USOO633315OB1 (12) United States Patent (10) Patent No.: US 6,333,150 B1 Roman0 et al. (45) Date of Patent: *Dec. 25, 2001

(54) ISOTHERMAL BASED 6,074,865 6/2000 Kelly et al.. ASSAY FOR THE DETECTION AND 6,190,859 * 2/2001 Putnal et al.. GENOTYPNG OF DENGUE VIRUS FOREIGN PATENT DOCUMENTS (75) Inventors: Joseph W. Romano, Derwood; Eun Mi 2279652 1/1995 (GB). Lee, Gaithersburg, both of MD (US); WO9306214 4/1993 (WO). Gregory J. Hurteau, Alexandria, VA WO 9640933 6/1995 (WO). (US) WO964O933 12/1996 (WO). WO9849351 11/1998 (WO).

(*) Notice: Subject to any disclaimer, the term of this R. Rico-Hesse et al., Virology, 230:244-251, 1997. patent is extended or adjusted under 35 E. Chungue et al., Journal of Medical Virology, 40: 142-145, U.S.C. 154(b) by 0 days. 1993. R. Lanciotti et al., Journal of Clinical Microbiology, This patent is Subject to a terminal dis- 30:3:545-551, 1992. claimer. V. Vorndam et al., Journal of Virological Methods, 48:237-244, 1994. (21) Appl. No.: 09/374,584 E. Henchal et al., Am. J. Trop. Med. Hyg., 45(4):418-428, 1991. (22) Filed: Aug. 13, 1999 T.M. Sudhiro, Am. J. Trop. Med. Hyg., 56(4):424-429, 1997. Lewis et al., Journal of Virological Methods, 38:11-23, Related U.S. Application Data 1992. Romano et al., Clinics in Laboratory , (63) Continuation of application No. 09/002,177, filed on Dec. X 31, 1977, now Pat. No. 5968,732. 16:1:89-103, 1996. 2 2 2 - - -2 Morita et al., Journal of Clinical Microbiology, (51) Int. Cl...... C12O 1/70; C12O 1/68; 29:10:2107-2110, 1991. C07H 21/02; CO7H 21/04; C12N 15/00 Killen et al., Journal of Virological Methods, 41:135-146, (52) U.S. Cl...... 435/5; 435/6; 536/23.1; 1986. 536/24.3; 935/76; 935/77; 935/78 Kerschner et al., Journal of General Virology, (58) Field of Search ...... 435/6, 91.2, 536/23.1, 67:2645–2661, 1986. 536/24.3; 935/76, 77, 78 Wattre, Jan.-Feb. 1997, Annales De Biologi Clinique, vol. 55, (1), p. 25–31. (Abstract). (56) References Cited sk - cited by examiner U.S. PATENT DOCUMENTS Primary Examiner Ethan Whisenant 5,234.809 8/1993 Boom et al. . (74) Attorney, Agent, or Firm-Rothwell, Figg, Ernst & 5,407,819 * 4/1995 Yahara et al.. Manbeck, p.c. 5,409,818 4/1995 Davey et al.. s 5,518,880 * 5/1996 Leonard et al.. (57) ABSTRACT 5,554,517 9/1996 Davey et al.. 5,712,385 1/1998 McDonough et al.. An isothermal transcription based amplification assay for 5,721,118 * 2/1998 Scheffler. dengue virus RNA uses primer combinations for Sequences 5,939,254 * 8/1999 Ennis et al.. within the envelope gene or the 3' non-coding region of the 5,968,732 - 10/1999 Romano et al.. Virus and a probe. Probes may be specific for a Serotype of 5,968,734 * 10/1999 Aurias et al.. dengue virus. 5,994,076 * 11/1999 Chenchik et al. . 6,015,892 * 1/2000 Bennett et al.. 6,017,535 * 1/2000 Fu et al.. 25 Claims, 3 Drawing Sheets U.S. Patent Dec. 25, 2001 Sheet 1 of 3 US 6,333,150 B1

