副溶血弧菌EMA-PCR检测技术的建立

Establishment of EMA-PCR detection method of Vibrio parahaemolyticus

摘要:

PCR技术被广泛应用于副溶血弧菌的检测中, 但传统的PCR技术无法区分样品中的死细菌与活细菌, 往往使检测结果出现较高的假阳性。因此, 将叠氮溴乙锭(Ethidium monoazide bromide, EMA)与PCR技术结合, 建立一种快速、准确的副溶血弧菌检测方法。以dnaJ基因为检测副溶血弧菌的靶基因, 分别用副溶血弧菌的纯培养细胞及其基因组DNA作模板进行PCR检测, 灵敏度分别为2.5×104 CFU/mL和6×102 fg/μL。在检测样品前处理过程中加入EMA, 当EMA的浓度小于5 mg/L时, EMA对活菌靶基因的扩增没有明显的抑制; 而终浓度为2 mg/L的EMA, 能有效抑制1×108 CFU/mL副溶血弧菌死菌的扩增。活菌和死菌混合体系的PCR结果表明, EMA-PCR能有效降低副溶血弧菌检测过程中的假阳性。

Abstract:

PCR technology has been widely used in the detection of Vibrio parahaemolyticus. However, traditional PCR appeared higher false-positive result because of the lack of differentiation between DNA from viable and dead microorganisms. Therefore, Ethidium Monoazide Bromide (EMA) was added in the process of PCR to establish a rapid and accurate detection method of V. parahaemolyticus. The dnaJ gene was used as the target gene for PCR detection of V. parahaemolyticus by utilizing its pure isolates and genomic DNA as the template, and the sensitivity was 2.5×104 CFU/mL and 6×102 fg/μL respectively. The use of 5 mg/L or less EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The PCR amplification of DNA derived from 1×108 CFU/mL V. parahaemolyticus dead cells can be inhibited by 2 mg/L EMA. The results show that EMA-PCR can be used to minimize the false-positive results by inhibiting the PCR amplification of V. parahaemolyticus dead cells from a mixed bacterial population.