676 94 bp – 335 bp – 287 by 218 by kTriticum aestivum AF438.191 95 bp – 336 bp 288 bp 219 bp kTriticum aestivum AF4381.88 94 bp 335 by 287 by kTriticum aestivum AF4381.87 94 bp 335 by 287 by 218 by kTriticum aestivum AF438186 94 bp 333 bp - 28S by 217 by kTriticum baeoiicum AJ238901, 94 bp kTriticum tirartu AJ3O18O3 94 bp = 334 bp - 286 bp 217 by k Triticum turgidium subsp. AJ301801 94 bp — 335 bp — dicoccum 287 by 218 by k Triticum monococcum AJ3O18OO 94 bp 286 bp - 217 by kTriticum aestivum AJ3O1799 94 bp 284 bp - 215 by kTriticum monococcum AJ245404 94 bp k Triticum turgidium AJ238919 94 bp k Triticum turgidium AJ238918, 94 bp k Triticum turgidium AJ238917 94 bp k Triticum turgidium AJ238.915 94 bp k Triticum turgidium AJ238913 94 bp

TABLE 8B Wheat, SEQ No. 28 & SEQ NOS: 29 and 30 (two types) primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Genus Trictim k Triticum turgidium AJ238912 94 bp – k Triticum turgidium AJ238911 94 bp – k Triticum timopheevi AJ238924 94 bp – k Triticum timopheevi AJ238923 94 bp – k Triticum timopheevi AJ238922 93 bp – k Triticum timopheevi AJ238921 94 bp – k Triticum timopheevi AJ238920 94 bp – k Triticum turgidium AJ238916 94 bp – k Triticum turgidium AJ238914. 94bp – kTriticum tirartu AJ238902 94 bp – kTriticum aestivum Z11761 94 bp — 335 bp — 287 bp 218 bp kTriticum monococcum L11581 94 bp — 286 bp — 217 bp Common Allergenic Plants Arachis hypogaea AF15667S — 342 bp (Peanut) 146 bp Fagopyrim escientiin ABOOO330 - (Buckwheat) Glycine max (Soybean) U60551 Jugians regia (Walnut) AF3O3809 - Tricholoma matsutake U62964 US 7,704,694 B2 67 68

TABLE 8B-continued Wheat, SEQ No. 28 & SEQ NOS: 29 and 30 (two types) primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 WS W4 W3 W2 (Matsutake mushroom) Prunus persica (Peach) AF185621 — 215 bp Maius x domestica AF186484 - (Apple) Citrus sp. E08821 (Valencia orange) Plants Widely Zea mays U46648 Used for a Food (Corn) Ingredient Oryza sativa (Brown rice) AF169230 — – 299 bp Piper nigrum (Pepper) AF275197 — 225 bp Sinapis alba (Mustard) X15915

TABLE 8C Wheat, SEQ No. 28 & SEQ NOS: 29 and 30 (two types) primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 WS W4 W3 W2 Rye, Barley Secale cereale (rye) L36504 and Oat Hordeum vulgare (Barley) AF44O678 – 287 bp — 218 by Avena sativa (Oat) Z96893 Related Species of the Genus Triticum k Aegilops sharonensis AF149195 94 bp - 335 bp - Belonging to Tribe Triticeae (Ancestral species of wheat) 287 by 218 by kTaeniaiherum L36505 94 bp - 287 b - captit-medusae 218 by y Agropyron pubertium L36482 94 bp - 287 b - 218 by k Thinopyrum intermedium AFSO7809 94 bp - 287 bp - 218 by st Lophopyri in elongattin L36495 94 bp — 334bp — 286 bp 217 by y Pseudoroegneria spicaia L36SO2 94 bp — 286 bp — 217 by k Peridictyon sanctum L36497 94 bp — 286 bp — 217 by kAustralopyrum pectinatum L36484 94 bp — 286 bp — 217 by kAmblyopyrum multicum AF1492O2 94 bp - 335 bp - 287 by 218 by kHenrardia persica L36491 94 bp — 334bp — 286 bp 217 by kEremopyrum bonaepartis L364.90 94 bp — 334bp — 286 bp 217 by k Crithopsis delileana L36487 94 bp - 335 by 287 by 218 by

