微滴式数字聚合酶链式反应定量检测水产品中溶藻弧菌

Detection of Vibrio alginolyticus in aquatic products by droplet digital polymerase chain reaction

目的建立水产品中溶藻弧菌微滴式数字聚合酶链式反应(ddPCR)快速定量检测方法。方法采用SN/T 1870—2016《出口食品中食源性致病菌检测方法实时荧光PCR法》中溶藻弧菌的引物探针,建立优化ddPCR反应体系,梯度稀释溶藻弧菌纯培养液,确定ddPCR方法的检出限(LOD)和定量限(LOQ);通过人工添加试验,比较ddPCR与实时荧光定量聚合酶链式反应(qPCR)方法定量的准确性,以验证ddPCR方法用于检测水产品中溶藻弧菌的可行性。结果所建立的水产品中溶藻弧菌ddPCR检测方法经过溶藻弧菌标准菌株、分离菌株以及其他近源菌株验证,具有良好的特异性;纯培养的LOD和LOQ一致,均为2.2拷贝/反应,相当于110 CFU/ml纯菌液,定值结果与传统平板计数方法相差0.04个对数值。在人工污染模拟试验中ddPCR的定量结果较qPCR更接近理论添加值,检测重复性以及对低浓度样品的检出情况也均优于qPCR。ddPCR对人工污染样品的LOQ可达120拷贝/g。结论溶藻弧菌ddPCR定量检测方法特异性好、灵敏度高、结果准确,可为快速定量检测溶藻弧菌提供参考。

Objective To establish a new accurate quantitative method for detection of Vibrio alginolyticus by droplet digital polymerase chain reaction PCR(ddPCR)in aquatic products.Methods The primer and probe of Vibrio alginolyticus in Method for the Detection of Pathogens in Food for Export—Real-time PCR Method(SN/T 1870-2016)were selected,and the ddPCR reaction system was established and optimized.The limit of detection(LOD)and the limit of quantitation(LQD)were tested based on ddPCR using pure bacterial genomic DNA and quantitative accuracy analysis was performed on the result.In order to verify the feasibility of the method,positive strain was added to pre-screening squid rings without background and the result obtained by ddPCR and quantitative real time polymerase chain reaction(qPCR)were compared.Results Vibrio alginolyticus performed good specificity in ddPCR detection in this experiment.In pure culture of Vibrio alginolyticus,the LOD and LOQ values overlapped each other,which was 2.2 copies/reaction,corresponding to 110 copies/ml.In the artificial contaminated experiment,the result of ddPCR for quantification of Vibrio alginolyticus were closer to theoretical value,and the repeatability and the detection rate of low concentration sample were also better than qPCR.The LOQ of artificially contaminated sample could reach 120 copies/g.Conclusion The result showed that quantitative detection of Vibrio alginolyticus by ddPCR had good specificity,high sensitivity and accuracy,which had practical application value.