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: r US 6,333,150 B1 1 2 ISOTHERMAL TRANSCRIPTION BASED SUMMARY OF THE INVENTION ASSAY FOR THE DETECTION AND The present invention provides isothermal transcription GENOTYPING OF DENGUE VIRUS based amplification assays for the detection and genotyping of dengue Virus. The detection assay may use primer pairs This application is a Continuation of Ser. No. 09/002,177 and probes for the envelope gene of dengue virus type 2. In filed Dec. 31, 1997 now U.S. Pat. No. 5,968,732. another embodiment of the invention, primer pairs and FIELD OF THE INVENTION probes from the 3' non-coding region of the virus are used The present invention is directed to an isothermal tran for the detection and genotyping of dengue virus. Scription based assay for the detection and genotyping of Amplification in an isothermal transcription based ampli dengue virus. The present invention is also directed to fication System is achieved through the coordinated activi oligonucleotides for amplifying dengue RNA and for type ties of three enzyme activities (reverse transcriptase, RNase Specific probes used in the detection of the amplification H, and RNA polymerase) and two DNA oligonucleotides product. (referred to herein as primers) specific for the target 15 sequence. The method starts with an RNA template and BACKGROUND OF THE INVENTION alternately synthesizes DNA and RNA. Using an RNA Dengue Virus infection is an arthropod-borne Viral disease template, a primer, and reverse transcriptase, an RNA/DNA which has very high morbidity and mortality in humans. hybrid is generated. The RNA is degraded from the hybrid Dengue is a member of Flaviviridae, and utilizes an 11 kb by the RNase H activity. A double stranded DNA is then Single Stranded, positive RNA genome. The genome encodes generated by the reverse transcriptase using another primer, 3 and contains additional non-coding regions at the and then the double stranded DNA is used as template for 5' and 3' ends. large amounts of RNA synthesis by the RNA polymerase. There are currently four different dengue virus Subtypes One of the primerS has, in addition to the Sequences comple known, which are distributed among geographically distinct mentary to the template, additional Sequences necessary for tropical and Subtropical regions. All four Subtypes can cause 25 generating an RNA polymerase and transcription an array of maladies, ranging from an acute, Self limited initiation site which can be used by the RNA polymerase. illness (dengue fever, DF) to the more severe and potentially The single stranded RNA product can be readily detected fatal dengue hemorrhagic fever or dengue shock Syndrome through the hybridization of an appropriately labeled oligo (dengue hemorrhagic fever, DHF). As many as 50 million nucleotide DNA probe, with or without an additional probe human cases occur annually, with an estimated 10,000 infant which can be used to immobilize the amplification product. deaths due to the hemorrhagic form of dengue. Immunity to Detection of an amplification product indicates that the one Serotype does not protect against infection by the others. target molecule (RNA) is present in the sample. In fact, Sequential infection by another Serotype Substan tially increases the probability of developing DHF (Rico BRIEF DESCRIPTION OF THE DRAWINGS Hesse et al, Virology 230, 244-251 (1997)). For this reason 35 FIG. 1 is an autoradiogram of the products of an isother it may be critical to be able to determine the genotype of the mal transcription based amplification assay of RNA extracts dengue virus for management of the disease. obtained from Virus StockS Spiked into blood, Serum, and Diagnosis and management of the disease, as well as plasma background using primers in the envelope region vector Surveillance and epidemiological Studies, would be with a probe as described in Example 2. facilitated by a rapid and Sensitive assay for the Specific type 40 FIG. 2 is an autoradiogram of the products of an isother of dengue involved. Extensive croSS reactions among the mal transcription based assay of RNA extracts obtained from flaviviruses and the existence of four distinct dengue virus Virus Stocks using primers in the 3' non-coding region with Serotypes makes Serotype identification difficult. Currently, type specific probes as described in Example 3. the most reliable method for identification involves isolation FIG. 3 is an autoradiogram of the products of an isother of the virus in a sensitive host followed by serotype iden 45 tification using reference antisera or monoclonal antibodies. mal transcription based assay using primers in the 3' non This method usually takes two or more weeks, however. coding region of human plasma Samples probed with a Alternatively, a four-fold or greater increase in antibody titer dengue type 1 probe as described in Example 4. by Standard Serological tests can be used, but generally DETAILED DESCRIPTION OF THE requires paired Samples. An ELISA for the detection of 50 INVENTION dengue virus-specific IgM in patient Serum has also been used in diagnosis, however, this assay has been shown to be An isothermal transcription based assay is used for the of very limited sensitivity. detection and genotyping of dengue RNA. Any isothermal AS a consequence