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...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.展开更多
目的建立一种基于叠氮溴化丙锭(Propidium monoazide,PMA)的定量反转录聚合酶链式反应(RT-qPCR)的方法,以快速检测甲型肝炎病毒(Hepatitis A virus,HAV)的感染性。方法将热灭活的HAV样品经PMA处理和曝光,通过RT-qPCR检测体系进行检测,优...目的建立一种基于叠氮溴化丙锭(Propidium monoazide,PMA)的定量反转录聚合酶链式反应(RT-qPCR)的方法,以快速检测甲型肝炎病毒(Hepatitis A virus,HAV)的感染性。方法将热灭活的HAV样品经PMA处理和曝光,通过RT-qPCR检测体系进行检测,优化PMA预处理浓度、孵育时间以及光解时间,建立甲肝病毒PMART-qPCR检测方法。以建立的检测方法对感染性病毒与灭活病毒混合样品、不同浓度的灭活病毒以及感染性病毒进行检测,评估其检测病毒感染性的效果。结果PMA-RT-qPCR检测方法的PMA浓度确定为50μmol/L,4℃避光孵育30min,光解30min。对热灭活HAV的RT-qPCR检测结果显示,PMA处理组与未处理组Ct值差异(ΔCt)>3.78;而对活病毒(10~2CCID_(50)/mL~10~7CCID_(50)/mL)的ΔCt值<0.5。该方法可最低分辨9 CCID_(50)的活病毒,在感染性病毒与灭活病毒混合物中检出最低27 CCID_(50)的活病毒。结论本研究建立的PMA-RT-qPCR无细胞培养方法为判断HAV的感染性提供了快速、便捷的检测方法。展开更多
Microalgae can be cultivated for producing high-valued products through the production of enzymes to offset the cost of CO_(2) sequestration,providing financial incentives.The viability of algae in the photobioreactor...Microalgae can be cultivated for producing high-valued products through the production of enzymes to offset the cost of CO_(2) sequestration,providing financial incentives.The viability of algae in the photobioreactor needs to be monitored to ensure biologically active live cells.In this study,we explored a simple fluorometry method for differentiation of live and dead algal cells in photobioreactors by fluorescein diacetate(FDA)and propidium iodide(PI)fluorescence staining.FDA stains fluorescent green to the living cells while PI stains the dead cells,allowing the discrimination of live and dead cells.The method was evaluated using two green algae and two strains of cyanobacteria grown in shake flasks and a continuously stirred photobioreactor.The method was found applicable for Chlorella pyrenoidosa and Synechococcus 7002 but was not applicable for the cultures of Scenedesmus dimorphus and Synechococcus elongatus 7942.We conclude that FDA is a good stain for monitoring live algal cells in photobioreactors but its applicability to individual species of algae must be evaluated.展开更多
基金supported by the National Natural Science Foundation of China (No. 51178242)the Tsinghua University Initiative Scientific Reserch Program (No. 20121087922)the Program of Changjiang Scholars and Innovation Research Team in University
文摘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.
基金the Natural Sciences and Engineering Research Council(NSERC)of Canada in the form of a strategic grant(STPGP 380768-09)。
文摘Microalgae can be cultivated for producing high-valued products through the production of enzymes to offset the cost of CO_(2) sequestration,providing financial incentives.The viability of algae in the photobioreactor needs to be monitored to ensure biologically active live cells.In this study,we explored a simple fluorometry method for differentiation of live and dead algal cells in photobioreactors by fluorescein diacetate(FDA)and propidium iodide(PI)fluorescence staining.FDA stains fluorescent green to the living cells while PI stains the dead cells,allowing the discrimination of live and dead cells.The method was evaluated using two green algae and two strains of cyanobacteria grown in shake flasks and a continuously stirred photobioreactor.The method was found applicable for Chlorella pyrenoidosa and Synechococcus 7002 but was not applicable for the cultures of Scenedesmus dimorphus and Synechococcus elongatus 7942.We conclude that FDA is a good stain for monitoring live algal cells in photobioreactors but its applicability to individual species of algae must be evaluated.
