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

Abstract

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.

Applied Barcoding: The Practicalities of DNA Testing for Herbals

“…Next-Generation Sequencing (NGS) technologies have been applied to herbal drug authentication using genome skimming or amplicon metabaroding approaches [ 1 , 8 , 87 , 88 , 89 , 90 , 91 , 92 ], including H. perforatum commercial products [ 16 ]. The quality of a herbal product is determined by the correct identity of its ingredients and its purity, measured by % contamination by a range of inorganic and biological contaminants.…”

Assessment of ITS1, ITS2, 5′-ETS, and trnL-F DNA Barcodes for Metabarcoding of Poaceae Pollen

“…Primers for nuclear DNA barcodes ITS1 and ITS2 were designed to anneal to conservative regions of plant rDNA selectively and not fungi (ITS1-F 5 -GGAAGGAGAAGTCGTAACAAGG-3 , ITS1-R 5 -AGATATCCGTTGCCGAGAGT-3 [35]; ITS2-F 5 -ATCGAGTYTTTGAACGCAAGTTG-3 , ITS2-R 5 -TCCTCCATGCTCTATTG-3 not published). Primers for the chloroplast inter-genic spacer trnL-F barcode were obtained from [36] (trnL_F 5 -GGTTCAAGTCCCTCTATCCC-3 ; trnF_R 5 -ATTTGAACTGGTGACACGAG-3 ).…”

Herbarium collections in molecular genetic studies

“…Колонки с сорбентом из диоксида кремния доступны в коммерческих наборах для выделения ДНК. Следует, однако, иметь в виду, что эффективность выделения из гербарных образцов ДНК-препаратов приемлемого качества при использовании наборов разных фирм различна, в некоторых случаях по выходу ДНК и по ее спектрофотометрическим характеристикам коммерческие наборы оказываются не способны превзойти результаты экстракции по стандартному ЦТАБ протоколу (Sarkinen et al, 2012;Gutaker et al, 2017;Omelchenko et al, 2019).…”