of the limitations of the above methods, transcription based assay may be used with the primerS and several attempts have been made to devise RT-PCR based 55 probes of the present invention. The isothermal transcription assays for the detection and genotyping of dengue virus based assay of the present invention is carried out under infection (Henchal et al, Am. J. Trop. Med. Hyg., 45(4), conditions that can be readily determined by a perSon of 1991, pp. 418-428; Lanciotti et al., Journal of Clinical ordinary skill in the art. Microbiology, 30(3), March 1992, p. 545–551; Chungue et The preferred amplification method of the present inven al, Journal of Medical Virology, 40: 142-145,1993; Vorndam 60 tion is the isothermal transcription based amplification Sys et al., Journal of Virological Methods, 48(1994) 237–244; tem referred to as NASBA. The NASBA method is disclosed and Sudiro et al, Am. J. Med. Hyg., 56(4), 1997, pp. in U.S. Pat. Nos. 5,409,818 and 5,554,527, which are herein 424-429). These assays have relied on alternative Strategies, incorporated by reference. NASBA includes the use of T7 including universal or Specific primer amplification and RNA polymerase to transcribe multiple copies of RNA from probe detection, differential nested amplification, and RFLP 65 a template including a T7 promoter. analysis of the PCR products. To date, isothermal amplifi Another technique for the amplification of is cation methods for dengue RNA have not been reported. the So-called transcription based amplification System US 6,333,150 B1 3 4 (TAS). The TAS method is described in International Patent The term “promoter Sequence' defines a region of a Application No. WO 88/10315. Transcription based ampli nucleic acid Sequence that is Specifically recognized by an fication techniques usually comprise treating target nucleic RNA polymerase that binds to a recognized Sequence and acid with two oligonucleotides one of which comprises a initiates the process of transcription by which an RNA promoter Sequence, to generate a template including a transcript is produced. In principle, any promoter Sequence functional promoter. Multiple copies of RNA are transcribed may be employed for which there is a known and available from Said template and can Serve as a basis for further polymerase that is capable of recognizing the initiation amplification. Sequence. Known and useful promoters are those that are Other transcription based amplification techniques are recognized by certain bacteriophage RNA polymerases Such described in EP 408295. EP 408295 is primarily concerned as bacteriophage T3, T7 or SP6. Their function as a primer, with a two-enzyme transcription based amplification e.g., the Starting point for an elongation reaction, however, method. Transcription based amplification methods, Such as may be blocked, as already mentioned above, or absent in the NASBA method described in EP 329822, are usually Some embodiments of transcription based amplification employed with a set of oligonucleotides, one of which is reactions. A particularly preferred promoter Sequence is the provided with a promoter Sequence that is recognized by an 15 sequence of the T7 RNA polymerase promoter. The pre enzyme with DNA dependent RNA polymerase activity such ferred downstream primer Sequences are shown below. as, for example, T7 polymerase. Several modifications of AATTCTAATA CGA CT CA CTATAG G GAC transcription based techniques are known in the art. These GAACCTTTTGTCCTGCTCTTCA (SEQ ID NO:18) modifications comprise, for example, the use of blocked AATTCTAATAC GA CT CA CTAT oligonucleotides (that may be provided with a promoter AGGGGCAGCGAGAATCTGTTGTTGTGTT (SEQ ID Sequence). These oligos are blocked So as to inhibit an NO:19) extension reaction proceeding therefrom (U.S. Pat. No. AATTCTAATA CGA CT CACTATAGGGAAA 5,554,516). One or more “promoter-primers” CACTCCTCCCAGGGATCCAAA (SEQ ID NO:20) (oligonucleotides provided with a promoter Sequence) may AATTCTAATAC GA CT CA CTAT be used in transcription based amplification techniques, 25 AGGGCAGCCCCATAGATTGCTCCGAAAA (SEQ ID optionally combined with the use of one or more oligonucle NO:21) otides that are not provided with a promoter Sequence. AATTCTAATA CGA CTCA CTATA GG G The term “oligonucleotide” as used herein refers to a GAGACAGCAGGATCTCTGGTCT (SEQ ID NO:22) molecule comprised of two or more deoxyribonucleotides or SEQ ID NOS 18-22 comprise the sequences of SEQ ID ribonucleotides. Such oligonucleotides may be used as prim NOs 1-2, 6-7, and 11 operably linked to the T7 promoter erS and probes. Sequence, shown in italics. This makes the Sequences espe cially Suitable for use as downstream primer in a transcrip Of course, based on the Sequences of the oligonucleotides tion based amplification technique Such as NASBA. of the present invention, analogues of oligonucleotides can A preferred embodiment of the present invention is a also be prepared. Such analogues may constitute alternative 35 combination of two oligonucleotides according to the structures such as “PNA' (molecules with a peptide-like invention, for use as a Set in nucleic acid amplification. backbone instead of the phosphate Sugar backbone of nor One of the oligonucleotides may serve as an "upstream mal nucleic acid) or the like. It is evident that these alter oligonucleotide', i.e., upstream primer, while the Second native Structures, representing the Sequences if the present oligonucleotide Serves as a "downstream oligonucleotide', invention are likewise part of the present invention. 