沙门氏菌微滴式数字PCR定量检测方法的建立

Establishment of Droplet Digital PCR Assay for Quantitative Detection of Salmonella

本研究通过对肠炎沙门氏菌PCR特异性扩增引物序列及探针序列进行优化,建立了沙门氏菌微滴式数字PCR快速定量检测方法。实验结果显示,该方法可实现检测沙门氏菌菌悬液浓度范围为5.3×10^(1)~5.3×10^(5)CFU/m L,可检测沙门氏菌有效基因组DNA浓度区间在2~20940拷贝/20μL。采用该方法测得结果与菌落总数测试片相比,两者无显著性差异(p>0.05);与荧光定量聚合酶链式反应(Quantitative polymerase chain reaction,q PCR)方法相比,该方法可检测菌悬液浓度更低,且能实现准确快速定量。实验结果证明,该方法特异性强、灵敏度高、检测范围广、定量结果准确可靠,可满足公共卫生事件快速应急处理的需要,具有广泛的应用前景。

In this study,a rapid quantitative detection method for Salmonella based on droplet digital PCR(ddPCR)was successfully established by designing synthetic Salmonella enteritidis specific PCR amplification primers and probe sequences.This method could detect Salmonella bacterial suspension concentrations ranging from 5.3×10^(1)to 5.3×10^(5)CFU/mL,and the effective genomic DNA concentration of Salmonella could be quantified in the range of 2 to 20940 copies/20μL.The results obtained by this method were not significantly different from the total colony count test strip(p>0.05).Compared with the quantitative polymerase chain reaction(qPCR)method,this method could detect lower concentrations of bacterial suspension and can achieve accurate and rapid quantification.The experimental results demonstrate that this method has strong specificity,high sensitivity,broad detection range,and reliable quantitative results.These attributes make it well-suited for rapid emergency response to public health events and have the potential for further development.