目的 沪地龙3个基原药材（通俗环毛蚓Pheretima vulgaris、威廉环毛蚓P. guillelmi、栉盲环毛蚓P. pectinifera）在外观性状方面相似度高，传统鉴定方法难以有效区分，通过建立多重位点特异性PCR方法同时鉴别沪地龙3个基原。方法 通过比对沪地龙和其他7个蚯蚓品种线粒体全基因序列的多态性位点差异，分别设计通俗环毛蚓、威廉环毛蚓及栉盲环毛蚓的特异性鉴别引物，建立单基原反应体系，并对耐受性和适用性进行考察和验证；而后优化特异性引物组合，构建多重位点特异性PCR鉴别体系；反应体系为25 μL：2×Rapid Taq Master Mix 12.5 μL，引物1.5 μL（通俗环毛蚓∶威廉环毛蚓∶栉盲环毛蚓＝1∶1.2∶0.8），DNA模板0.5 μL（50 ng/μL），灭菌水补足至25 μL。PCR扩增条件：95 ℃预变性4 min；95 ℃变性15 s，60 ℃退火15 s，72 ℃延伸20 s，循环33次；产物末端72 ℃延伸7 min。结果 通俗环毛蚓可得到535 bp条带，威廉环毛蚓可得到219 bp条带，栉盲环毛蚓可得到435 bp条带，其他品种蚯蚓样品均无条带。结论 多重位点特异性PCR鉴别体系可根据条带位置同时鉴别沪地龙3个基原。该方法可用于沪地龙基原和真伪鉴别，并为地龙的质量控制和研究提供参考。

Objective The three motif medicinal materials of Shanghai Pheretima (Pheretima vulgaris, P. guillelmi, P. pectinifera) have high similarity in appearance. It is difficult to distinguish by traditional identification methods. Multiple site-specific PCR method was established to identify the three motifs of Shanghai Pheretima simultaneously. Methods By comparing the differences of polymorphic sites in the mitochondrial whole gene sequences of Shanghai Pheretima and seven other earthworm species, specific identifying primers were designed for the P. vulgaris, P. guillelmi, P. pectinifera, respectively, and a single-motif reaction system was set up, and the tolerance and applicability were examined and verified; And then the specific primer combinations were optimized to construct a multiplex sitee-specific PCR identification system; The reaction system was 25 μL: 2×Rapid Taq Master Mix 12.5 μL, primers 1.5 μL (P. vulgaris∶P. guillelmi∶P. pectinifera = 1∶1.2∶0.8), DNA template 0.5 μL (50 ng/μL), and double-distilled water to make up to 25 μL. PCR amplification conditions: pre-denaturation at 95 ℃ for 4 min, denaturation at 95 ℃ for 15 s, annealing at 60 ℃ for 15 s, extension at 72 ℃ for 20 s, cycling 33 times; the product end was extended at 72 ℃ for 7 min. Results 535, 435 and 219 bp of fragments were amplified from DNA templates of P. vulgaris, P. guillelmi and P. pectinifera, respectively. There were no bands be found in other earthworms. Conclusion The multiplex site-specific PCR identification system can simultaneously identify three motifs of Shanghai Pheretima according to the site of the brand. It could be used to identify motif and authenticity of Shanghai Pheretimaand provide reference for the quality control and research of Pheretima.

R286.2

国家自然科学基金资助项目（82225047）;科技部“中医药现代化”重点研发计划（2022YFC3501703）