实时PCR检测食品中荧光假单胞菌方法的建立

Establishment of a Real-time PCR Method for Detecting Pseudomonas fluorescens in Food Samples

针对荧光假单胞菌分子检测靶点gyrB基因设计引物和探针,制备标准质粒,绘制标准曲线,建立荧光假单胞菌实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)检测体系。特异性评价表明该体系能够特异性检测荧光假单胞菌,其他细菌均无检出。灵敏性检测结果表明纯DNA水平灵敏度可达14.3 fg/μL,纯培养物水平上检测灵敏度为3.0×10^2 CFU/mL。抗干扰性实验结果表明当加入低浓度背景干扰菌时,对上述体系检测灵敏度没有影响。人工污染样品检测表明适当增菌可检测到荧光假单胞菌,说明TaqMan探针real-time PCR法特异性强、灵敏度高、抗干扰性能力强。

In this study,we designed primers and probes targeting the gyrB gene of Pseudomonas fluorescens,prepared standard plasmids,drew standard curves,and established a real-time polymerase chain reaction(PCR)system for the detection of P.fluorescens.Our results confirmed that the PCR system was specific to P.fluorescens without detection of other tested bacteria.The sensitivity was 14.3 fg/μL and 3.0×10^2 CFU/mL for pure DNA and pure culture,respectively,and was not affected by the background interference at low concentrations.In artificially contaminated samples,P.fluorescens could be detected by appropriate enrichment.The TaqMan-based real-time fluorescence quantitative PCR method proved to be highly specific,sensitive and resistant to interferences.