TABLE 8D Wheat, SEQ No. 28 & SEQ NOS: 29 and 30 (two types) primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Related Species of the Genus k Psathyrostachys L36498 94 bp – 335 bp Triticum Belonging to Tribe fragilis 287 bp Triticeae 218 bp Heteranihelium L364.92 94 bp — 286 bp piliferum 217 bp US 7,704,694 B2 69 70

TABLE 8D-continued Wheat, SEQ No. 28 & SEQ NOs: 29 and 30 (two types) primer: Amplification products

GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Critesion violiaceum L36488 — 335 bp 218 bp — 287 bp Secale Sylvestre AJ4O9210 Related Species of the Genus Triticum k Haynaldia villosa L36489 94 bp — 284 bp Not Belonging to Tribe Triticeae 215 bp Bromus tectorum L36485 94 bp — 286 bp 217 bp Heiictoirichon AJ389134 gervaisi Festica lasto AF3O3418 st Lagurus ovatus AJ389166 — 94 bp Poa pratensis AF1711.83 Pseudiarrhenaiherum AJ389.162 longifolium Alopectiris vaginiatus Z96921 Calamagrostis epigeios AJ306448 Tisetum spicatin AJ389168 Koeleria pyramidata Z96911 Beckmannia eruciformis AJ389164 Lolium persicum AF171157 Diarrhena americana AFO 19798 Arrhenaiherum elatius AFO 1979S Deschampsia AF486267 332 bp — Christophersenii 215 bp Piptochaetium L36523 fimbriatum Viipia fasciculata AF3O34O2 Phaiaris truncaia L36522 Holcus ianathis Z96919

TABLE 8E Wheat, SEQ No. 28 & SEQ No. 29 and 30 (two types) primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 WS WA. W3 W2 Related Species of the Genus Triticum Merxmuellera stricta AFO 19871 — 436 bp Not Belonging to Tribe Triticeae 280 bp 211 bp Brachypodium mexicanum AFO19805 AustroStipa nodosa AFO19804 - Ampelodesmos AFO 19799 mattritanica Nasseila viridulia L36521 Melica imperfecta L36519 – 114 bp Achnaitherum hymenoides L36507 – 286 bp 217 bp Austrodanthonia auriculata AF367604 — – 282 bp 213 bp Notodianthonia iaevis AFO 19875 - 283 by Oryzopsis exigua AFO198O1 Chionochloa rigida AF367597 – 281 bp 212 by Thysanolaena maxima AFO19854 - Monachather paradoxus AFO19852 – – 286 bp 217 bp 142 bp 73 bp Stipagrostis zeyheri AFO 19845 Arundo donax AFO19809 - – 289 bp 220 bp Zingeria biebersteiniana A428836 Centotheca lappacea AFO19814 - Briza minor L3651O Triboium hispidum AF3676O2 - – 282 bp 213 bp Rytidospermaputnium AFO 19878 – 282 bp 213 bp Karroochloa purpurea AFO19874 – - 282 by 213 by Centropodia giatica AFO 19861 — 281 bp 212 bp Coria deria archboidii AF36762O - — 280 bp 211 bp US 7,704,694 B2

TABLE 8E-continued Wheat, SEQ No. 28 & SEQ No. 29 and 30 (two types) primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 WS WA. W3 W2 Lamprothyrsus AF3676OS — 280 bp 211 bp peruviants Imperata cylindrica AF345653

TABLE 8F Wheat, SEQ No. 28 & SEQ No. 29 and 30 (two types) priner: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 WS W4 W3 W2 Related Species of the Genus Triticum Zizania latifolia AF169234 - — 311 bp Not Belonging to Tribe Triticeae 282 bp 213 bp Prionanthium eckioni AFO 19866 - — 471 bp 283 bp 214 bp Pentaschistis aspera AFO 1986S — 283 bp 214 bp Pentameris macrocalycina AFO 19864 - — 283 bp 214 bp Moinia caerulea AFO 19857 Dregeochloa pumilia AFO 19853 – 285 bp Diplopogon Setaceus AFO 19851 – 285 bp 216 bp Amphipogon amphopogonoides AFO 1985.0 — – 285 bp 216 bp Aristida purpurea AFO 19807

