虎掌南星超快速Real-time VPCR闭管检测方法的建立及应用

目的建立虎掌南星Pinellia pedatisecta特异性DNA的闭管式超快速检测方法,实现对虎掌南星样本的快速鉴别。方法针对虎掌南星特异性的DNA序列设计其特异性的引物和TaqMan探针,通过对扩增体系进行优化,建立虎掌南星的超快速实时荧光VPCR扩增体系,实现对虎掌南星特异性DNA的快速扩增和闭管式实时检测。同时对该方法的特异性、灵敏度和实用性进行考察。结果可以在24 min内完成对虎掌南星特异性DNA的闭管快速扩增检测;检测限低至pg级DNA;在同科属常见近缘品种中无交叉反应;对采集的7批56个"半夏"样本进行分析,检测出2个批次共16个样本为虎掌南星,且该结果与测序结果 100%符合。结论所建方法可以作为虎掌南星的特异性检测方法,具有灵敏、快速和不易污染等优点,可以为虎掌南星和半夏药材的质量控制提供参考。

Development and application of an ultra-fast closed-tube Real-time VPCR detection system for Pinellia pedatisecta

Objective To develop an ultra-fast closed-tube detection system for Pinellia pedatisecta specific DNA to realize fast identification of P. pedatisecta. Methods Species-specific primer pairs and probe were designed based on the P. pedatisecta unique sequence. The Real-time VPCR system was established by optimization of the concentrations of primers, probe, temperature, etc. Characters of a detection method including specificity, sensitivity and generality were systematically studied. Results The amplification and detection process for P. pedatisecta specific DNA could be finished within 24 min. The developed method was also proved to be with high specificity without any cross reaction in related species. The detection limit was as low as picogram of DNA. A total of 56 samples from seven batches of Pinelliae Rhizoma were evaluated by the developed method. It turned out that 16 samples from two batches were identified as rhizoma from P. pedatisecta. And all these results were 100% consistent with the sequencing results. Conclusion The proposed method which is sensitive, rapid and pollution-resistant can be used as a specific identification method for Pinelliae Pedatisectae Rhizoma. It could also provide a reference for quality control of Pinelliae Pedatisectae Rhizoma and Pinelliae Rhizoma.