Adulteration in meat products is a widespread issue that could lead to serious threats to public health and religious violations. Technology that offers rapid, sensitive, accurate and reliable detection of meat species is the key to an effectual monitoring and control against meat adulteration. In recent years, high-throughput sequencing-based DNA metabarcoding technology has developed rapidly. With the characteristics of being high-throughput, highly precise and high-speed, this technology can simultaneously identify multiple species in complex samples, thus offering pronounced advantages in the surveillance of adulteration in meat and meat products. Starting with an introduction of the major developments in the high-throughput sequencing technology in the past two decades, this review provides an overview of the technical characteristics and research methods of DNA metabarcoding, summarizes the application of DNA metabarcoding technology in meat adulteration detection over the last few years, discusses the challenges of using DNA metabarcoding technology in the detection of meat adulteration, and provides future prospects on the development of this technology.
DNA ; Food Contamination/analysis* ; High-Throughput Nucleotide Sequencing/methods* ; Meat/analysis* ; Meat Products ; Technology

BACKGROUND: Extraction of cell-free DNA (cfDNA) is a key step for determining the quality of cfDNA-related molecular diagnostics. We evaluated the effect of sample containers and sample storage conditions on cfDNA extraction. METHODS: The cfDNA extraction using the MagMAX Cell-Free DNA Isolation Kit from five healthy controls and five lung cancer patients was evaluated according to the type of sample container and storage conditions: K2-EDTA container, <1, 6, 24, and 48 hr storage at 4℃ after immediate plasma separation; and Cell-Free DNA BCT container, <1, 3, 7, and 14 days stored at room temperature. Mutation analysis of EGFR exons 18–21 was performed. To assess the effect of a delay in centrifugation, EDTA whole blood samples from five healthy individuals were stored at 4℃ for 6, 12, and 24 hr before plasma separation. RESULTS: There was no significant difference in the amount and nucleic acid size of cfDNA in both controls and patients with cancer when EDTA plasma was stored at 4℃ up to 48 hr. The amount and size of cfDNA in the BCT container were not different up to 7 days; however, the 14-day sample showed an increase in cfDNA concentration due to genomic DNA contamination. EGFR mutations were detected on EDTA containers up to 48 hr and with BCT containers up to 14 days. When EDTA whole blood was stored at 4℃ and plasma separation was delayed, the cfDNA concentration increased from 24 hr. CONCLUSIONS: The cfDNA extraction was affected by the sample containers and storage conditions.
Biopsy ; Centrifugation ; DNA Contamination ; DNA ; Edetic Acid ; Exons ; Humans ; Lung Neoplasms ; Pathology, Molecular ; Plasma

This research preliminarily discusses the relations of Dendrobium system growth through chloroplast gene rbcL, matK and the nuclear genome ITS2. The DNA barcoding universal sequence for authentication of the Dendrobium medical plants was slected and the possibility concerning utilizing the DNA barcoding to distinguish the D. huoshanenseand its adulterants was analyzed. Using the universal primer pair of ITS2, rbcL and matK, series of extended sequencing in the Dendrobium were conducted. Meanwhile, considering the different index about amplification and sequencing success rate of each sequence, the intraspecific and interspecific aberrance, the employment of BioEdit and MEGA 5.0 software were applied to establish the systematic tree of the NJ molecular and evaluate the diversified authentication capability of various sequences. The consequence demonstrates that the sequence of ITS2 is not only the largest one both in the intraspecific and interspecific aberrance of the Dendrobium but also has obvious barcoding gap. Considering the few overlap between the intraspecific and interspecific aberrance and the highest percentage regarding the formation of unilateral branch in diverse Dendrobium which have different ITS2 sequences, it can differentiate the species of Dendrobium. Furthermore, due to the inferior success rate of the rbcL and thematK and the lower reliability of NJ systematic tree, the percentage of the unilateral species which are generated by the systematic tree of rbcL and matK sequences is deficient. Therefore, the sequence of ITS2 can serves as DNA barcoding to distinguish the D. huoshanense, the D. moniliform and the D. officinale.
DNA Barcoding, Taxonomic ; DNA, Plant ; genetics ; Dendrobium ; classification ; Drug Contamination ; Plant Preparations ; standards ; Plants, Medicinal ; classification ; Reproducibility of Results

