EMA-PCR技术检测金黄色葡萄球菌活菌

Application of EMA-PCR Method in Detection of Live Staphylococcus Aureus

目的将叠氮溴化乙锭(Ethidium monoazide bromide,EMA)与聚合酶链反应(Polymerase Chain Reaction,PCR)相结合,建立一种快速、有效检测金黄色葡萄球菌活菌的方法。方法用EMA处理金葡菌培养物后提取模板,以金黄色葡萄球菌耐热核酸酶编码基因(nuc基因)为靶基因进行PCR反应,并对EMA浓度和曝光时间等条件进行优化。结果当检测2×106CFU/ml的金黄色葡萄球菌时,能有效抑制死菌DNA扩增的EMA最小浓度为0.3μg/ml,对活菌DNA扩增没有明显抑制的EMA最大浓度为2μg/ml;经EMA处理后,从含有1%金黄色葡萄球菌活菌混合悬液制备的DNA中可扩增出目的片段。结论 EMA-PCR能有效检测金黄色葡萄球菌活菌,在临床医学和公共卫生领域检测中具有重要的实用价值。

Objective To establish a rapid and effective method combining ethidium monoazide bromide （EMA） with Polymerase Chain Reaction （PCR） to detect live Staphylococcus aureus. Methods Heat resistant nucle- ase （nuc） encoding gene was used as the target gene for PCR amplification of Staphylococcus aureus by utili- zing its genomic DNA as the template after the treatment of EMA. The EMA concentration and irradiation time were optimized. Results The PCR amplification of DNA derived from 2×10^6CFU/mL Staphylococcus aureus dead cells could be inhibited by 0.3μg/mL or more EMA. The use of 2μg,/mL or less EMA did not inhibit the PCR amplification of DNA derived from live bacteria. The results demonstrated that it could detect 1% live bacteria from a mixture of live and dead bacteria on the effect of EMA. Conclusion EMA - PCR can effectively detect live Staphylococcus aureus, which shows great practical value both in clinical medicine and public health.