基于COI条形码的市售蝉蜕及其混淆品DNA分子鉴定研究

目的 利用COI序列对中药材蝉蜕Cicadae Periostracum及其混淆品进行DNA条形码鉴定研究，为蝉蜕药材的快速准确鉴定提供新的技术手段。方法 收集全国市售蝉蜕药材、基地自采蝉蜕及其混淆品总45份样本，同时采集江苏徐州黑蚱养殖基地不同树种中蝉卵样品5份作为对照。提取DNA，进行PCR扩增及双向测序，测序结果采用DNAMAN和SnapGene进行拼接校对，运用MEGA11.0进行比对分析，计算种内及种间Kimura 2-Parameter(K2P)遗传距离并构建邻接法（neighbor-joining,NJ）系统发育树。结果 黑蚱种内遗传距离远小于种间最小遗传距离，NJ树结果显示，黑蚱蝉蜕样品全部聚集为独立的一支，支持率为99%。混淆品样品分别聚集为单独的一支，支持率均大于90%，表明基于COI序列的DNA条形码技术可以有效鉴别蝉蜕药材真伪。结论 应用COI序列作为DNA条形码能够准确有效地鉴别蝉蜕及其混淆品。

Molecular identification of commercial Cicadae Periostracum and its adulterants based on COI barcode sequence

Objective To provide new technique for accurate identification of Chantui (Cicadae Periostracum) and its adulterants using DNA barcoding method based on the COI sequence. Methods Forty-five batches of the commercial Cicadae Periostracum and its adulterants, as well as self-harvested Cicadae Periostracum, were collected from different localities of China, and five batches of cicada eggs from different tree species in Xuzhou, Jiangsu Province were collected as controls. The total genomic DNAs of Cicadae Periostracum and its counterfeits were extracted. The COI fragments of these samples were amplified by PCR and sequenced in both directions to obtain the COI sequence. A total of 28 COI sequences of Cicadae Periostracum, 17 adulterants and 9 related species were downloaded from Genbank database. The obtained sequences were assembled using the DNAMAN and SnapGene. MEGA11.0 was applied to calculate intraspecific and interspecific Kimura 2-Parameter (K2P) genetic distances and construct the neighbor-joining (NJ) phylogenetic tree. Results The intraspecific genetic distance of Heizha (Cryptotympana pustulata Fabricius) was much smaller than the minimum genetic distance between C. pustulata and its adulterants. The NJ tree showed that the genuine C. pustulata samples were all gathered as a separate one, with a support rate of 99%. The counterfeit samples were aggregated into another separate unit, and the support rate was greater than 90%, indicating that the DNA barcoding technology based on COI sequence could effectively identify the authenticity of Cicadae Periostracum. Conclusion COI sequence can stably and accurately distinguish Cicadae Periostracum and its adulterants, which provide a new technique to ensure the quality and clinical safety in utilization of Cicadae Periostracum.