目的 基于线粒体cytochrome coxidase I（CO I）和16 S rRNA基因开发DNA双重条形码鉴定方法，由此验证并补充形态鉴定，以期建立一种准确、有效地鉴定地龙及混淆品原动物的方法。方法 对收集到的66份样品依据形态特征进行初步鉴定，使用优化后的引物同时扩增CO I和16 S rRNA序列。优化一步法双重PCR实验条件。用MEGA 5.1计算地龙及其混淆品的种内、种间遗传距离，基于K2P模型构建NJ树。结果 CO I和16 S rRNA双重DNA条形码鉴定与形态鉴定结合，可准确鉴别地龙及混淆品原动物。结论 形态鉴定是分子鉴定的基础，分子鉴定是形态鉴定的有力补充。二者结合可最大程度地实现地龙及其混淆品原动物的准确鉴定。DNA双重条形码鉴定方法也可为地龙药材鉴定以及其他动物药材的分子鉴定提供参考。

Objective To develop an accurate and effective method to identify the original animals of Chinese medicine Pheretima (Dilong in Chinese) and its adulterants by using mitochondrial genes, cytochrome coxidase I (CO I) and 16 S rRNA as DNA barcoding sequences to certify and improve morphological analysis. Methods A total of 66 samples were initially identified according to their morphological characteristics, and then their CO I and 16 S rRNA sequences were simultaneously amplified for DNA barcoding. The two gene sequences were amplified with improved primers. One-step double PCR was used and the experimental conditions were optimized. The genetic distance among and within species of Pheretima and its hybrids was calculated by MEGA 5.1. The N-J tree was constructed based on K2P model. Results Combined with morphological identification, CO I and 16S rRNA double DNA barcoding identification can accurately identify Pheretima and its adulterants. Conclusion Morphological identification is the basis of molecular identification. Molecular identification can be a powerful supplement to the morphological identification. The combination of molecular identification and morphological identification can increase the accuracy of the identification of the original animals of Pheretima and its adulterants. Double DNA barcoding can also provide a solution for the identification of the medicinal materials of Pheretima and other animal medicinal materials.

R286.12

国家科技基础性工作专项：“常用道地药材及及其产区的特征、标准及数字化”——道地药材知母、何首乌（夜交藤）、蔓荆子、蛇床子、地龙特征数据的收集和整理（2015FY111500-020，2015FY111500-021）；海军军医大学本科创新孵化基地项目（PH2019094）