微滴式数字PCR技术定量检测发酵乳中金黄色葡萄球菌

Quantitative Detection of Staphylococcus aureus in Yogurt by Droplet Digital PCR Assay

基于微滴式数字聚合酶链式反应(droplet digital polymerase chain reaction,dd PCR)技术,建立发酵乳中金黄色葡萄球菌定量检测方法。以金黄色葡萄球菌nuc基因为目的片段设计特异性的引物和探针,优化反应体系,通过活菌提取和ddPCR方法对靶标基因的检测特异性和灵敏度进行实验,并对定量结果进行分析。本研究建立了发酵乳中ddPCR技术定量检测金黄色葡萄球菌的方法,检测特异性良好,检测灵敏度为3.3×10~1 CFU/g,定量的偏差率为+10.18%,证明了ddPCR用于绝对定量检测的可行性,为其他食品污染菌和致病菌标准化控制体系提供有益的示范作用。

This study aimed to establish a new accurate quantitative method to detect Staphylococcus aureus in yogurt using droplet digital PCR(ddPCR).Specific primers and probes targeting the nuc gene of Staphylococcus aureus were designed,and the PCR reaction system was optimized.The specificity and sensitivity of ddPCR for detecting the target gene were tested using the DNA extracted viable bacterial cells,and the quantitative results were analyzed.The ddPCR method could be useful to quantitatively detect S.aureus in yogurt with good specificity.The limit of detection(LOD)of the method was3.3×101CFU/g,and the quantitative deviation was+10.18%,which proved the feasibility of using ddPCR for absolute quantitation of S.aureus.It will provide a useful demonstration for standardized control systems of other food-contaminating bacteria and foodborne pathogenic bacteria.