摘要
目的将叠氮溴化乙锭(ethidium monoazide bromide,EMA)与PCR技术相结合,建立一种有效、快速检测活肠出血性大肠杆菌O157∶H7的方法。方法以rfbE为PCR检测靶基因,O157∶H7培养物经EMA处理后制备模板进行PCR检测,并对EMA使用浓度、作用时间等进行优化。结果不抑制O157∶H7活菌PCR扩增的最大EMA浓度为10μg/mL;抑制2×107 CFU/mL死菌PCR反应的最小EMA浓度为0.5μg/mL;该法对O157∶H7检测的灵敏度为2×104 CFU/mL,结果显示能检出混合体系中含有的1%的活菌。结论 EMA-PCR技术能有效检测活的O157∶H7,在突发公共卫生事件的快速检测中具有良好的应用前景。
Objective A rapid and effective method with ethidium monoazide bromide （EMA） in combination with PCR （EMA-PCR） was established to detect live Enterohemorrhagic Eschrichia Coli O157 ：H7. Methods The rfbE gene was used as the target gene for PCR detection of Eschrichia Coli O157 ： H7 by utilizing its pure isolates after the treatment of EMA as the template. The EMA concentration and reaction time was optimized. Results The use of 10 μg/mL or less EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The PCR amplification of DNA derived from 2 × 10^7 CFU/mL dead cells can be inhibited by 0.5 μg/mL EMA. The sensitivity of the method was 2 × 10^4 CFU/mL. The results demonstrated that it could detect 1~ live bacteria from a mixed bacterial population. Conclusion EMA-PCR can effectively detect live bacteria of O157 ： H7, it could be a potential rapid detection method applied in public health emergent events.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2014年第1期152-155,共4页
Journal of Sichuan University(Medical Sciences)