PCR described above was conducted using primers 6: Glycine soia (U60550) designed in the present invention. In this case, target 93 to 95 35 7. Glycine soia (AF144653) bp amplification products, expected from the simulation 8. Glycine soia (AJO09790) results of the ITS-2 sequences of the genus Triticum, were 9. Glycine soia (AJO09791) obtained from 500 to 50 fg of wheat DNA. It is found from the 10: Glycine soia (AJ224109) results that even where 500 to 50 fg of wheat DNA is present, 11: Glycine max (AJO11337) the wheat can be detected. In this connection, this sensitivity 40 12: Glycine max (AJO09787) correspond to a sensitivity wherein there can be detected 10 to 13: Glycine max (AF144654) 1 ppm of peanut DNA contained in the sample DNA when 14: Glycine cyrtoloba (U60548) PCR was conducted with, as a template, 50 ng of DNA 15. Glycine tomentella (AF023447) isolated from Some samples. 16: Glycine tomentella (U60544) Consequently, in conjunction with the results of specificity 45 17: Glycine microphylla (U60537) studied by PCR simulation, and the results of sensitivity 18. Glycine tomentella (U60542) studied by PCR, it was confirmed that a wide range of the 19: Glycine arenaria (U60543) genus Triticum including wheat, ancestral species of wheat, 20: Glycine tabacina (U60539) and the majority of the tribe Triticeae were specifically 21: Glycine curvata (U60547) detectable at a high sensitivity using the present invention. 50 22. Glycine tomentella (AJO 11345) 23. Glycine pindanica (U60546) Example 7 24. Glycine lactovirens (U60540) 25. Glycine albicans (U60541) 26: Glycine argyrea (U60535) A. Design of Oligonucleotide Primers for Detection of DNA 55 from Wheat 27. Glycine tomentella (AF023446) 28: Glycine latifolia (U60538) (1) DNA Sequences of the Genus Glycine 29: Glycine clandestina (U60534) Regarding the genus Glycine, 5.8S rRNA gene. ITS-1 and 30: Glycine tomentella (AF023445) ITS-2 sequences in the following 50 DNA sequences regis 31. Glycine dolichocarpa (AJO 11340) tered in GenBank were examined to select suitable regions for 60 32. Glycine dolichocarpa (AJ2241 10) the primer. 33. Glycine canescens (AF023444) Glycine max (U60551) 34. Glycine hirticaulis (U60545) : Glycine max (L36612) 35. Glycine tomentella (AJO11342) : Glycine max (AF144652) 65 36. Glycine dolichocarpa (AJO 11341) : Glycine max (AF144651) 37: Glycine canescens (U60533) : Glycine max (BI674312) 38. Glycine canescens (AJO11348) US 7,704,694 B2 73 74 39. Glycine tabacina (AJO09788) 40. Glycine tabacina (AJO09789) - Continued 41. Glycine latrobeana (U60536) 42. Glycine tomentella (AJO11344) 5'-GCG TTG CTC ATC CAC CAT TTT GTC A-3 '' (SEO NO:37) 43. Glycine tomentella (AJO11343) 5'-GCA TTG CTC ATC CAC CAT TTT GTC A-3 '' (SEO NO:38) 44. Glycine tomentella (AJO11338) 45. Glycine tabacina (AJO 11346) 5'-GCG CTG CTC ATC CGC CAT TTT GTC A-3 '' (SEO NO:39) 46. Glycine dolichocarpa (AJO11339) 5'-GCG CTG CTC ATC CAC CAT TTT GTC A-3 '' (SEO NO: 40) 47. Glycine tabacina (AJ224111) 48. Glycine falcata (U60549) 10 s' - GCG, TGG CTC ATC CAT TTT ATC A-3' (SEQ NO: 41) 49. Glycine latifolia (AJO09786) 50: Glycine tabacina (AJO 11347) B. PCR Simulation (2) DNA Sequences of Other Common Allergenic Plants and Regarding the sense and antisense primer pairs, the simu Plants Widely Used for a Food Ingredient lation was conducted with PCR simulation software, Amplify The DNA sequences described in Example 1A (2) “DNA 15 1.0 (Bill Engels), which is the same as in Example 1 to Sequences of Other Common Allergenic Plants’ and (3) examine whether target size of amplification products are “DNA Sequences of Plants Widely Used for a Food Ingredi obtained from the 50 DNA sequences of the genus Glycine, ent” were selected. Regarding buckwheat, 5.8S rRNA gene, the 8 DNA sequences of common allergenic plants other than ITS-1 and ITS-2 sequences in the following DNA sequences Soybean (buckwheat, peanut, wheat, walnut, matsutake registered in GenBank were selected. mushroom, peach, apple and orange), the 4 DNA sequences 1: buckwheat: Fagopyrum esculentum (AB000330) of plants widely used for a food ingredient (corn, brown rice, pepper and mustard), the 6 DNA sequence of leguminous (3) DNA Sequences of Leguminous Plants Widely Used for a plants widely used for a food ingredient (French bean, lima Food Ingredient bean, lentil, chickpea, mung bean and adzuki bean) and the 5 The DNA sequences described in Example 3A (4) “DNA 25 DNA sequences of related species of the genus Glycine. The Sequences of Leguminous Plants Widely Used for a Food simulation results are shown in Tables 9A to 9C. The mean Ingredient' were selected. Regarding adzuki bean, ITS-2 ings of symbols and numerical values in Tables 9A to 9C are sequences in the following DNA sequences registered in explained below. GenBank were selected. k: An obtained amplification product whose size almost 1: adzuki bean: Vigna angularis var. nipponensis 30 