肠炎沙门氏菌EMA-PCR检测方法的建立

Detection of Viable Cells of Salmonella enteritidis by EMA-PCR

将荧光染料叠氮溴化乙锭(ethidium monoazide,EMA)与聚合酶链式反应(polymerase chain reaction,PCR)检测技术相结合,用于肠炎沙门氏菌活菌的检测。实验参数优化结果表明,当EMA终质量浓度50μg/mL、曝光时间10 min时,可以抑制约107 CFU/mL肠炎沙门氏菌死菌DNA的扩增;活菌灵敏度检测结果显示,EMA-PCR方法检测限与单一PCR方法一样均为27.5 CFU/mL,说明EMA处理既不会影响活菌DNA的扩增也不会影响PCR方法的灵敏度。利用EMA-PCR方法检测死活混合菌液时发现,添加EMA的实验组DNA条带亮度会随着活菌比例的降低而变暗,且当样品中全是死菌时,没有目标条带出现;而不添加EMA的对照组DNA条带亮度没有变化,当样品中全是死菌时,目标条带依然清晰可见。说明添加EMA可以达到区分死活菌的目的,EMA-PCR方法只检测样品中的活菌,避免了死菌DNA造成假阳性的可能性。

The combination of ethidium monoazide （EMA） and PCR was used for detecting viable cells of Salmonella enteritis. The optimization of experimental parameters showed that a final EMA concentration of 50μg/mL and exposure time of 10 min could restrain DNA amplification of dead bacteria in 107 CFU/mL of Salmonella enteritis. EMA-PCR and direct PCR methods showed the same detection limit of 27.5 CFU/mL for viable cells, suggesting that neither DNA amplification from viable cells nor PCR sensitivity is affected by the addition of EMA. As we observed for mixtures of viable and dead cells by the EMA-PCR method, the brightness of DNA stripes turned darker with reducing the proportion of viable cells, and when all bacteria were dead, no target stripe appeared. As for the control group without added EMA, the brightness of DNA stripe had no change, and even though the bacteria were all dead, the target stripe remained distinct. Therefore, the EMA-PCR method can distinguish dead bacteria from live ones, thereby avoiding false positive detection.