DNA-Based Identification of Spices: DNA Isolation, Whole Genome Amplification, and Polymerase Chain Reaction
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J. Agric. Food Chem. 2011, 59, 513–520 513 DOI:10.1021/jf103702s DNA-Based Identification of Spices: DNA Isolation, Whole Genome Amplification, and Polymerase Chain Reaction FELIX FOCKE,ILKA HAASE,* AND MARKUS FISCHER Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany Usually spices are identified morphologically using simple methods like magnifying glasses or microscopic instruments. On the other hand, molecular biological methods like the polymerase chain reaction (PCR) enable an accurate and specific detection also in complex matrices. Generally, the origins of spices are plants with diverse genetic backgrounds and relationships. The processing methods used for the production of spices are complex and individual. Consequently, the development of a reliable DNA-based method for spice analysis is a challenging intention. However, once estab- lished, this method will be easily adapted to less difficult food matrices. In the current study, several alternative methods for the isolation of DNA from spices have been developed and evaluated in detail with regard to (i) its purity (photometric), (ii) yield (fluorimetric methods), and (iii) its amplifiability (PCR). Whole genome amplification methods were used to preamplify isolates to improve the ratio between amplifiable DNA and inhibiting substances. Specific primer sets were designed, and the PCR conditions were optimized to detect 18 spices selectively. Assays of self-made spice mixtures were performed to proof the applicability of the developed methods. KEYWORDS: Spice; herb; DNA isolation; PCR; whole genome amplification; multiple displacement amplification INTRODUCTION came in the field of interest, was the different coumarin content in A spice is defined as a product of a single plant organism, the two cinnamon species Cinnamomum verum and Cinnamomum whereas spice mixtures are defined as products made of at least cassia. High concentrations of coumarin could only be detected two organisms. For spice production, all different parts of a plant in C. cassia (9). like the fruit (e.g., pepper), seed (e.g., mustard), bud or blossom Another question is the detection of toxic plant-derived con- (e.g., caper or clove), rhizome (e.g., ginger), root (e.g., horseradish), taminants in herbs. For example, undomesticated ramsons bark (e.g., cinnamon), spear (e.g., angelica), or the leaf (e.g., (Allium ursinum) can be contaminated or confused with lily of oregano) are used. the valley (Convallaria majalis)ormeadowsaffron(Colchicum Usually spices are identified and valued on the basis of their autumnale)(10, 11). Also possible is a contamination of roquette morphological differences, for example, characteristic cells or (Diplotaxis tenuifolia) with toxic ragwort species (Senecio spp.), tissues. For that purpose, trained staff is mandatory, and the quan- which could be detected via PCR. tity of a certain spice can only be roughly estimated. Especially in The most important prerequisite for a reliable PCR method is fine cut products or in processed food, characteristic attributes the isolation of amplifiable DNA from the sample. As described are hardly detected, making identification difficult or impossible. above, spices are made of very heterogenic parts of plants. Generally, the polymerase chain reaction (PCR) is a DNA- Furthermore, in most cases, spices are dried and/or fermented. based method enabling the control of food authenticity or the Consequently, the DNA of spices is embedded in different detection of genetically modified plants, allergens, or pathogens matrices and might be degraded during processing. Conse- in food (1-4). Currently used PCR-based methods for the au- quently, the first step in the analysis of spices is the development thentication of meat products, seafood products, dairy products, of effective DNA isolation methods, which crack the plant cell and foods of plant origin are summarized by Mafra et al. (4). For wall and carefully separate proteins, lipids, and carbohydrates in the quality control of spices, only a few DNA-based methods any of the spices under study. Additionally, phytochemicals have been published yet, for example, for celery and mustard, should also be removed during the isolation procedure, due to - which are known to contain proteins with allergenic poten- their potential to act as inhibitors for the PCR (12 14). tial (5, 6) and for the detection of adulterations of spices (7, 8). Furthermore, spices are very different in terms of their geno- Apart from that, the identification of spices might be important typic and phenotypic relationships. Some are members of the due to the fact that some of them contain harmful substances. same