高特异性PCR方法鉴别乌梢蛇及其混淆品

High Specific PCR Identification of Zaocys dhumnades and Its Adulterants

目的建立一种简便、准确的乌梢蛇药材DNA分子标记鉴别方法。方法根据乌梢蛇及10种常见混淆品线粒体12SrRNA基因序列,设计一对专用于乌梢蛇的鉴别引物HWL-1和HWH-1,用不同的复性温度PCR扩增,确立特异性反应条件,并利用此方法鉴别从市场上购买的各种乌梢蛇药材。结果PCR结果是在65℃复性温度下,正品乌梢蛇得到约320 bp的扩增带,而混淆品在同样的条件下无扩增带。为检验该方法的准确性,对市售乌梢蛇炮制品随机选取13块进行PCR鉴别验证,其中正品9块,混淆品4块,检出率达100%。结论所设计的鉴别引物对正品乌梢蛇有高度的特异性。PCR方法稳定性高,同种不同个体间的种内差异对鉴定结果无影响。本方法简单、准确、快速、灵敏度高、重现性好。

OBJECTIVE To develop a convenient and effective method for the identification of Zaocys dhumnades （black snake）. METHODS Based on the sequence of mitochondrial 12S rRNA gene fragment of Zaocys dhumnades and its adulterants, a pair of highly specific primer（ HWL-1 and HWH-1 ） were designed for distinguishing Zaocys dhumnades from other species of snake. A specific PCR reaction condition was established. The primers were employed to amplify the DNA templates extracted from Zaocys dhumhades and 10 other species of snake, under different annealing temperatures. Using this method,Zaocys dhumnades from thirteen samples bought from drugstores was identified. RESULTS About 320 bp DNA fragment was amplified from Zaocys dhumnades in PCR with annealed temperature at 65 ℃, whereas no any DNA fragment was amplified from other snake samples under the same reaction condition. Zaocys dhumnades was clearly distinguished from other by PCR reaction with the highly specific primers. In the present study, thirteen samples, bought from different drugstores, were also identified by the highly specific PCR with the primers. The results indicated that 9 samples were Zaocys dhumnades and the others were adulterants, which was consistent with the conlusion of authentication based on morphology. CONCLUSION The primers designed in the present study are highly specific for Zaocys dhumnades. This method is simple, fast and reproducable.