双重实时荧光定量PCR技术检测中药制剂中沙门氏菌和大肠埃希菌

Detection of Salmonella and Escherichia coli in Traditional Chinese Medicine Preparations by Dual Real-time Fluorescence Quantitative PCR

目的:建立双重实时荧光定量PCR(multiplex quantitative real-time PCR,multiplex qPCR)快速检测中药制剂中沙门氏菌和大肠埃希菌2种常见控制菌的方法。方法:筛选两种目标菌株的特异性引物与探针,扩增沙门氏菌的invA基因和大肠埃希菌的uidA基因,优化反应体系,建立双重荧光定量PCR方法。结果:两对引物探针对沙门氏菌和大肠埃希菌均有较好地扩增效率,人工污染中药制剂中沙门氏菌的检出限为101 CFU·mL^(-1),大肠埃希菌的检出限为101 CFU·mL^(-1)。结论:本研究建立的双重实时荧光定量PCR方法具有特异性强、重复性好,灵敏度高等特点、适用于中药制剂中沙门氏菌和大肠埃希菌的快速检测。

Objective:To establish a method for rapid detection of Salmonella and Escherichia coli in traditional Chinese medicine preparations by multiplex quantitative real-time PCR(multiplex qPCR).Methods:The specific primers and probes of two target strains were screened,the invA gene of Salmonella and uidA gene of Escherichia coli were amplified,the reaction system was optimized,and the dual fluorescence quantitative PCR method was established.Results:Two pairs of primer probes can amplify Salmonella and Escherichia coli effectively.The detection limit of Salmonella and Escherichia coli in Artificially Polluted traditional Chinese medicine preparations is 101 CFU·mL^(-1).Conclusion:The dual real-time fluorescence quantitative PCR method established in this study has the characteristics of strong specificity,good repeatability and high sensitivity.It is suitable for the rapid detection of Salmonella and Escherichia coli in traditional Chinese medicine preparations.