净水技术2025，Vol.44Issue(5)：206-215,10.DOI:10.15890/j.cnki.jsjs.2025.05.023
PMA-qPCR定量检测饮用水中铜绿假单胞菌的方法
Quantitative Determination of Pseudomonas aeruginosa in Drinking Water by PMA-qPCR Method
摘要
Abstract
[Objective]To prevent waterborne disease outbreaks and ensure water quality safety,it is crucial to establish a rapid and accurate method for detecting active pathogenic bacteria in water.In view of the low bacterial content of domestic drinking water,enrichment concentration and propidium monoazide-real time fluorescence quantitative polymerase chain reaction(PMA-qPCR)method that suitable for the detection of Pseudomonas aeruginosa in large-volume drinking water is established.[Methods]The enrichment and concentration method was optimized by adjusting the vortex oscillation duration and calculating the spiked recovery rate.L16(43)orthogonal array design was employed to optimize the PMA treatment conditions for effective removal of DNA from non-viable Pseudomonas aeruginosa cells,followed by experimental validation.[Results]After enriching Pseudomonas aeruginosa from 10 L of drinking water onto the membrane by membrane filtration,the bacteria on the membrane were eluted into 1 mL phosphate buffer solution(PBS)by vortex shaking(the rotational speed was 3 000 r/min,shaking for 15 min,repeated twice)and centrifugation(8 000 g,10 minutes).The method was completed within 50 minutes and the recovery rate was 80.95％±7.43％.The optimal PMA treatment conditions for the PMA-qPCR method:PMA concentration was 30 μmol/L,incubation time in the dark was 20 min,light exposure time was 20 minutes.The method was consistent with the detection result of the plate counting method,which could effectively inhibit the amplification of dead bacterial DNA.The linear relationship between the number of viable bacteria and the number of PMA-qPCR amplification cycles was good in the range of 1×102 CFU/mL to 1×106 CFU/mL(R2=0.99),and the standard curve equation(y=-3.541x+44.11)was established to quantitatively detect the number of viable bacteria.The limit of quantification of PMA-qPCR was 1× 102 CFU/mL,and the sensitivity could be improved 1× 104 fold by combining with enrichment method.[Conclusion]The assay established in this study is suitable for the quantitative analysis of Pseudomonas aeruginosa viable bacteria in 10 L large-volume drinking water,which provides an effective technical means for subsequent in-depth research.关键词
铜绿假单胞菌/叠氮溴化丙锭(PMA)/荧光定量聚合酶链式反应(PCR)/活菌计数/饮用水Key words
Pseudomonas aeruginosa/propidium monoazide(PMA)/quantitative polymerase chain reaction(PCR)/viable bacteria count/drinking water分类
建筑与水利引用本文复制引用
张瑞停,王园媛,邢方潇,张晓,张岚..PMA-qPCR定量检测饮用水中铜绿假单胞菌的方法[J].净水技术,2025,44(5):206-215,10.基金项目
国家重点研发计划(2022YFC3204702) （2022YFC3204702）
