基于DNA染料EMA的RT-PCR技术定量检测海产品中病原性副溶血弧菌活细胞

将一种DNA染料叠氮溴化乙锭(ethidium bromide monoazide，EMA)与实时定量聚合酶链式反应(RT-PCR)技术相结合，建立一种能选择性定量检测牡蛎中trh阳性副溶血弧菌活细胞的新方法。结果表明：使EMA成功插入死细胞DNA并且光解溶液中游离EMA的最佳曝光时间为20min；不抑制副溶血弧菌活细胞DNA扩增的最大EMA质量浓度为2.0μg/mL；完全抑制热致死细胞DNA扩增的最小EMA质量浓度为1.0μg/mL；人工污染牡蛎样品，不经过富集，在2.0×103～2.0×107CFU范围内细胞数的常用对数值与Ct值之间呈严格的负相关性，并且纯培养与人工污染牡蛎样品的RT-PCR检测限均为2×103CFU，即人工污染牡蛎样品的RT-PCR检测灵敏度为每克牡蛎样品400个活细胞；冻融实验表明，在温度低于55℃的水浴中对冷冻海产品进行解冻时，冻融过程对副溶血弧菌活细胞几乎没有影响。该方法是一种快速、灵敏且能有效鉴别并定量检测病原活细胞的新方法。

Quantification of Pathogenic Viable Cells of Vibrio parahaemolyticus in Seafood by Ethidium Bromide Monoazide Staining and Real-time Polymerase Chain Reaction

A new method for selectively quantitative detection of trh-positive viable cells of Vibrio parahaemolyticus in oysters was developed using ethidium bromide monoazide (EMA) staining in combination with real-time PCR (RT-PCR, real-time polymerase chain reaction). The results showed the optimum light exposure time to achieve DNA crosslinking by EMA in dead cells and to photolyze the free EMA in solution was 20 min. The use of 2.0μg/mL EMA or less did not inhibit the RT-PCR amplification of DNA derived from viable cells of Vibrio parahaemolyticus. The minimum amount of EMA to completely inhibit the RT-PCR amplification of DNA derived from heat-killed cells was 1.0μg/mL. In artificial contaminated oyster samples without enrichment process, there was a strict negative correlation between the log cell number and the Ct values in the range of 2.0 × 103 to 2.0 × 107 CFU. The detection limit of the real-time PCR assay was 2 × 103 CFU in both pure cultures and artificial contaminated oyster samples, which indicated the sensitivity of RT-PCR was 400 CFU/g in artificial contaminated oyster sample. The freezing/thawing experiments showed thawing in water bath at temperature below 55 ℃ had little effect on viable cells of Vibrio parahaemolyticus. Therefore, this method avoided the defection in traditional PCR, which could not distinguish viable bacteria cells from dead ones. It may offer a fast, sensitive method to identify and quantify pathogenic viable cells effectively.