40 i.e., downstream primer, in the amplification reaction. The term “primer' as used herein refers to an oligonucle Preferably, the reverse transcriptase activity is provided otide either naturally occurring (e.g., as a restriction by avian myeloblastosis virus (AMV) reverse transcriptase fragment) or produced Synthetically, which is capable of and the RNA polymerase is provided by T7 RNA poly acting as a point of initiation of Synthesis of a primer CSC. extension product which is complementary to a nucleic acid 45 One of the advantages of an isothermal transcription Strand (template or target Sequence) when placed under based amplification method, as compared to other amplifi Suitable conditions (e.g., buffer, Salt, temperature and pH) in cation methods such as PCR, is that by being essentially the presence of nucleotides and an agent for nucleic acid isothermal, it requires few manipulations by the experi polymerization, such as DNA dependent or RNA dependent menter. However, the absence of a high temperature Step polymerase. A primer must be Sufficiently long to prime the 50 does make it Somewhat more difficult to find appropriate Synthesis of extension products in the presence of an agent primers (see below). for polymerization. A typical primer contains at least 10 The amplification method of the present invention may be nucleotides in length of a sequence Substantially comple applied to extracts of Samples comprising nucleic acid, or mentary or homologous to the target Sequence, but Some whole cells or tissues for in Situ amplification. The Samples what longer primers are preferred. Usually primers contain 55 may be various body fluids, particularly blood, plasma, and about 15-26 nucleotides but longer primerS may also be Serum, from humans. The Samples may also be tissue employed, especially when the primers contain additional Samples from humans. For use in epidemiological and Sequences Such as a promoter Sequence for a particular Surveillance Studies, the Samples may also be mosquito polymerase. tissue, tissue culture media or cultured cells, or body fluids Normally a set of primers will consist of at least two 60 from non-human animals. primers, one “upstream” (P2) and one “downstream” (P1) If the method is applied to extracts of Samples comprising primer which together define the amplificate (the sequence nucleic acids, the sample may be total RNA extracts (Such that will be amplified using said primers). One of the primers as those described in Chomczynski and Sacchi, Anal. Bio is understood to contain, in addition to Sequences that will chem. 162:156, 1987) or “Boom” extracts (Boom et al., J. hybridize to the target Sequence, Sequences which provide 65 Clin. Micro.: 28, No. 3, March 1990, p. 495-503), which is promoter activity. Most often the P1 primer will include the herein incorporated by reference. The method is preferably promoter Sequence. applied to “Boom extracts”. US 6,333,150 B1 S 6 The amplificate is detected by hybridization with an method is used in concert with a particular RNA extraction appropriately labeled A oligonucleotide probe. The label technique ("Boom extraction', Boom et al., J. Clin. Micro.: may contain a radioactive moiety, a detectable enzyme, or 28, No. 3, March 1990, p. 495-503), and ECL detection any other moiety capable of generating a detectable Signal, (electrochemiluminescence). The advantages of the System Such as a calorimetric, fluorescent, chemiluminescent or are those associated with an amplification based assay electrochemiluminescent (ECL) signal. Blot based hybrid capable of providing Sequence level data. Although Some of ization analysis and liquid hybridization based ECL analysis these same advantages exist for the RT-PCR (i.e., increased are preferably used, although other analysis Systems. Such as Sensitivity over ELISA, gene Sequence specificity), there are ELGA (enzyme-linked gel assay) and in Situ hybridization advantages of NASBA for RNA over RT-PCR. These can also be used. include isothermal amplification, incorporation of reverse In one embodiment of the present invention, the ampli transcription into the amplification, application to wider fication products are resolved by agarose gel array of Specimen types (via Boom extract), and the Sensi electrophoresis, then transferred to nylon membranes and tivity and dynamic range of the ECL detection. hybridized to a probe that is 5'-end labeled with “P using The isothermal amplification is of particular importance standard methods. The products are then visualized by 15 to applications of the assay in developing regions or under autoradiography. In a Second embodiment of the present field assay conditions, two important considerations relevant invention, the amplification products can be detected using to dengue epidemiology. In light of these considerations, a the ELGA. In this method the products of the amplification colorimetric detection System applied to a dried blood Spot reaction are resolved on a polyacrylamide gel. A probe that collection would be ideal for a field assay for dengue. is Specific for