荧光定量PCR结合EMA/PMA染色法快速检测军团菌活菌

Quick detection of viable legionella by real-time PCR combined with EMA/PMA

目的:利用EMA/PMA经膜核酸染色结合荧光定量PCR技术快速检测军团菌活菌,比较两者的检测效率。方法:军团菌分别经加热灭活和氯消毒剂灭活,加入EMA/PMA处理,确定EMA/PCR、PMA/PCR体系和反应条件,之后进行快速检测。利用BacLight Live/Dead bacterial viability kit验证活菌,同时用培养法做比对。结果:EMA和PMA工作浓度分别为0μg/ml,5μg/ml,10μg/ml,25μg/ml,50μg/ml,100μg/ml,将菌液与之混合后用卤素灯照射,时间分别为0 min,1 min,5 min,15 min,经测试确定EMA工作浓度为50μg/ml,而PMA为25μg/ml,光照时间选用15 min。模拟环境样本检测,EMA/PMA real-time PCR检测限均为104cfu/ml,但培养法PMA处理后平板上菌落多于EMA处理组。结论:EMA/PMA real-time PCR可快速检测军团菌活菌,检测效率相当(均低于不加染料组),PMA效果优于EMA。

Objective： To detect viable Legionella based on Real - time PCR combined with EMA/PMA, and compare the both detection efficiency. Methods： Legionella was inactivated with heating and chlorine disinfectant respectively, then EMA/PMA were added to establish the experimental system of EMA./PMA, and real - time PCR was used to detect viable Legionella. The simulation environment samples were tested and verified with this method. BacLight Live/Dead bacterial viability kit was used to verify the state of bacteria, and cultivation was used at the same time for comparison. Results： The working concentrations of EMA/PMA were 0 μg/ml, 5 μg/ml, 10 μg/ml, 25 μg/ml, 50 μg/ml, 100μg/ml, then the mixture of EMA/PMA and bacteria were exposed to halogen lamp with the exposure time for 0 min, 1 min, 5 min, 15 min. By test under different conditions, the optimal concentration of EMA was 50 C/ml, while PMA was 25 C/ml. The time of exposure was better set at 15 min. Both of the detection limits were 104 cfu/ml, while after cultivation, the bacterial colony in plate treated with PMA were more than that with EMA. Conclusion： EMA/PMA real - time PCR can detect viable Legionella rapidly, and the efficiency of EMA/PMA real - time PCR was almost the same （ both were lower than that without dye）. The effect of PMA was better than EMA.