Improved Protocols of ITS1-Based Metabarcoding and Their Application in the Analysis of Plant-Containing Products

Plants are widely used for food and beverage preparation, most often in the form of complex mixtures of dried and ground parts, such as teas, spices or herbal medicines. Quality control of such products is important due to the potential health risks from the presence of unlabelled components or absence of claimed ones. A promising approach to analyse such products is DNA metabarcoding due to its high resolution and sensitivity. However, this method’s application in food analysis requires several methodology optimizations in DNA extraction, amplification and library preparation. In this study, we present such optimizations. The most important methodological outcomes are the following: (1) the DNA extraction method greatly influences amplification success; (2) the main problem for the application of metabarcoding is DNA purity, not integrity or quantity; and (3) the “non-amplifiable” samples can be amplified with polymerases resistant to inhibitors. Using this optimized workflow, we analysed a broad set of plant products (teas, spices and herbal remedies) using two NGS platforms. The analysis revealed the problem of both the presence of extraneous components and the absence of labelled ones. Notably, for teas, no correlation was found between the price and either the absence of labelled components or presence of unlabelled ones; for spices, a negative correlation was found between the price and presence of unlabelled components.

[1]  V. S. Melo,et al.  Tracing tree nut allergens in chocolate: A comparison of DNA extraction protocols. , 2015, Food chemistry.

[2]  Damon P. Little,et al.  Commercial Teas Highlight Plant DNA Barcode Identification Successes and Obstacles , 2011, Scientific reports.

[3]  P. D. Gudkova,et al.  Optimization of Polymerase Chain Reaction for Inter Simple Sequence Repeat Technique for Four Species of Plants , 2016 .

[4]  Donna S. Smith,et al.  Comparison of several methods for the extraction of DNA from potatoes and potato-derived products. , 2005, Journal of agricultural and food chemistry.

[5]  A. Seregin.  Making the Russian flora visible: fast digitisation of the Moscow University Herbarium (MW) in 2015 , 2016 .

[6]  Martijn Staats,et al.  DNA Damage in Plant Herbarium Tissue , 2011, PloS one.

[7]  P. Finglas,et al.  Adverse effects of plant food supplements and botanical preparations: a systematic review with critical evaluation of causality. , 2015, British journal of clinical pharmacology.

[8]  Steven G. Newmaster,et al.  DNA Barcoding and Pharmacovigilance of Herbal Medicines , 2015, Drug Safety.

[9]  Ting Gao,et al.  Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species , 2010, PloS one.

[10]  Amit Kumar,et al.  DNA barcoding: an efficient tool to overcome authentication challenges in the herbal market. , 2016, Plant biotechnology journal.

[11]  Paul Turner,et al.  Reagent and laboratory contamination can critically impact sequence-based microbiome analyses , 2014, BMC Biology.

[12]  K. Everstine,et al.  Economically motivated adulteration (EMA) of food: common characteristics of EMA incidents. , 2013, Journal of food protection.

[13]  Shiliang Zhou,et al.  Barcoding the kingdom Plantae: new PCR primers for ITS regions of plants with improved universality and specificity , 2016, Molecular ecology resources.

[14]  M. J. Cupp.  Toxicology and Clinical Pharmacology of Herbal Products , 2000 .

[15]  J. Mano,et al.  Comparison of DNA extraction methods for sweet corn and processed sweet corns. , 2013, Shokuhin eiseigaku zasshi. Journal of the Food Hygienic Society of Japan.

[16]  S. Kuusienė,et al.  Comparison of ten DNA extraction protocols from wood of European aspen (Populus tremula L.). , 2010 .

[17]  R. DeSalle,et al.  Testing the Effectiveness of an International Conservation Agreement: Marketplace Forensics and CITES Caviar Trade Regulation , 2012, PloS one.

[18]  A. P. Sukhorukov,et al.  A rapid and cost-effective method for DNA extraction from archival herbarium specimens , 2015, Biochemistry (Moscow).

[19]  P. Hebert,et al.  Rapid identification of the botanical and entomological sources of honey using DNA metabarcoding. , 2017, Food chemistry.

[20]  S. Kareh.  Toxicology and Clinical Pharmacology of Herbal Products , 2000, Forensic Science and Medicine.

[21]  Steven G Newmaster,et al.  DNA barcoding detects contamination and substitution in North American herbal products , 2013, BMC Medicine.

[22]  J. Bengtsson-Palme,et al.  ITS1: a DNA barcode better than ITS2 in eukaryotes? , 2015, Molecular ecology resources.