matched the target 87 to 89 bp (+10 bp). (AB060088) W 2-6. Probability to obtain amplification products (4) DNA Sequences of Related Species of the Genus Glycine High Probability W6>W5>W4>W3>W2 Low Prob As representatives of the DNA sequences of related species ability of the genus Glycine, 5.8S rRNA gene, ITS-1 and ITS-2 Numerical values followed by bp: sequences in the following 5 DNA sequences registered in 35 Each value was obtained by subtracting 2 from the value GenBank were selected. In this connection, the 5 DNA obtained in the simulation. sequences were selected as representatives of the DNA (-): No amplification product was predicted. sequences of related species of the genus Glycine, each of Related Species of the genus Arachis: which had the highest score in the corresponding genus other Sequences similar to the ITS-2 sequence of Glycine max than genus Glycine and a score of 60 bits or more among 40 (U60551) were searched by means of a BLAST homol sequences of species belonging to the corresponding genus ogy search and the sequences having a score of 60 bits or selected from sequences registrated in GenBank through a more were selected from among them. Each sequence BLAST homology search using the ITS-2 sequence of soy having the highest score in each genus and having a bean (Glycine max U60551). score of 60 bits or more is shown in the following Table 1: Ophrestia radicosa (AF467-484) 45 9C as the representative of the DNA sequences of related 2: Myrospermum sousanum (AF187086) species of the genus Glycine. 3. Amphicarpaea bracteata (AF417019) 4. Amphicarpaea edgeworthii (AF417013) C. Preparation of Template DNA for PCR 5. Strophostyles umbellata (AFO69115) 50 (1) Samples Used for DNA Extraction (5) Oligonucleotide Primer Synthesis Soybean: Among ITS-2 sequences of the aforementioned 50 DNA Commercially available seeds of soybean were used. sequences of the genus Glycine, there was determined nucle otide sequences which would specifically hybridize to all of (2) DNA Isolation from Soybean 55 A DNA was isolated from seeds of soybean in the same the 50 DNA sequences of the genus Glycine through the study way as in Example 1B (3). The isolated DNA preparation of of the sequences. The thus determined nucleotide sequences soybean was diluted stepwise with sterilized ultrapure water are indicated as SEQ NOS:34 to 41. Subsequently, the oligo nucleotide primers with SEQNOS:34 to 41 were synthesized. to use as template DNA for PCR. D. PCR 60 PCR was conducted in the substantially same way as Sense primer: Example 1C, except for use of the following primers and PCR s' - CTG ACC TCC CGC GAG CAC-3' (SEO NO:34) program. Anti sense primer: Primer: 5'-GCG TGG CTC ATC CAC CAT TTT ATC A-3 '' (SEO NO:35) 65 The primer of SEQ NO:34 was used at 0.5 M of final 5'-GCG TTG CTC ATC CAC CAT TTT ATC A-3 '' (SEO NO:36) concentration and each primer of SEQ NOS:36 and 37 was used at 0.25uM of final concentration. US 7,704,694 B2 75 76 PCR Program: the genus Glycine. On the other hand, it was predicted that no PCR was conducted according to the following PCR pro target 87 to 89bp amplification product would be obtained gram. from the 8 DNA sequences of other common allergenic plants Pre-incubation at 95°C. for 15 min...: thereafter 45 cycles (buckwheat, peanut, wheat, walnut, matsutake mushroom, consisting of denaturation at 95°C. for 1 min., annealing at peach, apple and orange), the 4 DNA sequences of plants 68°C. for 1 min. and extension at 72°C. for 1 min.; followed widely used for a food ingredient (corn, brown rice, pepper by a final extension at 72°C. for 4 mm. and mustard), the 6 DNA sequence of leguminous plants The quality of each of the template DNA used here was widely used for a food ingredient (French bean, lima bean, sufficient enough to be used for PCR based on the result of a lentil, chickpea, mung bean and adzuki bean) and the 3 DNA separate PCR, in which target products were obtained using a 10 sequences of related species of the genus Glycine belonging primer pair to amplify a part of plant chloroplast DNA. to leguminous plants. In this connection, it was predicted by E. Results simulation that amplification products having almost 87 to 89 Regarding the primers of the present invention, the simu bp would be obtained from the DNA sequences of Amphicar lation was conducted with PCR simulation software, Amplify paea edgeworthii and Ophrestia radicosa, but the former can 1.0 (Bill Engels) to examine the specificity to ITS-2 sequence be identified by sequence analysis whether the genus Glycine of each plant. As a result, as shown in Tables 9A to 9C, it was or not and the latter may be able to be identified by appropri predicted that target 87 to 89bp amplification products would ately using a commercially available ELISA kit of soybeans be obtained from the aforementioned 50 DNA sequences of and various types of PCR and the like reported.