genus, and some are even different on the family level One recent example, where an identification of a certain spice (Table 1). The former is challenging regarding the identification of specific primer hybridization regions. As a prerequisite, the *To whom correspondence should be addressed. Tel: þ49- respective sequence must be known from every spice under study 40-428384379. Fax: þ49-40-428384342. E-mail: ilka.haase@ to avoid false positive reactions. In the rDNA region, which is chemie.uni-hamburg.de. part of each eukaryotic genome, internal transcribed spacers © 2010 American Chemical Society Published on Web 12/23/2010 pubs.acs.org/JAFC 514 J. Agric. Food Chem., Vol. 59, No. 2, 2011 Focke et al. Table 1. Spices Used for Isolation of DNA MATERIALS AND METHODS spice family Spice Samples. Commercially available samples of 39 spices from 13 families were used in this study. An overview is given in Table 1. A contami- chive (Allium schoenoprasum L.) nation with other spices, plants, or microorganisms is possible but unlikely. garlic (Allium sativum L.) Alliaceae DNA Isolation Method 1: Precipitation (Modified CTAB Proto- leek (Allium porrum L.) col, § 64 LFGB L 00.00-31). Asampleamountof50-100 mg was mixed onion (Allium cepa L.) with 1 mL of extraction buffer [55 mM cetyltrimethylammonium bromide ramson (A. ursinum L.) (CTAB), 1.4 M NaCl, 0.1 M tris(hydroxymethyl)-aminomethan (Tris/ HCl), and 20 mM ethylenediaminetetraacetic acid disodium salt (Na - anise L. 2 (Pimpinella anisum ) EDTA), pH 8.0] in a 2 mL cap. Two steel balls (1/800) were added, and the caraway L. (C. carvi ) sample was mechanically ground for 5 min at 30 Hz using the TissueLyser celery L. (Apium graveolens ) system (Qiagen, Hilden, Germany) followed by an incubation at 65 °Cfor chervil L. Hoffm. [Anthriscus cerefolium ( ) ] 30 min (water bath). A 500 μL amount of chloroform was added, and the coriander L. Apiaceae (Coriandrum sativum ) suspension was mixed. After centrifugation (5 min, 10000g), 700 μLofthe cumin L. (Cuminum cyminum ) aqueous supernatant was carefully removed. dill L. (Anethum graveolens ) For DNA precipitation, the solution was mixed with 1 mL of lovage W. Koch (Levisticum officinale ) precipitation buffer (14 mM CTAB and 40 mM NaCl) and incubated parsley Mill. Nym. [Petroselinum crispum ( ) ] for 1 h at room temperature. To pelletize the precipitated DNA, a centri- fugation step (5 min, 10000g) was performed, and the supernatant was mugwort (Artemisia vulgaris L.) Asteraceae carefully removed. The DNA pellet was resolved in 350 μL of NaCl tarragon (A. dracunculus L.) solution (1.2 M) and mixed with 350 μL of chloroform. After centrifuga- μ black mustard [Brassica nigra (L.) Koch] tion (2 min, 10000g), the aqueous supernatant was mixed with 200 Lof Brassicaceae ° white mustard (Sinapis alba L.) isopropanol. After incubation (30 min, 4 C) and centrifugation (5 min, 10000g), the supernatant was removed, and the DNA pellet was washed saffron (Crocus sativus L.) Iridaceae with ethanol (70% v/v). Remaining ethanol was removed by incubation of the sample at 300 mbar for 5 min. The DNA pellet was finally resolved in basil (O. basilicum L.) 50 μL of water. marjoram (Origanum majorana L.) DNA Isolation Method 2: Adsorption on Silica. The first steps for oregano (Origanum vulgare L.) Lamiaceae the isolation of DNA by adsorption on silica were performed as described rosemary (Rosmarinus officinalis L.) above. However, the DNA was not precipitated in the aqueous solution sage (S. officinalis L.) but was adsorbed on a silica containing EconoSpin column (Epoch thyme (Thymus vulgaris L.) Biolabs, Sugar Land, United States). After 2 min of centrifugation (10000g), the bound DNA was washed with 500 μL of washing buffer [50 cassia (Cinnamomum aromaticum Nees) mM NaCl, 20 mM Tris/HCl, and 1 mM Na -EDTA, in ethanol (50% v/v), Lauraceae 2 cinnamon (C. verum J. S. Presl) pH 7.4] and subsequently with 500 μL of ethanol (70% v/v). Ethanol laurel (Laurus nobilis L.) should be removed completely by an additional centrifugation step (1 min, 10000g).The DNA was eluted with 50 μL of water by centrifugation nutmeg Hout (Myristica fragrans ) Myristicaceae (2 min, 10000g). Spectroscopic Characterization of DNA Isolates. The DNA purity allspice [P. dioica (L.) Merr.] Myrtaceae μ clove L. Merr. and Perry was determined photometrically. The absorbance of 20 LofDNA [S. aromaticum ( ) ] solution was measured at 260 and 280 nm in 384 well plates in a plate reader (SpectraMax M2, Molecular Devices, Ismaning, Germany). The pepper (Piper nigrum L.) Piperaceae concentration of the DNA solution was calculated fluorometrically star anise (I. verum J. D. Hook f.) Schisandraceae (SpectraMax M2) using SYBR Green I and an external calibration curve (dilution series of plasmid standard). cayenne (Capsicum frutescens L.) MDA (18, 19).