[23]  W. Symondson,et al.  New universal ITS2 primers for high-resolution herbivory analyses using DNA metabarcoding in both tropical and temperate zones , 2018, Scientific Reports.

[24]  Spatial distribution of intron 2 of nad1 gene haplotypes in populations of Norway and Siberian spruce (Picea abies–P. obovata) species complex , 2015, Russian Journal of Genetics.

[25]  R. Shepherd,et al.  An overview of consumer attitudes and beliefs about plant food supplements. , 2011, Food & function.

[26]  T. Wadden,et al.  Use of Dietary Supplements for Weight Loss in the United States: Results of a National Survey , 2008, Obesity.

[27]  P. Hufnagl,et al.  Development of a DNA metabarcoding method for the identification of fifteen mammalian and six poultry species in food. , 2019, Food chemistry.

[28]  Nicolas Gryson,et al.  Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review , 2010, Analytical and bioanalytical chemistry.

[29]  Barbara Gravendeel,et al.  DNA metabarcoding of orchid-derived products reveals widespread illegal orchid trade , 2017, Proceedings of the Royal Society B: Biological Sciences.

[30]  Sophie Lorraine Vassou,et al.  DNA barcoding for species identification from dried and powdered plant parts: a case study with authentication of the raw drug market samples of Sida cordifolia. , 2015, Gene.

[31]  M. Meira,et al.  Review of the genus Ipomoea: traditional uses, chemistry and biological activities , 2012 .

[32]  De‐Zhu Li,et al.  Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants , 2011, Proceedings of the National Academy of Sciences.

[33]  Ning Ma,et al.  BLAST+: architecture and applications , 2009, BMC Bioinformatics.

[34]  A. Schwarzbach,et al.  Comparing DNA Extraction Methods for Analysis of Botanical Materials Found in Anti-Diabetic Supplements , 2013, Molecular Biotechnology.

[35]  C. Sánchez-Hernández,et al.  Two mini-preparation protocols to DNA extraction from plants with high polysaccharide and secondary metabolites , 2006 .

[36]  C. Poschenrieder,et al.  High Quality DNA Obtained from a Single Seed of Vitis vinifera L. Using Rapid DNA Extraction Method , 2014 .

[37]  Carmela Martino,et al.  A comparison of DNA extraction methods for food analysis , 2007 .

[38]  M. Cipollaro,et al.  Comparative Evaluation of Different DNA Extraction Procedures from Food Samples , 2007, Biotechnology progress.

[39]  Bernard R. Baum,et al.  Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components , 1997, Plant Molecular Biology Reporter.

[40]  C. Quave,et al.  Plants used for making recreational tea in Europe: a review based on specific research sites , 2013, Journal of Ethnobiology and Ethnomedicine.

[41]  Kang Ning,et al.  Biological ingredient analysis of traditional Chinese medicine preparation based on high-throughput sequencing: the story for Liuwei Dihuang Wan , 2014, Scientific Reports.

[42]  Samantha H. Cheng,et al.  Using DNA barcoding to track seafood mislabeling in Los Angeles restaurants , 2017, Conservation biology : the journal of the Society for Conservation Biology.

[43]  A. P. Sukhorukov,et al.  Diagnostics, taxonomy, nomenclature and distribution of perennial Sesuvium (Aizoaceae) in Africa , 2018, PhytoKeys.

[44]  E. Pahlich,et al.  A rapid DNA isolation procedure for small quantities of fresh leaf tissue , 1980 .

[45]  A. Shmakov,et al.  Taxonomic rank of Tanacetum boreale Fisch. ex DC. (Asteraceae) , 2017 .

[46]  Rosalee S. Hellberg,et al.  Identification of Species in Ground Meat Products Sold on the U.S. Commercial Market Using DNA-Based Methods , 2016 .

[47]  R. DeSalle,et al.  PCR identification of black caviar , 1996, Nature.

[48]  Anne Krag Brysting,et al.  Comparative authentication of Hypericum perforatum herbal products using DNA metabarcoding, TLC and HPLC-MS , 2017, Scientific Reports.

[49]  German A. Shipulin,et al.  Comparative analysis of Illumina and Ion Torrent high-throughput sequencing platforms for identification of plant components in herbal teas , 2018, Food Control.

[50]  Nelson Marmiroli,et al.  Comparison of DNA extraction methods and development of duplex PCR and real-time PCR to detect tomato, carrot, and celery in food. , 2011, Journal of agricultural and food chemistry.