A Nucleotide Signature for the Identification of Angelicae Sinensis Radix (Danggui) and Its Products.

Xiaoyue Wang,Jianping Han,Yang Liu,Lili Wang,Shilin Chen

doi:10.1038/srep34940

Abstract

It is very difficult to identify Angelicae sinensis radix (Danggui) when it is processed into Chinese patent medicines. The proposed internal transcribed spacer 2 (ITS2) is not sufficient to resolve heavily processed materials. Therefore, a short barcode for the identification of processed materials is urgently needed. In this study, 265 samples of Angelicae sinensis radix and adulterants were collected. The ITS2 region was sequenced, and based on one single nucleotide polymorphism(SNP) site unique to Angelica sinensis, a nucleotide signature consisting of 37-bp (5′-aatccgcgtc atcttagtga gctcaaggac ccttagg-3′) was developed. It is highly conserved and specific within Angelica sinensis while divergent among other species. Then, we designed primers (DG01F/DG01R) to amplify the nucleotide signature region from processed materials. 15 samples procured online were analysed. By seeking the signature, we found that 7 of them were counterfeits. 28 batches of Chinese patent medicines containing Danggui were amplified. 19 of them were found to contain the signature, and adulterants such as Ligusticum sinense, Notopterygium incisum, Angelica decursiva and Angelica gigas were detected in other batches. Thus, this nucleotide signature, with only 37-bp, will broaden the application of DNA barcoding to identify the components in decoctions, Chinese patent medicines and other products with degraded DNA.

Highlights

DNA barcoding technology uses a standard gene fragment for rapid identification of unknown specimens to species level
The results showed that 126 sequences of the A. sinensis species were highly conserved
Based on this SNP site, we developed a 37-bp nucleotide signature (5′-aatccgcgtc atcttagtga gctcaaggac ccttagg-3′) exclusively specific for A. sinensis

Summary

Introduction

DNA barcoding technology uses a standard gene fragment for rapid identification of unknown specimens to species level. Meusnier et al proposed a “mini-barcode” sequence to overcome the amplification problem of degraded DNA20 They found that short lengths of 150-bp and 100-bp in the CO1 region could achieve 95% and 90% successful identification, respectively. While the 650-bp length (CO1) could distinguish only 20.5% of the species tested, mini-barcoding achieved 88.6% successful identification, indicating that shorter fragment lengths are more likely to be amplified from degraded DNA23. Liu et al successfully developed a 23-bp signature for identification of ginseng products and found some adulterants in Chinese patent medicines[25] These studies have inspired us to develop a short nucleotide signature for Angelicae sinensis radix within the ITS2 region. We aimed to develop a short gene identifier for the identification of Angelicae sinensis radix and its Chinese patent medicines. The method was used for analysis of Chinese patent medicines containing Angelicae sinensis radix

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