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
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
为可视化检测海产品中的活副溶血性弧菌,该研究将叠氮溴化丙锭(propidium monoazide,PMA)染料与跨越式滚环等温扩增(saltatory rolling circle amplification,SRCA)技术相结合,以toxR为靶基因,设计并筛选引物,成功建立可视化PMA⁃SRCA检...为可视化检测海产品中的活副溶血性弧菌,该研究将叠氮溴化丙锭(propidium monoazide,PMA)染料与跨越式滚环等温扩增(saltatory rolling circle amplification,SRCA)技术相结合,以toxR为靶基因,设计并筛选引物,成功建立可视化PMA⁃SRCA检测方法,并评估所建立的方法。研究表明,12株副溶血性弧菌样品呈现阳性,29株非副溶血性弧菌样品呈现阴性,表明该方法特异性良好。该方法灵敏度为3.2×100 CFU/mL,高于可视化PMA⁃环介导等温扩增(3.2×101 CFU/mL)和可视化PMA⁃聚合酶链式反应(3.2×102 CFU/mL)的灵敏度。对76份市售海鲜样品进行检测,该方法的敏感性为100.00%,特异性为92.00%,符合率为97.37%。综上所述,可视化PMA⁃SRCA检测方法可检测活的副溶血性弧菌,具有良好的特异性、较优异的灵敏度。展开更多
【目的】将改良版叠氮溴化丙锭(improved propidium monoazide,PMAxx)与微滴式数字PCR(droplet digital PCR,ddPCR)技术相结合,建立一种针对单核细胞增生李斯特氏菌(Listeria monocytogenes,LM)活菌的检测方法。【方法】对PMAxx终浓度...【目的】将改良版叠氮溴化丙锭(improved propidium monoazide,PMAxx)与微滴式数字PCR(droplet digital PCR,ddPCR)技术相结合,建立一种针对单核细胞增生李斯特氏菌(Listeria monocytogenes,LM)活菌的检测方法。【方法】对PMAxx终浓度、暗处理时间及曝光时间进行优化,建立最佳的PMAxx-ddPCR检测体系,并用不同体积比的LM活菌验证其准确性,考察PMAxx-ddPCR检测方法的特异性、灵敏性和重复性。用不同浓度LM活菌与死菌对奶酪棒样品进行人工污染,比较传统的平板计数法和作者建立的PMAxx-ddPCR检测方法的结果;进一步对市售金针菇和火腿肠进行检测,评估PMAxx-ddPCR检测方法的实际效果。【结果】在PMAxx终浓度为10μmol/L、暗处理时间和曝光时间均为10 min时,PMAxx可显著抑制死菌DNA扩增,但对活菌DNA扩增没有显著影响。建立的PMAxx-ddPCR检测方法仅对LM产生特异性扩增,定量限为147 CFU/mL,相对标准偏差(relative standard deviation,RSD)均小于20%,重复性较好。人工污染奶酪棒试验结果表明,平板计数法和PMAxx-ddPCR检测方法对LM的定量结果差异不显著(P>0.05)。进一步分析17份金针菇和16份火腿肠样品检测结果,PMAxx-ddPCR检测法和平板计数法均从7份金针菇样品中检出LM,且2种方法定量结果没有显著差异(P>0.05)。【结论】作者建立的LM活菌的PMAxx-ddPCR检测方法可对食品中LM活菌进行特异性定量检测。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.
文摘为可视化检测海产品中的活副溶血性弧菌,该研究将叠氮溴化丙锭(propidium monoazide,PMA)染料与跨越式滚环等温扩增(saltatory rolling circle amplification,SRCA)技术相结合,以toxR为靶基因,设计并筛选引物,成功建立可视化PMA⁃SRCA检测方法,并评估所建立的方法。研究表明,12株副溶血性弧菌样品呈现阳性,29株非副溶血性弧菌样品呈现阴性,表明该方法特异性良好。该方法灵敏度为3.2×100 CFU/mL,高于可视化PMA⁃环介导等温扩增(3.2×101 CFU/mL)和可视化PMA⁃聚合酶链式反应(3.2×102 CFU/mL)的灵敏度。对76份市售海鲜样品进行检测,该方法的敏感性为100.00%,特异性为92.00%,符合率为97.37%。综上所述,可视化PMA⁃SRCA检测方法可检测活的副溶血性弧菌,具有良好的特异性、较优异的灵敏度。
文摘【目的】将改良版叠氮溴化丙锭(improved propidium monoazide,PMAxx)与微滴式数字PCR(droplet digital PCR,ddPCR)技术相结合,建立一种针对单核细胞增生李斯特氏菌(Listeria monocytogenes,LM)活菌的检测方法。【方法】对PMAxx终浓度、暗处理时间及曝光时间进行优化,建立最佳的PMAxx-ddPCR检测体系,并用不同体积比的LM活菌验证其准确性,考察PMAxx-ddPCR检测方法的特异性、灵敏性和重复性。用不同浓度LM活菌与死菌对奶酪棒样品进行人工污染,比较传统的平板计数法和作者建立的PMAxx-ddPCR检测方法的结果;进一步对市售金针菇和火腿肠进行检测,评估PMAxx-ddPCR检测方法的实际效果。【结果】在PMAxx终浓度为10μmol/L、暗处理时间和曝光时间均为10 min时,PMAxx可显著抑制死菌DNA扩增,但对活菌DNA扩增没有显著影响。建立的PMAxx-ddPCR检测方法仅对LM产生特异性扩增,定量限为147 CFU/mL,相对标准偏差(relative standard deviation,RSD)均小于20%,重复性较好。人工污染奶酪棒试验结果表明,平板计数法和PMAxx-ddPCR检测方法对LM的定量结果差异不显著(P>0.05)。进一步分析17份金针菇和16份火腿肠样品检测结果,PMAxx-ddPCR检测法和平板计数法均从7份金针菇样品中检出LM,且2种方法定量结果没有显著差异(P>0.05)。【结论】作者建立的LM活菌的PMAxx-ddPCR检测方法可对食品中LM活菌进行特异性定量检测。