TABLE 9A Soybean, SEQ NO:34 & SEQ NOs: 35 to 41 (7 types) primer: Amplification products

GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Genus Glycine kGlycine max (soybean) U60551 89 bp - 62 bp — kGlycine max (soybean) L36612 89 bp - 62 bp — kGlycine max (soybean). AF144652 89 bp — 62 bp — kGlycine max (soybean). AF144651 89 bp — 62 bp — kGlycine max (soybean) BI674312 89 bp — 62 bp — kGlycine soia U60550 89 bp - 62 bp — kGlycine soia AF144653 89bp kGlycine soia AJOO9790 89 bp 61 bp — kGlycine soia AJOO9791, 89bp 60 bp — kGlycine soia AJ224109 89bp 60 bp — kGlycine max (soybean) AJO11337 89 bp — 60 bp — kGlycine max (soybean) AJO09787 89 bp — 60 bp — kGlycine max (soybean). AF144654 87 bp — 55 by kGlycine cyrtoloba U60548 89 bp - 62 bp — kGlycine tomentella AFO23447 89bp 62 bp — kGlycine tomentella U60544 89 bp - 62 bp — kGlycine microphylla U60537 89 bp - 62 bp — kGlycine tomentella U60542 89 bp - 62 bp — kGlycine arenaria U60543 89 bp - 62 bp — kGlycine tabacina U60539 89 bp - 357 b - 62 bp kGlycine curvata U60547 89 bp - 62 bp — kGlycine tomentella AJO11345 89bp 61 bp — kGlycine pindanica U6OS46 89 bp - 62 bp — kGlycine lactovirens U60540 89 bp - 62 bp — kGlycine albicans U60541 89 bp - 62 bp — kGlycine argyrea U60535 89 bp - 62 bp — kGlycine tomentella AFO23446 89bp 62 bp — kGlycine latifolia U60538 89 bp – 357 bp, – 62 bp kGlycine clandestina U60534 89 bp - 62 bp — kGlycine tomentella AFO23445 89bp 62 bp — kGlycine dolichocarpa AJO11340 89 bp — 61 bp —

