金属离子指示剂介导的核酸扩增技术检测食品中的副溶血弧菌

Detection of Vibrio Parahaemolyticus by fluorescent metal indicator mediated isothermal amplification method

建立了金属离子指示剂介导的核酸扩增技术用于水产品中副溶血弧菌的检测,同时将检测结果与PCR方法进行比较。结果表明,金属离子指示剂介导的核酸扩增技术能够在恒温65℃、60min内完成对副溶血弧菌tlh基因的特异性检测,检出限达到1.35CFU/mL,是PCR检测限的10倍;阳性检测结果呈亮绿色,明显区别于阴性橘黄色,通过目视颜色的改变即可判断;检测48份样品过程中,金属离子指示剂介导核酸扩增方法检出45份,PCR方法检出34份阳性样品,灵敏度分别为93.8%和70.8%,阴性预测率分别为76.9%和41.7%。金属离子指示剂介导恒核酸扩增法具有高效、高特异性、高灵敏度特性,可满足实地检测需要。

Fluorescent metal indicator mediated isothermal amplification method was used for Vibrio parahaemolyticus detection which was isolated from seafoods,and the sensitivities of the method and PCR were compared.The results showed that the target DNA was amplified in 60min at isothermal temperature 65℃ and the detection limit was 1.35CFU/mL,which was 10-fold greater than PCR.The positive result was light green color while negative was orange,and the resulting amplicons were visualized by naked eyes.Application of fluorescent metal indicator mediated isothermal amplification method and PCR on 48 V.parahaemolyticus strains from seafood samples,the fluorescent metal indicator mediated isothermal amplification method detected 45 samples while PCR 34 samples,the sensitivity and specificity with regard to PCR were 93.8% and 70.8%,respectively,and the positive predictive value（PPV）were 76.9% and 100%,respectively.So,the fluorescent metal indicator mediated isothermal amplification method reported here demonstrates a potential and valuable means for detection of Vibrio parahaemolyticus especially for its rapidity,high simplicity and specificity.