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目的比较传统的平板培养法、普通聚合酶链反应(polymerase chain reaction,PCR)、实时荧光定量PCR(realtime fluorescence quantitative PCR,real-time PCR)和荧光染料叠氮溴化乙锭(ethidium monoazide,EMA)结合荧光定量PCR的方法(EMA-荧光定量PCR)对管道水中军团菌的检测效果。方法于2010年对上海市2个口岸管道水进行采样,共采集132份。分别采用传统的平板培养法、普通PCR、荧光定量PCR和EMA-荧光定量PCR对水样中军团菌进行定性和/或定量分析。结果对132份管道水标本进行军团菌定性检测,平板培养法、普通PCR、荧光定量PCR和EMA-荧光定量PCR检出军团菌污染率分别为18.9%、20.5%、24.2%和24.2%。3种定量方法(平板培养法、荧光定量PCR和EMA-荧光定量PCR)对阳性标本中军团菌定量数值分别为50~1320菌落形成单位/升(cfu/L)、20~13 085基因单位/升(GU/L)和10~4480 GU/L。对于132份水样,3种方法对军团菌含量分析的定量数值大小差异有统计学意义(χ2=193.6,P<0.01),其中荧光定量PCR的定量数值最高,EMA-荧光定量PCR的数值次之,平板培养的数值最低。结论上海市口岸管道水中军团菌污染率和含菌量高。荧光定量PCR和EMA-荧光定量PCR方法检测管道水中军团菌的灵敏度优于传统的培养方法,EMA-荧光定量PCR方法适合用于环境水体中军团菌活菌监测。
Objective To compare the effects of traditional culture methods such as plate culture, polymerase chain reaction (PCR), realtime fluorescence quantitative PCR (real-time PCR) and ethidium monoazide (EMA) ) Combined with fluorescence quantitative PCR method (EMA-fluorescence quantitative PCR) to detect Legionella in pipeline water. Methods In 2010, a total of 132 samples of pipeline water were collected from two ports in Shanghai. Legionella in water samples were qualitatively and / or quantitatively analyzed by conventional plate culture method, ordinary PCR, real-time PCR and EMA-fluorescence quantitative PCR, respectively. Results 132 Legionella samples were detected qualitatively by Legionella pneumophila. The contamination rates of Legionella were 18.9%, 20.5%, 24.2% and 24.2% respectively by plate culture, normal PCR, fluorescence quantitative PCR and EMA-PCR. The results of three quantitative methods (plate culture, fluorescence quantitative PCR and EMA-fluorescence quantitative PCR) showed that the quantitative values of Legionella in positive samples were 50 ~ 1320 cfu / L, 20 ~ 13 085 gene units / L (GU / L) and 10 ~ 4480 GU / L. For 132 water samples, the quantitative values of the three methods for analyzing the content of Legionella were statistically significant (χ2 = 193.6, P <0.01), of which the quantitative value of fluorescence quantitative PCR was the highest, the value of EMA- The lowest value of plate culture. Conclusion The water pollution of Legionella in Shanghai port was high and the bacteria content was high. Fluorescent quantitative PCR and EMA-fluorescence quantitative PCR method for detection of legionella in pipeline water is superior to traditional culture methods, and EMA-fluorescence quantitative PCR method is suitable for the monitoring of Legionella viable bacteria in environmental water.