EMA real-time PCR method for detection of alive Salmonella

Abstract

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 10 ⁶, 10 ⁵, 10 ⁴, 10 ³ 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 50 μg/mL. The optimal concentration of EMA was 10 μg/mL. The results of pretreatment with 10 μg/mL EMA were consistent in the concentration of 10 ⁶, 10 ⁵, 10 ⁴, 10 ³ 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 10 ³-10 ⁶ CFU/mL, with the pretreatment of 10 μg/mL EMA, the ratio of alive Salmonella was probably below 50% when the ACt≥1 between EMA treated sample and EMA untreated sample.

Abstract

摘要： 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 10 ⁶、10 ⁵、10 ⁴、10 ³ 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 50 μg/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 10 μg/mL。采用 10 μg/mL EMA 进行预处理, 混合菌量为10 ⁶、10 ⁵、10 ⁴、10 ³ 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 10 ³~10 ⁶ CFU/mL 时, 通过 10μg/mL EMA 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%。