基于PCR技术可视化鉴别金钱白花蛇方法的建立

目的 基于聚合酶链式反应(PCR)技术,利用核酸扩增产物,建立一种可以快速鉴定金钱白花蛇中药材真伪的可视化试纸条检测方法。方法 提取金钱白花蛇、赤链蛇及市售样本的基因;根据mtDNA Cytb序列进行对比分析,设计1对特异性鉴别引物,进行修饰物标记;建立鉴别PCR反应体系,使用核酸扩增产物,利用可视化试纸条对市售样本进行真伪鉴定。结果 鉴别引物可将正品金钱白花蛇中药材扩增出500~600 bp之间的特异性条带,而伪混品则不出现相应的特异性条带;可视化试纸条对市售样本进行检测,鉴别效果良好,灵敏度高,可准确鉴定中药材真伪。结论 可视化试纸条可以快速鉴别金钱白花蛇中药材真伪,稳定性高、特异性强,可应用于市场出售金钱白花蛇中药材的鉴定。

Establishment of a Visual Identification Method Based on PCR Technique to Identify Bungarus multicinctus

OBJECTIVE To develop a rapid visual test strip detection method based on polymerase chain reaction(PCR)technology to identify Chinese medicinal authenticity of Bungarus multicinctus, using the products of nucleic acid amplification. METHODS The total DNA of the genome of Bungarus multicinctus, Dinodon rufozonatum and commercial samples was extracted. Comparative analysis was performed according to mtDNA Cytb sequence. One pair of specific identification primers were designed and the decoration was marked. A differential PCR reaction system was established. Visual test strip was used to authenticate the commercial samples. RESULTS The distinguishing primers of Bungarus multicinctus could obtain the specific amplification band between 500 bp to 600 bp, but adulterants had no specific bands. Visual test strip could accurately identify the authenticity of Chinese medicinal materials with excellent sensitivity. CONCLUSION A rapid visual test strip detection method is established to identify the authenticity of Chinese medicinal Bungarus multicinctus, which has high stability and good specificity, and can be used to identify commercial medicinal materials of Bungarus multicinctus.