Fructus Arctii is a traditional Chinese medicine. The main counterfeit species are the seeds of Arctium tomentosum, Onopordum acanthium, Silybum marianum, Saussurea costus, Amorpha fruticosa. Traditional identification methods or molecular barcoding techniques can identify Fructus Arctii and its counterfeit species. However, the identification of the mixture of it and its spurious species is rarely reported. In this paper, we sequenced the ITS2 sequences of Fructus Arctii and 5 kinds of spurious species mix powder by high-throughput sequencing to identify the mixed powder species and providing new ideas for the identification of Fructus Arctii mix powder. The total DNA in mixed powder was extracted, and the ITS2 sequences in total DNA was amplified. Paired-end sequencing was performed on the DNA fragment of the community using the Illumina MiSeq platform. The sequence was analyzed by the software FLASH, QIIME and GraPhlAn etc. The results showed that the high quality ITS2 sequences of 39910 mix samples were obtained from the mixed samples, of which the total ITS2 sequence of the samples genus was 34 935. Phylogenetic analysis showed that the samples contained Fructus Arctii, A. tomentosum, O. acanthium, S. marianum, S. costus and A. fruticosa. Using ITS2 sequences as DNA barcodes, high-throughput sequencing technology can be used to detect the Fructus Arctii and its spurious specie in mixed powder, which can provide reference for the quality control, safe use of medicinal materials of Fructus Arctii and the identification of mixed powder of traditional Chinese medicine.
Arctium ; chemistry ; classification ; DNA Barcoding, Taxonomic ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drug Contamination ; Drugs, Chinese Herbal ; standards ; Fabaceae ; Fruit ; High-Throughput Nucleotide Sequencing ; Milk Thistle ; Onopordum ; Phylogeny ; Saussurea

The adulteration of herbal products is a threat to consumer safety. In the present study, we surveyed the species composition of commercial Radix Clerodendri Japonicum products using DNA barcoding as a supervisory method. A reference database for plant-material DNA-barcode was successfully constructed with 48 voucher samples from 12 Clerodendrum species. The database was used to identify 27 Radix Clerodendri Japonicum decoction piece samples purchased from drug stores and hospitals. The DNA sequencing results revealed that only 1 decoction piece (3.70%) was authentic C. japonicum, as recorded in the Dai Pharmacopeia, whereas the other samples were all adulterants, indicating a potential safety issue. The results indicate that decoction pieces that are available in the market have complex origins and that DNA barcoding is a suitable tool for regulation of Dai medicines.
Clerodendrum ; classification ; genetics ; DNA Barcoding, Taxonomic ; Drug Contamination ; Medicine, Chinese Traditional ; adverse effects ; Polymerase Chain Reaction

Hyoscyami Semen, the mature dried seed of Hyoscyamus niger L., has long been used as a traditional Chinese medicine to treat human diseases. Hyoscyami Semen is found in local markets in China. In markets, sellers and buyers commonly inadvertently mix the seeds of H. niger with the seeds of related species such as Hygrophila salicifolia (Vahl) Nees, Astragalus complanatus R. Br., Cuscuta australis R. Br., Cuscuta chinensis Lam., and Impatiens balsamina L. because of their similar morphologies or similar names. Thus, developing a reliable method for discriminating H. niger seeds from its adulterants is necessary to reduce confusion and ensure the safe use of Hyoscyami Semen. The present study was designed to evaluate the efficiency of high-resolution melting analysis combined with DNA barcoding (Bar-HRM) with internal transcribed spacer 2 to discriminate H. niger. Our results show that Bar-HRM successfully identified the adulterants and detected the proportion of H. niger DNA extract within an admixture. In particular, HRM detected H. niger DNA extract in A. complanatus DNA extract at concentrations as low as 1%. In conclusion, the Bar-HRM method developed in the present study for authenticating H. niger is rapid and cost-effective. It can be used in the future to guarantee the purity of Hyoscyami Semen for the clinical use.
China ; DNA Barcoding, Taxonomic ; methods ; DNA, Intergenic ; chemistry ; genetics ; DNA, Plant ; chemistry ; genetics ; Discriminant Analysis ; Drug Contamination ; Drugs, Chinese Herbal ; chemistry ; Hyoscyamus ; genetics ; growth & development ; Seeds ; genetics ; growth & development ; Transition Temperature

To establish a molecular identification method for Bletillae Rhizoma, this paper extracted genome DNA from Bletillae Rhizoma and its adulterants. The sequences of rDNA ITS2 were sequenced after amplifying. Then multiple alignments of ITS2 were constructed phylogenetic tree with Neighbor Joining by MEGA 5. 1 and found out SNPs loci. The result showed that rDNA ITS2 region could identify Bletillae Rhizoma and its adulterants. There existed the SNPs loci, which could identify Bletilla striata and B. ochracea. Furthermore, we designed specific primers against the SNPs loci of B. striata and B. ochracea, then screened primers and optimized the PCR amplification conditions. Finally, the DNA of B. striata and B. ochracea were specifically amplified by BJ59-412F, BJ59-412R and HHBJ-225R. The length of amplification products were respectively about 350 bp and 520 bp that were effectively identified of B. striata and B. ochracea. While, the adulterants of Bletillae Rhizoma were no-reaction occurring. To sum up, the amplification conditions of the primers can identify B. striata, B. ochracea and their adulterants successfully at the same time. This method was easy, time-saving, and reliable, which can be used as a rapid method for molecular identification of Bletillae Rhizoma.
Base Sequence ; DNA Primers ; genetics ; DNA, Intergenic ; genetics ; DNA, Plant ; genetics ; Drug Contamination ; prevention & control ; Molecular Sequence Data ; Orchidaceae ; classification ; genetics ; Phylogeny ; Polymorphism, Single Nucleotide ; Rhizome ; classification ; genetics