the amplification reaction product and conju Boom extracts are purified preparations of DNA and gated at its 5' end with horseradish peroxidase (HRP) is then RNA. The Boom method is based on the lysing and nuclease hybridized. A colorimetric enzyme reaction allows for the inactivating properties of the guanidinium Visualization of the reaction product in the gel. A third thiocyanate (GuSCN) together with the nucleic acid binding embodiment of the present invention makes use of electro properties of Silica particles or diatoms. By using Size chemiluminescence chemistry (or ECL). This embodiment 25 fractionated Silica particles, nucleic acids, including uses a biotinylated capture probe immobilized onto the covalently closed circular, relaxed circular, linear double Surface of a Streptavidin-coated magnetic bead via the stranded DNA, single stranded DNA, tRNA, mRNA, and biotin-avidin interaction. This System also requires an oli rRNA, can be purified from a Sample in less than one hour gonucleotide detector probe, which can hybridize to an and recovered in the original reaction vessel. independent region of the amplification product. This detec A Small Sample is pipetted into a reaction vessel contain tor probe is labeled with Ruthenium, the substance that is ing a Solid nucleic acid carrier and a GuSCN containing lysis responsible for generating an ECL signal. buffer. Lysis of the cells occurs and the released nucleic The amplification methods of the invention may be used acids bind to the carrier. The carrier-nucleic acid complexes with one or more internal controls to monitor the efficiency can be separated by centrifugation. Several wash Steps of the extraction process and the amplification assay itself. 35 follow and the complexes are then dried. The nucleic acids The detection Systems are described in detail in Romano et are eluted in an aqueous low-salt buffer in the initial reaction al, DNA Technology 16:89-103 (1996), and van Gemen et vessel and used for the amplification reaction. al., J. of Virol. Methods, 49:157-168 (1994), which are In a preferred embodiment of the present invention, herein incorporated by reference. Methods for internal con amplification is achieved in a 20 till reaction containing 5 ul trols are described in van Gemen et al, Reviews in Medical 40 of the nucleic acid extract material in 10 ul of premix Tris Virology, 5:205–211 (1995), which is herein incorporated by (40 mM) pH8.5; MgCl, (12 mM); KCl (70 mM); DTT (5 reference. mM); dNTPs (each) (1 mM); raTP, rUTP, rCTP (2 mM); If the method is to be practiced on fixed preparations for rGTP (1.5 mM); ITP (0.5 mM); DMSO (15%); P1 and P2, in Situ analysis, the method is performed as follows. (0.2 uM); Sorbitol (1.5 M)). This is then added to 5 uL of Samples may include various body fluids or tissue Samples. 45 enzyme mix BSA (2.1 lug/NASBA); RNase H (0.08 unit/ Lymph tissue is a preferred tissue for in Situ analysis. The NASBA); T7 RNA Polymerase (32 units/NASBA); and cells are fixed and then permeabilized to optimize perme AMV-RT (6.4 units/NASBA)). (The enzyme mixture must ability of the cell membranes. The fixatives are those stan not be vortexed). If the nucleic acid Sample decreases (5 ul), dardly used in the art for cell or tissue preparations, Such as then the water Volume increases accordingly So that the total acetone and methanol, ethanol, formalin, formaldehyde, 50 Volume stayS 15 ul when the nucleic acid is added. paraformaldehyde, or Permafix(R), and the permeabilization The method can be carried out as follows. is done by proteinases, Such as proteinase K or pepsinogen. 1. Mix premix. The cells are then washed to remove all reagents that might 2. Add 10 ul of premix to 5 ul of nucleic acid in an inhibit the transcription based reaction. Permeabilization is Eppendorf tube. done to the point that the cells allow entry of all necessary 55 3. Incubate at 65 C. for 5 minutes. amplification reaction components, yet retain the targets and 4. Transfer to 41 C. heat block, incubate for 5 minutes. amplification products within the cells. In addition, coSol 5. Add 5 ul of enzyme mix. vents such as glycerol or DMSO may be added to optimize 6. Mix without vortexing. the NASBA reaction. 7. Incubate at 41 C. for 5 minutes. Detection of amplification products may be by direct 60 8. If the tops of the tubes have condensation from the labelling (with, for instance, biotin or digoxigenin-UTP) or cooling, they may be spun. by in situ hybridization with labelled probe. The direct 9. Incubate at 41° C. for 90 minutes. labelling method requires that conditions can be optimized 10. Spin down samples and store at -20° C. to remove unincorporated label while maintaining the ampli In the method of the present invention NASBA primers fication products. 65 were designed using the Standard approach for the envelope In a particularly preferred embodiment of the present gene of dengue virus type 2. A total of eight primers were invention, the isothermal transcription based amplification designed and Synthesized; there were two primer combina US 6,333,150 B1 7 8 tions for two different target Sequences within the envelope analysis indicated that all eight primer combinations were gene. The primerS and probes are listed on Table 1. functional in NASBA amplification (data not shown).

