荧光定量PCR结合PMA染料法评价口岸消毒效果的研究

Application of propidiummonoazide based Real-time quantitative PCR for evaluation of disinfection efficacy at ports

目的利用叠氮溴化丙锭(PMA)结合实时荧光定量PCR(qPCR)技术,建立应用于口岸消毒效果的快速评价方法。方法针对细菌16S rRNA基因建立qPCR反应体系,以铜绿假单胞菌为对象,采用菌片浸泡定量杀菌试验对口岸常用含氯消毒剂(泰胜)进行消毒效果评价,分别比较培养计数法、qPCR和PMA-qPCR法评价消毒效果的有效性。结果基于16S rRNA基因的q PCR法重复性好,灵敏度高,细菌最低检测浓度为63 cfu/ml;PMA处理对活菌无显著影响。对比3种检测方法发现,未经PMA处理的q PCR法检测消毒前后细菌数量变化较小,而PMA-qPCR法检测结果显示消毒后细菌数量显著下降(下降3.52个log值),且PMA-qPCR法的检测结果与传统培养法相近,说明PMAq PCR法能有效抑制死亡细菌的扩增,真实反映消毒后的细菌存活情况。结论建立的PMA-qPCR法进行口岸消毒效果评价具有快速、灵敏度高等优点,有望发展为口岸适用的快速评价消毒效果的有利工具。

Objective To establish a method of propidiummonoazide based Real-time quantitative PCR(PMA-qPCR)for evaluating disinfection efficacy,and to explore its applicability at ports.Methods The 16 S rRNA gene was used as a target for q PCR.Pseudomonas aeruginosa was used as a representative bacteria,and quantitative germicidal test was applied to evaluate the disinfection efficacy of chlorine disinfectants which was commonly used at ports.The e-valuation results obtained by PMA-qPCR method were compared with those obtained by qPCR and plate counts.Results The established 16 S rRNA gene based qPCR showed high sensitivity with a quantitative limit of 63 cfu/ml.PMA showed no significant effect on living cells.The disinfection efficacy evaluation showed that the cell number decreased significantly after PMA treatment of samples compared to non-PMA treated samples.The result from PMA-qPCR was close to that from plate counts,which indicating that the method,in combination of PMA couldeffectively eliminate the dead cells and showed better accuracy.Conclusion PMA-qPCR is a rapid,convenient and sensitive method whichhas potential to be a promising diagnostic tool for disinfection efficacy evaluation at ports.