Combination of c oxidase subunit I based deoxyribonucleic acid barcoding and HPLC techniques for the identification and quality evaluation of Pheretima aspergillum

Abstract

This study aimed to identify Pheretima aspergillum (Guang‐Pheretima) and its adulterants using the cytochrome c oxidase subunit I based deoxyribonucleic acid barcoding technology, and further to evaluate their quality using an optimized high‐performance liquid chromatography method. For deoxyribonucleic acid barcoding identification, the Kimura‐2‐Parameter model was used to analyze genetic distance, and phylogenetic neighbor‐joining tree was constructed for species identification of 20 labeled Guang‐Pheretima samples. A high‐performance liquid chromatography method was developed for the simultaneous determination of seven nucleoside components for quality evaluation. Compared with the GenBank database, 10 samples were identified as real Guang‐Pheretima (P. aspergillum), and the others as the adulterants‐Metaphire magna. The maximum intraspecific genetic distances of c oxidase subunit I sequence for P. aspergillum were smaller than the minimum interspecific genetic distances between P. aspergillum and M. magna. Ten P. aspergillum and 10 M. magna samples were clearly clustered in the neighbor‐joining tree. The contents of seven nucleosides components in P. aspergillum were significantly higher than that in its adulterant‐M. magna. The incidence of adulterants for Guang‐Pheretima was high (up to 50%) with an alarming quality. This study provided a powerful idea for the quality evaluation of other highly valuable plant‐ or animal‐derived products for safety concerns to avoid misidentification.

A novel biological sources consistency evaluation method reveals high level of biodiversity within wild natural medicine: A case study of Amynthas earthworms as “Guang Dilong”

“…Previous reports have validated 658 bp Folmer region of COI gene amplified by LCO1490/HCO2198 as the “standard” marker for the DNA barcoding of earthworms 3 , 37 . To set up a local reference database, 1575 Amynthas and Metaphire COI sequences were first collected from BARCODE OF LIFE DATA (BOLD; http://boldsystems.org ) and GenBank.…”

“…Over the past decade, molecular methods such as DNA barcoding have provided an accurate and efficient strategy for identifying species and distinguishing the authentic ones from the adulterants in various types of traditional medicines 2 , 3 , 4 , 5 . Furthermore, DNA barcoding can also speed up the identification of distinct lineages, and accelerate the discovery of the cryptic/undescribed inter-/intra-species level biodiversity 6 , 7 .…”

“…Nevertheless, while the alpha taxonomic classification of Megascolecidae in south China is still ongoing, information on the sympatric distribution of multiple cryptic species 32 and relative taxa abundance is still too limited to inform the selection of suitable collection or breeding sites. On the other hand, although multiple methods were developed for the authentication of Amynthas species 3 , 33 , 34 , 35 , 36 , so far there is no reasonable biological composition assessment approach suitable for batch consistency evaluation of “Guang Dilong”.…”