TABLE 1.

TARGET AREA A:

1B 5ACGAACCTTTTGTCCTGCTCTTCA 3" (249-272) S

P1A: 5 'GCAGCGAGAATCTGTTGTTGTGTT 3' (199-222) S D NO

P2A: 5'TTTGTAGAAGGGGTTTCAGGAGGA 3' (31-54) S D NO 3

P2B: 5'AACATGGAAGCTGTGTGACGACGA 3' (77-100) S D NO 4

Probe A: 5 'GAAACAGAAGCCAAACAACCTGCC 3' (139-162) S D NO

TARGET AREA B

P1C: 5'AAACACTCCTCCCAGGGATCCAAA 3' (1264-1287) S D NO 6

P1D: 5"CAGCCCCATAGATTGCTCCGAAAA 3' (1316-1339) S D NO 7

P2C: 5'AGAAGCAGAACCTCCATTCGGAGA 3' (1101-1124) S D NO 8

P2D: 5'AGGAGTAGAGCCGGGACAATTGAA 3' (1140-1163) S D NO 9

Probe: 5'ATTGAGACAACAATGAGGGGAGCG 3' (1204-1227) S D NO 10

ProbeC: 5 'GGTGACACAGCTTGGGATTTTGGA 3' (1246-1269) S 23

An additional Set of primers and probes was Selected for EXAMPLE 2 the detection and genotyping of dengue. These oligonucle otides are derived from the 3' non-coding region of the Because dengue infection causes Viremia, and Samples of dengue genome. The primers will amplify all four Serotypes blood may therefore be used as samples for the NASBA of dengue virus, the probes D1–D4 will specifically bind to assay for dengue Virus, it was desirable to know if the assay each type of dengue virus, and the capture probe can be used was reliable in the background of blood samples. Serial as a capture probe in a genotyping reaction, or alternatively dilutions of the dengue virus Stock material were Spiked into as a detection probe in a detection assay. These oligonucle 35 normal human whole blood, Serum, and plasma, extracted otides are shown on Table 2. by the Boom method, and used in a NASBA assay with the envelope gene primers P1D and P2D. The amplification TABLE 2 products were resolved on a slot blot and probed with the envelope gene probe C. Results demonstrated that it was Primers 40 possible to amplify as little as 10 uL of original dengue P1 GAGACAGCAGGATCTCTGGTCT SEQ ID NO : 11 virus stock material in a whole blood or blood component P2 GGTTAGAGGAGACCCCTCCC SEQ ID NO : 12 background (see FIG. 1). Thus, initial model system evalu PROBES ation of the NASBA-based dengue virus assay demonstrated the feasibility of the assay for application on the type of D1 (type 1) GGGAAGCTGTATCCTGGTGGTAAGG SEQ ID NO : 13 45 D2 (type 2) ATGAAGCTGTAGTCTCACTGGAAGG SEQ ID NO: 14 clinical Samples which are most likely to be used in clinical D3 (type 3) AGGGAAGCTGTACCTCCTTGCAAAG SEQ ID NO: 15 practice. D4 (type 4) GAGGAAGCTGTACTCCTGGTGGAAG SEQ ID NO: 16 CAPTURE AAACAGCATATTGACGCTGGG SEQ ID NO : 17 EXAMPLE 3 Genotyping 50 EXAMPLE 1. The oligonucleotides shown on Table 2 from the 3' NASBA-Initial Evaluation non-coding region of the dengue genome were used for the The NASBA primers of Table 1 were tested as follows. genotyping of virus Stock material. RNA was extracted by the Boom method from dengue virus FIG. 2 shows the results of a NASBA assay of RNA type 2 (New Guinea) Stock produced from an in vitro culture Samples prepared from Virus StockS using primers P1 and P2 system. The primers were used in four different combina 55 of Table 2, with probes D1, D2, D3, and D4. The upper left tions per target site in standard NASBA reactions with 5 till quadrant shows hybridization with the type 1 probe to the of 1x Boom extract. products of the NASBA reaction using the indicated virus Amplification was achieved in a 20 till reaction containing Strains. The upper right quadrant shows hybridization with 5 uL of the nucleic acid extract material in Tris (40 mM) the type 2 probe to the products of the NASBA reaction pH8.5; MgCl, (12 mM); KCl (70 mM); DTT (5 mM); 60 using the indicated virus Strains. The lower left quadrant dNTPs (each) (1 mM); raTP, rUTP, rCTP (2 mM); rGTP (1.5 shows hybridization with the type 3 probe to the products of mM); ITP (0.5 mM); DMSO (15%); P1 and P2, (02 uM); the NASBA reaction using the indicated virus strains. The Sorbitol (1.5 M); BSA (2.1 lug/NASBA); RNase H (0.08 lower right quadrant ShowS hybridization with the type 4 unit/NASBA); T7 RNA Polymerase (32 units/NASBA); and probe to the products of the NASBA reaction using the AMV-RT (6.4 units/NASBA). 65 indicated virus Strains. Thus, each of the probes reacts only The NASBA products were detected using Southern Blot with the appropriate Samples using proper hybridization analysis and P-labeled versions of the probes. The initial conditions. US 6,333,150 B1 9 EXAMPLE 4 TABLE 3-continued The type specific probe D1 was also used to test NASBA reaction products from clinical Samples. Ten Specimens SEROTYPE PLAOUE ASSAY which were previously identified as dengue type 1 by viral SAMPLE (IFA) (PFU/ml) RT/PCR NASBA isolation and IFA were also tested by the method of the 9 1. O -- present invention. 1O 1. 25 ND -- The Samples were from patients diagnosed with dengue ND = not done type 1. Cell cultures were inoculated with Six of the Samples + = positive and two of the six showed plaque formation. Five of the ++ = strongly positive samples were tested by reverse transcriptase-PCR (RT/PCR) These results show that the amplification assay of the and none of the Samples tested were positive in this assay. present invention is extremely Sensitive in the detection of These 10 samples were then tested by the method of the dengue virus RNA in clinical Samples. present invention using the Boom extraction method, the The biggest problem encountered in the development of NASBA amplification method and detection with radioac 15 NASBA assays is the selection of primers. It has often been tively labeled probe. Seven of the 10 were positive when the case that primers which are Selected from Sequence data, probed with dengue type 1 probe, including three which and meet all the known requirements for primers, do not actually function in practice. In addition, in Some cases were previously found to be negative in the RT/PCR assay. primerS have been developed using model Systems Sush as Samples 1, 5, 9, and 10 were considered positive, and in vitro transcribed RNA, virus stocks, or cells lines with Samples 2, 4, and 6 were considered strongly positive (See very high expression of the target gene, but those primers FIG.3). The amplification products were also tested with the were found to be nonfunctional when the target molecule is dengue type 2 probe and all were negative (data not shown). in a background of clinical Samples. The exact mechanism The results are shown in TABLE 3. underlying this problem is not understood, but is believed to arise due to the lower temperature of the NASBA reaction, 25 which does not entirely melt Secondary Structure of the TABLE 3 target molecule and may permit nonspecific annealing of SEROTYPE PLAOUE ASSAY primer to background nucleic acids in the Sample. It is SAMPLE (IFA) (PFU/ml) RT/PCR NASBA essential for the application of the NASBA System to clinical Samples that the primerS be not absorbed by background 1. 1. 2 x 10 -- 2 1. O ---- nucleic acids, but rather be available for Specific binding to 3 1. O the target molecule. 4 1. ND ND ---- The results shown in the present application demonstrate 5 1. ND ND -- that the primers and probes of the present invention can 6 1. ND ND ---- Specifically detect low levels of target molecule, even in the 7 1. ND ND background of clinical Samples. Thus, the primers used in 8 1. O the present invention provide unexpectedly good results for the detection and genotyping of dengue virus.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 23 <210> SEQ ID NO 1 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Dengue virus <400 SEQUENCE: 1

