克罗诺阪崎肠杆菌微滴数字PCR定量方法的建立

Establishment of Droplet Digital PCR System for Absolute Quantitative Detection of Cronobacter sakazakii

为建立克罗诺阪崎肠杆菌的微滴式数字聚合酶链式反应(droplet digital polymerase chain reaction,ddPCR)快速定量检测方法,针对克罗诺阪崎肠杆菌的特异性单拷贝PapC基因设计引物探针,进行特异性、灵敏度和重复性实验,本文通过对纯菌液进行检测,比较平板计数法、实时PCR和ddPCR方法的定值效果。结果表明,所建立的克罗诺阪崎肠杆菌ddPCR检测方法具有良好的特异性、灵敏性和重复性。克罗诺阪崎肠杆菌纯菌液中最低定量限为104 CFU/mL,最低检出限为16 CFU/mL。不同浓度的纯菌液的测定,ddPCR与平板计数的测定值结果偏差小于10%,比实时PCR方法的定值结果更加准确。因此,本研究建立的ddPCR方法能够快速、准确、灵敏、特异地定量检测克罗诺阪崎肠杆菌。

A droplet digital polymerase chain reaction(ddPCR)method was developed for the rapid quantitative detection of Cronobacter sakazakii,a pair of primers and probe was designed according to the specific single copy gene of PapC in Cronobacter sakazakii,the specificity,sensitivity and repeatability of this method was analyzed.The constant value effect of plate counting method、real-time quantitative PCR(qPCR)and ddPCR method was compared through the detection of pure bacterial solution.The results indicated that the ddPCR had the characteristics of excellent specificity,sensitivity and repeatability in Cronobacter sakazakii detection.The limit of detection(LOD)of pure bacterial culture was 104 CFU/mL,the limit of quantification(LOQ)of pure bacterial culture was 26 CFU/mL.In different levels of pure bacterial solution,the deviation between ddPCR and plate count was less than 10%,which was more accurate than the results of qPCR.Therefore,the established ddPCR method is more rapid,accurate,sensitive and specific for the quantitative detection of Cronobacter sakazakii.