Abstract
In this study, the second-generation high-throughput sequencing and DNA barcoding were combined to manually prepare multi-species mixed samples, and the mitochondrial gene CO І was used as a barcode to simultaneously identify the animal species in the mixed samples and identify endangered species. The results showed that under the family and genus level, the simultaneous detection rate of the species in the mixed samples was as high as 100%, and the species identification rate was as high as 89% at the species level, and with high sensitivity, as little as 1% of the trace species could be detected. However, nearly 30% of non-target classification annotations appeared at the species level. It can be concluded that the mini CO I barcoding can be applied to the simultaneous identification of animal species in mixed biological samples, and the species identification rate is high. Non-target classification match existing at the species level can be further improved by increasing the length of the barcoding, improving the sequencing technology, reference database and so on. In this study, DNA metabarcoding technology was used to evaluate the feasibility of identification of endangered animals in multi-species mixed biological samples with CO І, in order to lay a preliminary foundation for the advancement of DNA metabarcoding method in the field of wildlife forensic identification.
Highlights
Species genetic identification plays a key role in the investigation of illegal trade in endangered wildlife and food adulteration
In the case of species identification of traditional Chinese medicine, a mixed sample containing more than one component, DNA barcoding technology based on the generation of Sanger sequencing is not applicable
The animal samples used in the preparation of artificial mixed samples involved 11 genus of 9 families, including China's Cand secondary key protected animals, beneficial species with economic and scientific research values and animals supervised by CITES appendix
Summary
Species genetic identification plays a key role in the investigation of illegal trade in endangered wildlife and food adulteration. The correct identification of the species contained in the food is essential to protect consumers from potential food adulteration, mislabeling of ingredients or food poisoning Another mature application of DNA barcoding in forensic science is to investigate illegal criminal activities such as illegal acquisitions, transportation, trading and smuggling of endangered wildlife. For samples that are unrecognizable in this form and do not know the composition of their species, a standardized, fast, and reliable method of identification is helpful and necessary for law enforcement It is these advantages that make DNA barcoding the preferred method of identifying endangered species in animal and plant products. In the case of species identification of traditional Chinese medicine, a mixed sample containing more than one component, DNA barcoding technology based on the generation of Sanger sequencing is not applicable. This study combined the second generation of high-throughput sequencing with DNA barcoding technology (metabarcoding: DNA metabarcoding), and manually prepared mixed samples of three different species and different proportions, and used the mitochondrial gene CO І as a barcode to initially explore the feasibility and application prospect of DNA metabarcoding technology in rapidly identification of endangered species in complex samples
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