acgaaccttt totcctgctc titca 24

SEQ ID NO 2 LENGTH 24 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 2

gcago gaga a totgttgttg tott 24

SEQ ID NO 3 LENGTH 24 TYPE DNA ORGANISM: Dengue virus

<400 SEQUENCE: 3

tttgtagaag gg gtttcagg agga 24 US 6,333,150 B1 11 12

-continued

SEQ ID NO 4 LENGTH 24 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 4 aacatggaag citgtgttgacg acga 24

SEQ ID NO 5 LENGTH 24 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 5 gaaacagaag ccaaacaa.cc tocc 24

SEQ ID NO 6 LENGTH 24 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 6 aaac acto ct cocagg gatc caaa 24

SEQ ID NO 7 LENGTH 24 TYPE DNA ORGANISM: Dengue virus SEQUENCE: 7 cagocc cata gattgctc.cg aaaa 24

SEQ ID NO 8 LENGTH 24 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 8 agaa.gcagaa ccto cattcg gaga 24

SEQ ID NO 9 LENGTH 24 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 9 aggagtagag ccgggacaat tdaa 24

SEQ ID NO 10 LENGTH 24 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 10 attgagacaa caatgagggg agcg 24

SEQ ID NO 11 LENGTH 22 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 11 US 6,333,150 B1 13 14

-continued gag acagoag gatctotggit ct 22

SEQ ID NO 12 LENGTH 2.0 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 12 ggittagagga gaccc.citc.cc 20

SEQ ID NO 13 LENGTH 25 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 13 gggaagctgt atcCtggtgg talagg 25

SEQ ID NO 14 LENGTH 25 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 14 atgaagctgt agt citcactg. galagg 25

SEQ ID NO 15 LENGTH 25 TYPE DNA ORGANISM: Dengue virus

<400 SEQUENCE: 15 agggaagctg tacctoctitg caaag 25

SEQ ID NO 16 LENGTH 25 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 16 gaggaagctg tacticcitggt ggaag 25

SEQ ID NO 17 LENGTH 21 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 17 aaac agcata ttgacgctgg g 21

SEQ ID NO 18 LENGTH 49 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 18 aattctaata cqactcacta tagggacgaa ccttttgtcc td citcttca 49

SEQ ID NO 19 LENGTH 49 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 19 US 6,333,150 B1 15 16

-continued aattictaata cqactcacta taggggcago gagaatctgt tgttgttgtt 49

SEQ ID NO 20 LENGTH 49 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 20 aattictaata cqactcacta tagggaaa.ca citcct cocag ggatccaaa 49

SEQ ID NO 21 LENGTH 49 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 21 aattictaata cqactcacta taggg cagcc ccatagattg citc.cgaaaa 49

SEQ ID NO 22 LENGTH 47 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 22 aattictaata cqactcacta taggggagac agcaggatct citggtot 47

SEQ ID NO 23 LENGTH 24 TYPE DNA ORGANISM: Dengue virus <400 SEQUENCE: 23 ggtgacacag cittgggattt toga 24