TABLE 9B Soybean, SEQ NO. 34 & SEQ NOs: 35 to 41 (7 types) primer: Amplification products GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2 Genus Glycine Glycine doichocarpa AJ224110 89 bp — 161 bp — kGlycine canescens AFO23444 89 bp – 161 bp – kGlycine hiricaulis U60545 89 bp — 162 bp — kGlycine tomentella AJO11342 89 bp — 161 bp — US 7,704,694 B2 77 78

TABLE 9B-continued

Soybean, SEQNO:34 & SEQ NOS: 35 to 41 (7 types) primer: Amplification products

GenBank Scientific Name Accession (Common Name) No. W6 W5 W4 W3 W2

kGlycine doichocarpa AJO11341 89 bp — 161 bp — kGlycine canescens U60533 89 bp — 162 bp — kGlycine canescens AJO11348 89 bp – 162 bp – kGlycine tabacina AJOO9788 89 bp – 160 bp – kGlycine tabacina AJOO9789 89 bp – 161 bp – kGlycine latrobeana U60536 89 bp — 162 bp — kGlycine tomentella AJO11344 89bp kGlycine tomentella AJO11343 89bp kGlycine tomentella AJO11338 89bp kGlycine tabacina AJO11346 89 bp – 161 bp – kGlycine doichocarpa AJO11339 89bp kGlycine tabacina AJ224111 89 bp — 161 bp — kGlycine falcata U60549 89 bp — 439 bp — 184 bp kGlycine latifolia AJOO9786 89 bp – 357 bp – kGlycine tabacina AJO11347 89 bp – 161 bp –

TABLE 9C Soybean, SEQ NO. 34 & SEQ NOS: 35 to 41 (7 types) primer: Amplification products GenBank Scientific Name Accession (Common Name) No. Other Common Allergenic Plants Arachis hypogaea (Peanut) AF1566.75 Fagopyrim esculenium ABOOO330 (Buckwheat) Triticum aestivum (Wheat) AJ301799 Jugians regia (Walnut) AF303809 Trichoioma matsutake U62964 (Matsutake mushroom) Prunus persica (Peach) AF185621 Maius X domestica AF1864.84 (Apple)

Citrus sp. E08821 (Valencia orange) Plants Widely Zea mays (Corn) U46648 Used for a Oryza sativa (Brown rice) F169230 Food Piper nigrum (Pepper) F2751.97 Ingredient Sinapis alba 5915 (Mustard) Leguminous Plants Widely Phaseolus vulgaris F115169 Used for a Food Ingredient (French bean) Phaseolus iunatus F115175 (Lima bean) Lens clinaris Subsp. F228.066 culinaris (Lentil) Cicer arietinum (Chickpea) 237698 Vigna radiata (Mung bean) 4337 Vigna angularis var. BO60O88 nipponensis (Adzuki bean) Related Species of k Ophirestia radicosa F467484 the Genus Glycine Myrospermain Sousant in F187086 Belonging to Amphicarpaea bracteata F417019 Leguminous Plants kAmphicarpaea edgeworthii F417013 (Wild bean) Strophostyles umbellata FO6911S US 7,704,694 B2 79 80 PCR described above was conducted using primers detected 10 to 1 ppm of soybean DNA contained in the sample designed in the present invention. In this case, target 87 to 89 DNA when PCR was conducted with, as a template, 50 ng of bp amplification products, expected from the simulation DNA isolated from some samples. results of the ITS-2 sequences of the genus Glycine, were Consequently, in conjunction with the results of specificity obtained from 500 to 50 fg of soybean DNA. It is found from studied by PCR simulation, and the results of sensitivity the results that even where 500 to 50 fg of soybean DNA is studied by PCR, it was confirmed that a wide range of the present, the Soybean can be detected. In this connection, this genus Glycine including soybean were specifically detectable sensitivity corresponds to a sensitivity wherein there can be at a high sensitivity using the present invention.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 41

<21 Os SEQ ID NO 1 &211s LENGTH: 73 &212s. TYPE: DNA <213> ORGANISM: Fagopyrum esculentum

