为实现活的非可培养(Viable but nonculturable state,VBNC)态沙门氏菌的定量检测,文章根据沙门氏菌的特异性毒力基因设计引物和探针,优化微滴数字聚合酶链反应(Droplet digital polymerase chain reaction,ddPCR)反应条件及叠氮丙咬(Propidium monoazide,PMA)处理方式,构建PMA-ddPCR快速定量检测方法.结果表明,拷贝数与菌液浓度在103～107 CFU/mL范围内呈现良好的线性关系,PMA-ddPCR方法检出限为102 CFU/mL,定量限为103 CFU/mL.所建立的PMA-ddPCR快速定量检测冷冻肉制品中VBNC态沙门氏菌方法,弥补了现有标准检测方法的不足.
A Quantitative Assay for Measuring VBNC Salmonella by Droplet Digital PCR Combined with Propidium Monoazide
In order to quantitatively detect viable but nonculturable state(VBNC)Salmonella,primers and probes were designed based on the specific virulence gene of Salmonella,and droplet digital polymerase chain reaction(ddPCR)reaction conditions and propidium monoazide(PMA)treatment were optimized.A rapid quantitative detection method of PMA-ddPCR was established.The results showed a good linear relationship between copy number and bacterial solution concentration in the range of 103-107 CFU/mL.The detection limit and quantitation limit of PMA-ddPCR were 102 CFU/mL and 103 CFU/mL respectively.The established PMA-ddPCR method for rapid and quantitative detection of VBNC Salmonella in frozen meat products addresses the shortcomings of existing standard detection methods.
propidium monoazidedroplet digital PCRSalmonellaviable but nonculturable statequantitative assay
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维普
