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目的:探讨鉴别金钱白花蛇药材及其伪品的PCR方法。方法:利用PCR方法对金钱白花蛇正品药材及四种伪品进行鉴别分析。结果:利用PCR方法进行检测得出以下结果:第一,上述5种样本的收集无误符合实验要求,基于细胞色素C氧化酶I基因序列所进行的引物设计均能够顺利实现实验样品的扩增。第二,当退火温度保持在55℃与63℃之间时58℃以下的各样品检测条带较为清晰;当Mix的用量处于27μl到33μl之间时,29μl用量下可以较为清晰的反映各样品检测条带并且均为特异性扩增。优化后的反应体系为:2×Taq PCR Master Mix29μl、正反向引物8μl、ddH2O9μl、模板DNA共4μl。第三,在进行灵敏度与特异性检测后可知:5种蛇类样品均出现单一模板下的特异性单一条带以及在混合模板下出现了5条条带。当样品的DNA模板浓度为0.1ng/μl时尚可发现有扩增的条带,即,可推断其灵敏度已达到皮克级。结论:PCR鉴别方法的特异性与灵敏度均较高,能够快速进行检测,值得在临床应用上进行推广。
Objective: To explore the PCR method for identifying the medicinal materials and their counterfeit money. Methods: The authenticated medicinal materials and four kinds of counterfeit goods were analyzed by PCR. Results: The following results were obtained by PCR: First, the collection of the above five samples was in good agreement with the experimental requirements, and the primer design based on the sequence of cytochrome C oxidase I gene was able to successfully amplify the experimental samples. Second, when the annealing temperature is maintained between 55 ℃ and 63 ℃, the detection bands of each sample below 58 ℃ are clearer; when the dosage of Mix is between 27μl and 33μl, the amount of 29μl can clearly reflect each sample The bands were detected and both amplified specifically. The optimized reaction system was: 2 × Taq PCR Master Mix 29μl, forward and reverse primers 8μl, ddH2O 9μl, template DNA 4μl. Thirdly, after detecting the sensitivity and specificity, we can see that there are 5 single bands under the single template and 5 bands under the mixed template. When the sample DNA template concentration of 0.1ng / μl can still be found with amplified bands, that is, it can be inferred that the sensitivity has reached Pico grade. Conclusion: The specificity and sensitivity of PCR identification method are high, can be quickly tested, it is worth in the clinical application of promotion.