<4 OOs SEQUENCE: 1 caacggatat ct cqgct ct c goatcgatga agaacgtagc galaatgcgat acttggtgtg 60

aattgcagaa to c 73

<21 Os SEQ ID NO 2 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: PCR primer <4 OOs SEQUENCE: 2

gcatttcqct acgttct tca to gatgc 27

<21 Os SEQ ID NO 3 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: PCR primer <4 OOs SEQUENCE: 3

atcgcatttic got acgttct tcatcg 26

<21 Os SEQ ID NO 4 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: PCR primer <4 OOs SEQUENCE: 4

agtat cqcat titcgctacgt tott catc 28

<21 Os SEQ ID NO 5 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: PCR primer <4 OOs SEQUENCE: 5

gcatcgatga agaacgtagc galaatgc 27

<21 Os SEQ ID NO 6 &211s LENGTH: 26 &212s. TYPE: DNA

US 7,704,694 B2 83 84

- Continued SEQ ID NO 13 LENGTH: 21 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: PCR primer SEQUENCE: 13 ggaccacgaa Cagaag.cgcg t 21

SEQ ID NO 14 LENGTH: 22 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: PCR primer SEQUENCE: 14 cgc.caaggac cacgalacaga ag 22

SEO ID NO 15 LENGTH: 23 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: PCR primer SEQUENCE: 15 cgttgc.cgag agt cqttctg titt 23

SEQ ID NO 16 LENGTH: 26 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: PCR primer SEQUENCE: 16 gtcgttctgt titmiktagaaa coacgc 26

SEO ID NO 17 LENGTH: 99 TYPE: DNA ORGANISM: Arachis willosa

< 4 OOs SEQUENCE: 17 aacaagaa.ca aaa.ccc.cggc gcggaaag.cg C caaggaagc caaacgttt C totctic ccc 6 O

99

SEQ ID NO 18 LENGTH: 2O TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: PCR primer SEQUENCE: 18 gcggaaag.cg C caaggaagc

SEQ ID NO 19 LENGTH: 18 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: PCR primer

US 7,704,694 B2 89 90

- Continued

SEQ ID NO 34 LENGTH: 18 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: PCR primer SEQUENCE: 34

Ctgacctic cc gcgagcac 18

SEO ID NO 35 LENGTH: 25 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: PCR primer SEQUENCE: 35 gcqtggct catccaccattt tat ca 25

SEQ ID NO 36 LENGTH: 25 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: PCR primer SEQUENCE: 36 gcqttgct catccaccattt tat ca 25

SEO ID NO 37 LENGTH: 25 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: PCR primer SEQUENCE: 37 gcqttgct catccaccattt tdtca 25

SEQ ID NO 38 LENGTH: 25 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: PCR primer SEQUENCE: 38 gcattgct catccaccattt tdtca 25

SEO ID NO 39 LENGTH: 25 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: PCR primer SEQUENCE: 39 gcqctgct catcc.gc.cattt tdtca 25

SEQ ID NO 4 O LENGTH: 25 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: PCR primer US 7,704,694 B2 91 92

- Continued

<4 OOs, SEQUENCE: 4 O gcqctgct catccaccattt tdtca 25

SEQ ID NO 41 LENGTH: 22 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: PCR primer <4 OOs, SEQUENCE: 41 gcqtggct catccatttitat ca 22

