EMA-PCR方法快速检测铜绿假单胞菌活菌研究  

Study on Rapid Detecting of Live Pseudomonas Aeruginosa by EMA-PCR

作　　者：方梅[1] 陆巧荣[1] 王国庆[2] 张宏斌[1] 李建[3] 

机构地区：[1]江苏省昆山市疾病预防控制中心,昆山215300 [2]四川大学华西公共卫生学院,成都610041 [3]江苏省苏州市疾病预防控制中心,苏州215004

出　　处：《四川大学学报（医学版）》2014年第6期1015-1018,共4页Journal of Sichuan University(Medical Sciences)

基　　金：苏州市饮用水安全与水性疾病监测公共服务平台2012年开放课题(No.SZPT2012002)资助

摘　　要：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3×103 CFU/mL;曝光处理时间为10 min;EMA浓度≤5μg/mL对活菌DNA扩增没有明显抑制,终浓度为1μg/mL EMA能有效抑制3×106 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。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 5μg/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 1μg/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.

关 键 词：铜绿假单胞菌 EMA-PCR 活菌检测 

分 类 号：R446.5[医药卫生—诊断学]