EMA-qPCR检测活沙门菌方法研究

EMA real-time PCR method for detection of alive Salmonella

目的为解决荧光定量PCR(qPCR)方法不能区分死菌和活菌的问题,探索一种快速定量检测“活的”沙门菌的技术路线。方法将不同浓度叠氮溴化乙锭(EMA)作用于不同浓度的死/活沙门菌后,用qPCR检测沙门菌Ct值,确定EMA的最优使用浓度。在混合菌量为106、105、104、103 CFU/mL,活菌比例为0%、10%、30%、50%、70%、100%条件下,确定EMA处理过的活菌+死菌,未加EMA的活菌+死菌,未加EMA的活菌+生理盐水三组菌的Ct值,研究EMA-qPCR法对死/活沙门菌的区分能力。结果EMA浓度低于50μg/mL,EMA处理活菌组与EMA未处理活菌组的Ct值差异无统计学意义。综合考虑,EMA最优使用浓度为10μg/mL。采用10μg/mL EMA进行预处理,混合菌量为106、105、104、103 CFU/mL时得出的结果一致:活菌比例为0%,≤50%,>50%时,EMA处理过的活菌+死菌与未加EMA的活菌+死菌两组Ct值差异(ΔCt)分别为≥5,≥1,<1。同时,除活菌比例0%外,其他活菌浓度下,EMA处理过的活菌+死菌与未加EMA的活菌+生理盐水两组Ct值无明显差异。结论菌量为103~106 CFU/mL时,通过10μg/mLEMA作用,EMA处理过样品与未加EMA处理样品的ΔCt值≥1,可考虑样品中沙门菌活菌比例≤50%。

Objective To probe into the problem of dead bacteria and alive bacteria couldn't be distinguished by fluorescence quantitative PCR(qPCR),a rapid quantitative detection method for alive Salmonella was explored.Methods Different concentrations of dead/alive Salmonella were pretreated with different concentrations of ethidiummonoazide(EMA),the Ct value of Salmonella was detected by qPCR,and the optimal concentration of EMA was determined.In the concentration of 106,105,104,103 CFU/mL and alive bacteria proportion of 0%,10%,30%,50%,70%and 100%,the Ct values of three groups,alive+dead bacteria(EMA treatment),alive+dead bacteria(without EMA),alive bacteria+saline(without EMA),were determined to distinguish the dead and alive salmonella byEMA-qPCR.Results There was no significant difference in Ct value between EMA treated alive Samonella group and EMA untreated alive Samonella group when the EMA concentration was below 50μg/mL.The optimal concentration of EMA was 10μg/mL.The results of pretreatment with 10μg/mL EMA were consistent in the concentration of 106,105,104,103 CFU/mL.When the proportion of alive Samonella was 0%,≤50%,>50%,the difference of Ct value(ΔCt)was≥5,≥1,<1between alive+dead bacteria(EMA treatment)and alive+dead bacteria(without EMA).Except alive bacteria proportion of 0%,there was no significant difference in Ct value between the alive+dead bacteria(EMA treatment)and alive bacteria+saline(without EMA)group.Conclusion In the concentration of 103-106 CFU/mL,with the pretreatment of 10μg/mL EMA,the ratio of alive Salmonella was probably below 50%when theΔCt≥1 between EMA treated sample and EMA untreated sample.