广地龙特异性引物序列的设计及其混伪品的鉴别

目的?通过设计特异性引物来鉴定广地龙及其混伪品。方法?提取广地龙及其混伪品的DNA。利用通用引物COⅠ序列对广地龙及其混伪品进行PCR扩增，并对扩增产物进行双向测序。通过比对广地龙及其混伪品的COⅠ序列，找出广地龙的变异位点，设计出广地龙的特异性引物，用于广地龙及其混伪品的鉴定。对PCR反应条件退火温度、循环次数、DNA模板量和Taq酶用量进行适应性考察。结果?COⅠ序列不存在特异性，不同物种、不同品种的动物药均可扩增出750?bp大小的片段，无法鉴定广地龙及其混伪品。通过比对广地龙及其混伪品的COⅠ序列，设计出广地龙的特异性引物PA-f/PA-r。在建立的PCR反应体系中，只有广地龙可以获得574?bp的基因片段，其他混伪品未扩增出相应条带。结论?设计的广地龙特异性引物可以准确、成功鉴别广地龙，与其他混伪品区分，为广地龙的品种鉴别提供了有效的方法。 

The Design of Specific Primers of Pheretima aspergillum<\i> and the Identification of Adulterants

OBJECTIVE?The specific primers were designed to identify Pheretima aspergillum and its adulterants. METHODS?The DNA of Pheretima aspergillum and its adulterants were extracted and amplified using the COⅠsequences as primers. The amplification products were sequenced afterwards. By comparing the COⅠsequences of Pheretima aspergillum with its adulterants， the variation sites were found in Pheretima aspergillum and the specific primers were designed to identify Pheretima aspergillum and its adulterants. PCR reaction system was optimized. RESULTS?750 bp fragments could be detected in the products of other animal medicinal materials using the COⅠsequences as primers， which meant the primers were not specific enough to distinguish the Pheretima aspergillum from its adulterants. Based on the COⅠsequences of amplification products， the specific primers PA-f/PA-r were designed. Through the established PCR reaction system， 574 bp fragments were amplified from DNA templates of Pheretima aspergillum. All the adulterants had no bands. CONCLUSION?The designed primers sequences could accurately and successfully distinguish the Pheretima aspergillum from its adulterants， providing an effective method for the identification of Pheretima aspergillum.