Quantification of viable bacteria in wastewater treatment plants by using propidium monoazide combined with quantitative PCR （PMA-qPCR）

• Published in: 环境科学学报：英文版 no. 2; pp. 299 - 306
• Main Author: Dan Li Tiezheng Tong Siyu Zeng Yiwen Lin Shuxu Wu Miao He
• Format: Journal Article
• Language: English
• Published: 2014
• Subjects: PMA; 定量PCR; 氮丙啶; 污水处理厂; 特异性引物; 环境样品; 细菌细胞; 聚合酶链反应
• ISSN: 1001-0742; 1878-7320

The detection of viable bacteria in wastewater treatment plants （WWTPs） is very important for public health, as WWTPs are a medium with a high potential for waterborne disease transmission. The aim of this study was to use propidium monoazide （PMA） combined with the quantitative polymerase chain reaction （PMA-qPCR） to selectively detect and quantify viable bacteria cells in full-scale WWTPs in China. PMA was added to the concentrated WWTP samples at a final concentration of 100 μmol/L and the samples were incubated in the dark for 5 min, and then lighted for 4 min prior to DNA extraction and qPCR with specific primers for Escherichia coli and Enterococci, respectively. The results showed that PMA treatment removed more than 99% of DNA from non-viable cells in all the WWTP samples, while matrices in sludge samples markedly reduced the effectiveness of PMA treatment. Compared to qPCR, PMA-qPCR results were similar and highly linearly correlated to those obtained by culture assay, indicating that DNA from non-viable cells present in WWTP samples can be eliminated by PMA treatment, and that PMA-qPCR is a reliable method for detection of viable bacteria in environmental samples. This study demonstrated that PMA-qPCR is a rapid and selective detection method for viable bacteria in WWTP samples, and that WWTPs have an obvious function in removing both viable and non-viable bacteria. The results proved that PMA-qPCR is a promising detection method that has a high potential for application as a complementary method to the standard culture-based method in the future.