脱氧胆酸钠在副溶血性弧菌死活细胞叠氮溴乙锭PCR鉴别中的应用

The application of sodium deoxycholate incorperation into ethidium bromide monoazide PCR for discrimination of viable and dead cells of Vibrio parahaemolyticus

摘要:

【目的】通过将表面活性剂脱氧胆酸钠(Sodium deoxycholate, SD)与叠氮溴乙锭(Ethidium bromide monoazide, EMA)-PCR反应体系相结合, 建立SD-EMA-PCR鉴别副溶血性弧菌死活细胞的检测方法。【方法】依次对加入检测体系中的脱氧胆酸钠最适浓度、EMA区分死活细胞DNA的浓度范围、EMA激活光解最佳曝光时间进行优化; 确定SD-EMA-PCR方法检测副溶血性弧菌死活细胞混合体系中活细胞的最低检出限。【结果】当脱氧胆酸钠浓度≤0.5 g/L, EMA的浓度为3.2?34.0 mg/L, 曝光时间为25 min时, SD-EMA-PCR检测体系仅对死细胞DNA扩增产生抑制作用。SD-EMA-PCR检测活菌细胞的最低检出限为10 CFU/mL。【结论】死活细胞混合体系的SD-EMA-PCR检测证明该方法能够明显降低EMA-PCR漏检的死菌对检测结果造成的影响, 为完善食源性致病菌检测中死活菌细胞鉴别方法提供了一种有效途径。

Abstract:

[Objective] The SD-EMA-PCR was established by incorporating sodium deoxycholate (SD) into EMA-PCR assay for discrimination viable and dead cells of Vibrio parahaemolyticus. [Methods] The optimal concentration of the sodium deoxycholate, the concentration of ethidium bromide monoazide (EMA) for discrimination viable and dead cells, the optimal light exposure time for acativating and photolyzing EMA were determined respectively. And the detection limit value of V. parahaemolyticus in the mixtures of viable and dead cells was obtained by SD-EMA-PCR. [Results] The results showed that when the concentration of sodium deoxycholate is less than or equal to 0.5 g/L, the concentration of EMA is ranged from 3.2 to 34.0 mg/L, the light exposure time is 25 min, the DNA amplification from the dead cells by SD-EMA-PCR were inhibited. The detection limit of the SD-EMA-PCR assay for the viable cells was 10 CFU/mL. [Conclusion] SD-EMA-PCR can be used to minimize the false-positive results by inhibiting the EMA-PCR amplification of V. parahaemolyticus dead cells from a mixed bacterial population. It is an efficient new approach for distinction between the viable and dead cells and efficiently avoid false positive results.