OBJECTIVETo establish a method for detecting 3 common toxigenic molds (Aspergillus, Penicillium, and Fusarium) based on non-modified magnetic beads coupled with multiple real-time PCR (NMB-multiple qPCR).

METHODSThe primers and genus-specific probes were designed based on the rDNA sequences to develop a multiple real-time PCR using non-modified magnetic bead to enrichment of fungal spores. The sensitivity, specificity and repeatability of this assay were evaluated.

RESULTSThe detection limit of this assay for spiked samples was 10(4) CFU/g, demonstrating a 10-fold greater detection sensitivity of this assay than that of real-time PCR. The NMB-multiple qPCR assay also showed good specificity and reproducibility and yielded comparable results with those by traditional colony counting method for spiked samples (P>0.05).

CONCLUSIONNMB-multiple qPCR assay we established allows rapid and sensitive detection of common mycotoxigenic fungi in paprika.

Aspergillus ; Capsicum ; microbiology ; DNA Primers ; Food Contamination ; analysis ; Food Microbiology ; Fungi ; isolation & purification ; Fusarium ; Magnetic Phenomena ; Penicillium ; Real-Time Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Sensitivity and Specificity

Maca (Lepidium meyenii) is an herbaceous plant that grows in high plateaus and has been used as both food and folk medicine for centuries because of its benefits to human health. In the present study, ITS (internal transcribed spacer) sequences of forty-three maca samples, collected from different regions or vendors, were amplified and analyzed. The ITS sequences of nineteen potential adulterants of maca were also collected and analyzed. The results indicated that the ITS sequence of maca was consistent in all samples and unique when compared with its adulterants. Therefore, this DNA-barcoding approach based on the ITS sequence can be used for the molecular identification of maca and its adulterants.
DNA Barcoding, Taxonomic ; methods ; DNA, Intergenic ; analysis ; DNA, Plant ; analysis ; Drug Contamination ; prevention & control ; Humans ; Lepidium ; genetics ; Phytotherapy

The fruit of Lycium ruthenicum is a common folk medicine in China. Now it is popular for its antioxidative effect and other medical functions. The adulterants of the herb confuse consumers. In order to identify a new adulterant of L. ruthenicum, a research was performed based on NCBI Nucleotide Database ITS Sequence, combined analysis of the origin and morphology of the adulterant to traceable varieties. Total genomic DNA was isolated from the materials, and nuclear DNA ITS sequences were amplified and sequenced; DNA fragments were collated and matched by using ContingExpress. Similarity identification of BLAST analysis was performed. Besides, the distribution of plant origin and morphology were considered to further identification and verification. Families and genera were identified by molecular identification method. The adulterant was identified as plant belonging to Berberis. Origin analysis narrowed the range of sample identification. Seven different kinds of plants in Berberis were potential sources of the sample. Adulterants variety was traced by morphological analysis. The united molecular identification-origin-morphology research proves to be a preceding way to medical herbs traceability with time-saving and economic advantages and the results showed the new adulterant of L. ruthenicum was B. kaschgarica. The main differences between B. kaschgarica and L. ruthenicum are as follows: in terms of the traits, the surface of B. kaschgarica is smooth and crispy, and that of L. ruthenicum is shrinkage, solid and hard. In microscopic characteristics, epicarp cells of B. aschgarica thickening like a string of beads, stone cells as the rectangle, and the stone cell walls of L. ruthenicum is wavy, obvious grain layer. In molecular sequences, the length of ITS sequence of B. kaschgarica is 606 bp, L. ruthenicum is 654 bp, the similarity of the two sequences is 53.32%.
Berberis ; classification ; cytology ; genetics ; China ; DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; chemistry ; genetics ; DNA, Ribosomal Spacer ; chemistry ; genetics ; Drug Contamination ; Drugs, Chinese Herbal ; isolation & purification ; standards ; Lycium ; classification ; cytology ; genetics ; Medicine, Chinese Traditional ; Phylogeny ; Sequence Analysis, DNA ; Species Specificity