EMA-PCR方法快速检测铜绿假单胞菌活菌研究

目的探讨将叠氮溴乙锭(ethidium monoazide bromide，EMA)选择渗透性与PCR技术相结合（EMA-PCR），建立有效快速检测铜绿假单胞菌活菌的方法。方法以铜绿假单胞菌oprI基因为PCR检测靶基因，纯培养物提取模板进行PCR，检测PCR灵敏度、EMA使用浓度和曝光时间优化试验。结果 PCR检测灵敏度为3×103 CFU/mL；曝光处理时间为10 min；EMA浓度≤5 μg/mL对活菌DNA扩增没有明显抑制，终浓度为1 μg/mL EMA能有效抑制3×106 CFU/mL死菌扩增；1%活菌混合体系检测结果阳性。结论 EMA-PCR方法能有效快速检测铜绿假单胞菌活菌，能避免PCR检测可能造成的假阳性结果。

英文摘要:

Objective To establish a effective and rapid method by Ethidium Monoazide Bromide(EMA) in combination with PCR(EMA-PCR) for thedetection of live Pseudomonas aeruginosa. Methods The oprI gene was used as the target gene for PCR detection of Pseudomonas aeruginosa, and PCR amplification was carried out by utilizing its pure isolates as the template. Sensitivity, EMA concentration and exposure time were optimized. Results The sensitivity of PCR detection was 3×103 CFU/mL, exposure time was 10 min. when the EMA concentration was not more than 5 μg/mL, no obvious inhibition to the amplification of DNA derived from viable bacteria was observed. The PCR amplification of DNA derived from 3×106 CFU/mL dead cells could be inhibited effectively by EMA at the final concentration of 1 μg/mL. The results demonstrated the establised method could detect 1% live bacteria from a mixed bacterial population. Conclusion EMA-PCR can detect live bacteria of Pseudomonas aeruginosa effectively and avoid false positive result of the PCR detection.

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