基于PMA-定量PCR选择性检测技术的病原菌消毒特性研究

建立了一种核酸染料propidium monoazide(PMA)与定量PCR技术联合选择性检测活性病原菌的技术(PMA-qPCR)，以大肠杆菌作为模式菌，研究了氯和一氯胺消毒对病原菌的灭活特性.结果表明，PMA染料能够分别去除99.94%和99.99%的来自非活性大肠杆菌和沙门氏菌的DNA，PMA-qPCR技术能够有效区分活性菌与非活性菌；PMA-qPCR技术得到的氯和一氯胺消毒对大肠杆菌的灭活曲线符合一级动力学方程，灭活速率常数分别为 2.24 L·(mg·min)^-1和0.0175 L·(mg·min)^-1，低于平板培养法得到的灭活速率常数；当大肠杆菌的去除率达到99%时，采用PMA-qPCR技术检测需要的ct值相比于平板培养法从0.6 mg·L^-1·min上升到0.9 mg·L^-1·min(氯消毒)和从20 mg·L^-1·min上升到超过100 mg·L^-1·min(一氯胺消毒)；随着ct值的升高，常规qPCR的检测结果基本不变，因此常规qPCR不能够反映氯和一氯胺消毒对病原菌的灭活效果.作为一种新的表征消毒特性的检测技术，PMA-qPCR技术有助于更为准确地评价氯和一氯胺消毒对病原菌的灭活效果.

Evaluation of Pathogen Disinfection Efficacy by Chlorine and Monochloramine Disinfection Based on Quantitative PCR Combined with Propidium Monoazide (PMA-qPCR)

A novel detection method of quantitative PCR combined with a DNA intercalating dye propidium monoazide (PMA-qPCR) was developed and then applied to analyze inactivation efficacy of chlorine and monochloramine on E.coli as a representative organism. The results shows that PMA removed 99.94% and 99.99% DNA from non-viable E.coli and Salmonella cells respectively and PMA-qPCR could effectively differentiate viable bacteria from non-viable bacteria; According to the first-order kinetic model, the inactivation coefficients on E.coli obtained by PMA-qPCR were 2.24 L·(mg·min)^-1 and 0.0175 L·(mg·min)^-1 for chlorine and monochloramine respectively, both of which were lower than those obtained by traditional plating counting method. In order to inactivate 99% of E.coli, the ct values by PMA-qPCR were 0.9 mg·L^-1·min and more than 100 mg·L^-1·min for chlorine and monochloramine while those by plating counting method were only 0.6 mg·L^-1·min and 20 mg·L^-1·min, respectively; E. coliconcentration detected by conventional qPCR kept almost the same when ct value increased, indicating that conventional qPCR was unable to evaluate inactivation efficacy of both chlorine and monochloramine disinfection. In summary, PMA-qPCR shows to be a promising method for evaluating disinfection efficacy by chlorine and monochloramine more accurately.