We claim: 40 5. The method of claim 1, wherein the probe is selected 1. A method for the detection of dengue virus RNA, from the group consisting of SEQ ID NO:5 and SEQ ID comprising: NO:10. a) obtaining a sample which may contain dengue virus 6. A method for the detection or genotyping of dengue RNA; Virus RNA in a Sample comprising: b) performing an isothermal transcription based amplifi 45 a) obtaining a sample which may contain dengue virus cation on the Sample with two oligonucleotide primers, RNA; a first primer which comprises at least 10 consecutive b) performing an isothermal transcription based amplifi nucleotides of a Sequence Selected from the group cation on the Sample with two primers, a first primer consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID which comprises at least 10 consecutive nucleotides of NO:6 and SEQ ID NO:7, and a second primer which 50 a Sequence according to SEQ ID NO:11, and a Second comprises at least 10 consecutive nucleotides of a primer which comprises at least 10 consecutive nucle Sequence Selected from the group consisting of SEQ ID otides of a sequence according to SEQ ID NO:12; and NO:3, SEQ ID NO:4, SEQID NO:8 and SEOID NO:9; c) detecting or genotyping the amplification product using and one or more probes. c) detecting the amplification product using a labeled 55 7. The method of claim 6, wherein the first primer further probe, whereby hybridization of the probe to the ampli comprises an RNA polymerase promoter Sequence fication product indicates the presence of dengue virus covalently bonded to the 5' end thereof. RNA in the sample. 8. The method of claim 7, wherein the RNA polymerase 2. The method of claim 1, wherein the first primer further promoter Sequence is a T7 RNA polymerase promoter comprises an RNA polymerase promotor Sequence 60 Sequence. covalently bonded to the 5' end thereof. 9. The method of claim 6, wherein the sample comprises 3. The method of claim 2, wherein the RNA polymerase cells or virus and RNA is extracted from the cells or virus in promoter Sequence is a T7 RNA polymerase promoter the sample prior to step (b). Sequence. 10. The method of claim 6, wherein the probe is selected 4. The method of claim 1, wherein the Sample comprises 65 from the group consisting or SEQ ID NO:13, SEQ ID cells or virus and RNA is extracted from the cells or virus in NO:14, SEQID NO:15, SEQ ID NO:16 and SEOID NO:17, the sample prior to step (b). whereby hybridization of the amplification product with US 6,333,150 B1 17 18 SEQ ID NO:13 indicates the virus RNA is type 1, hybrid group consisting of SEQ ID NO:1, SEQ ID NO:4 and SEQ ization of the amplification product with SEQ ID NO:14 ID NO:6, and SEQ ID NO:9. indicates the virus RNA is type 2, hybridization of the 17. The oligonucleotide of claim 15, comprising at least amplification product with SEQ ID NO:15 indicates the 10 consecutive nucleotides of a Sequence Selected from SEQ virus RNA is type 3, hybridization of the amplification ID NO:1 and SEQ ID NO:6, further comprising an RNA product with SEQID NO:16 indicates the virus RNA is type polymerase promoter Sequence covalently bonded to the 5' 4, and wherein hybridization of the amplification product end thereof. 18. The oligonucleotide of claim 17, wherein the RNA with SEQ ID NO:17 indicates the presence of dengue virus polymerase promoter Sequence is the T7 RNA polymerase RNA. promoter Sequence. 11. A kit for the detection or genotyping of dengue virus 19. An oligonucleotide of about 15-26 nucleotides in RNA in a Sample, comprising two oligonucleotide primers, length, comprising at least 10 consecutive nucleotides of a a first primer being about 15-26 nucleotides in length and Sequence Selected from the group consisting of SEQ ID comprising at least 10 consecutive nucleotides of a Sequence NO:11, SEQ ID NO:12, and SEQ ID NO:14. according to SEQ ID NO:11, and a second primer being 20. The oligonucleotide of claim 19, comprising at least about 15-26 nucleotides in length and comprising at least 10 15 10 consecutive nucleotides of a Sequence according to SEQ consectutive nucleotides of a Sequence according to SEQID ID NO:11 or SEO ID NO:12. NO:12, and at least one probe. 21. The oligonucleotide of claim 19, comprising at least 12. A kit according to claim 11, wherein the at least one 10 consecutive nucleotides of a Sequence according to SEQ probe is selected from the group consisting of SEQ ID ID NO:11, further comprising an RNA polymerase promoter NO:13, SEQ ID NO:14, SEQID NO:15, SEQ ID NO:16 and sequences covalently bonded to the 5' end thereof. SEO ID NO:17. 22. The oligonucleotide of claim 21, wherein the RNA 13. A kit for the detection of dengue virus type 2 RNA in polymerase promoter Sequence is the T7 RNA polymerase a Sample, Said kit comprising two oligonucleotide primers, promoter Sequence. a first primer being about 15-26 nucleotides in length and 23. The oligonucleotide of claim 22, wherein the oligo comprising at least 10 consecutive nucleotides of a Sequence 25 nucleotide is a sequence according to SEQ ID NO:22. selected from the group consisting of SEQID NO:1, SEQID 24. An oligonucleotide of 15-26 nucleotides in length NO:2, SEQ ID NO:6 and SEQ ID NO:7, and a second which is a Subsequence of Dengue virus and which com primer being about 15-26 nucleotides in length and com prises at least 15 consecutive nucleotides of a Sequence prising at least 10 consecutive nucleotides of a Sequence selected from the group consisting of SEQID NO:2, SEQID selected from the group consisting of SEQID NO:3, SEQID NO:3, SEQ ID NO:7, and SEQ ID NO:8, wherein said NO:4, SEQ ID NO:8, and SEQ ID NO:9; and a probe. consecutive nucleotides are identical throughout their length 14. A kit according to claim 13, wherein the probe is to a Dengue virus genome. selected from the group consisting of SEQID NO:5, SEQID 25. An oligonucleotide of 15-26 nucleotides in length NO:10, and SEQ ID NO:23. which is a Subsequence of Dengue virus and which com 15. An oligonucleotide of about 15-26 nucleotides in 35 prises at least 15 consecutive nucleotides of a Sequence length comprising at least 10 consecutive nucleotides of a selected from the group consisting of SEQ ID NO:13, SEQ Sequence Selected from the group consisting of SEQ ID ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, wherein NO:1, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQID Said consecutive nucleotides are identical throughout their NO:9, SEQ ID NO:10, and SEQ ID NO:23. length to a Dengue virus genome. 16. The oligonucleotide of claim 15, comprising at least 40 10 consecutive nucleotides of a Sequence Selected from the k k k k k