What is claimed is: ITS-1, 5.8S rRNA gene, ITS-2 and LSU rRNA gene 1. A method for detecting species in the genus Glycine in sequences of the genus Glycine are continuously bonded. cases where even one species of the genus Glycine is con 5. The method of claim 3, wherein the step of conducting tained in a subject to which the method is to be applied such PCR comprises using the primers (B) and (D), which primer as a food ingredient or product, which comprises the steps of (D) can hybridize under Stringent conditions to a nucleic acid a) collecting 45S rRNA precursor gene sequences contain molecule having a part of a nucleotide sequence in which ing an ITS sequence of the genus Glycine and plants 25 SSU rRNA gene, ITS-1, 5.8S rRNA gene and ITS-2 thought to be related species thereof and then selecting sequences of the genus Glycine are continuously bonded. the region common to the genus Glycine in the 45S 6. The method of claim 2, wherein the step of conducting rRNA precursor gene sequences; PCR comprises using the primers (A) and (E), which primer b) selecting the base specific to the genus Glycine from the (E) can hybridize under stringent conditions to a nucleic acid region; 30 molecule having a part of a nucleotide sequence in which c) designing primers (A) and/or (B) having the specific SSU rRNA gene and ITS-I sequences of the genus Glycine base at the 3' end thereof, which primers can hybridize are continuously bonded. under the stringent conditions to a nucleic acid molecule 7. The method of claim 3, wherein the step of conducing having a common nucleotide base sequence for all spe PCT comprises using the primers (B) and (F), which primer cies in the genus Glycine in the 45S rRNA precursor 35 (F) can hybridize under stringent conditions to a nucleic acid gene sequence thereof, wherein the 3' end of primer (A) molecule having a part of a nucleotide sequence in which can complementarily bind to a base in the ITS-1 ITS-2 and LSU rRNA sequences of the genus Glycine are Sequence of the genus Glycine when the primer hybrid continuously bonded. izes to the nucleic acid molecule while the 3' end of 8. The method of claim 3, wherein the common specific primer (B) can complementarily bind to a base in the 40 nucleotide sequence for all species in the genus Glycine in ITS-2 sequence of the genus Glycine when the primer ITS-2 sequence thereof is selected from the group consisting hybridizes to the nucleic acid molecule: of SEQ NOS:31, 32 and 33, and complementary nucleotide d) isolating DNA from the subject to which the method is sequences thereof. to be applied; 9. The method of claim 1, wherein the primer (B) is e) conducting PCR amplification for the isolated DNA 45 selected from the group consisting of SEQ NOS:34, 35, 36, using at least one member selected from the group con 37, 38, 39, 40 and 41. sisting of the primers (A) and (B); and 10. The method of claim 4, wherein the primer (C) can f) identifying the presence of the resulting amplification hybridize under Stringent conditions to a nucleic acid mol product from PCR containing at least a part of ITS-1 or ecule having a part of a nucleotide sequence in 5.8S rRNA ITS-2 sequence of the genus Glycine. 50 gene sequence of the genus Glycine, and 3' end of primer (C) 2. The method of claim 1, wherein the nucleic acid mol can complementarily bind to a base in 5.8S rRNA gene ecule having a common nucleotide sequence for all species in sequence of the genus Glycine when the primer hybridizes to the genus Glycine in 45S rRNA precursor gene sequence the nucleic acid molecule. thereof is a nucleic acid molecule having a common specific 55 11. The method of claim 5, wherein the primer (D) can nucleotide sequence for all species in the genus Glycine in hybridize under Stringent conditions to a nucleic acid mol 1-ITS-1 sequence thereof. ecule having a part of a nucleotide sequence in 5.8S rRNA 3. The method of claim 1, wherein the nucleic acid mol gene sequence of the genus Glycine, and 3' end of primer (D) ecule having a common nucleotide sequence for all species in can complementarily bind to a base in 5.8S rRNA gene the genus Glycine in 45S rRNA precursor gene sequence 60 sequence of the genus Glycine when the primer hybridizes to thereof is a nucleic acid molecule having a common specific the nucleic acid molecule. nucleotide sequence for all species in the genus Glycine in 12. The method of claim 10, wherein the part of nucleotide ITS-2 sequence thereof. sequence in 5.8S rRNA gene sequence of the genus Glycine is 4. The method of claim 2, wherein the step of conducting selected from the group consisting of SEQ NO:1 and a PCR comprises using the primers (A) and (C), which primer 65 complementary nucleotide sequence thereof. (C) can hybridize under Stringent conditions to a nucleic acid 13. The method of claim 11, wherein the part of nucleotide molecule having a part of a nucleotide sequence in which sequence in 5.8S rRNA gene sequence of the genus Glycine is US 7,704,694 B2 93 94 selected from the group consisting of SEQ NO:1 and a 16. The method of claim 1, wherein the step of conducting complementary nucleotide sequence thereof. PCR comprises using a combination of a primer of SEQ 14. The method of claim 4, wherein the primer (C) is NO:28 and a primer selected from the group consisting of selected from the group consisting of SEQ NOs:2, 3 and 4. SEQ NOS:35, 36, 37,38, 39, 40 and 41 as the primer (B). 15. The method of claim 5, wherein the primer (D) is 5 selected from the group consisting of SEQ NOs:5, 6 and 7. k . . . .