Scientifically sound methods to rapidly measure fecal indicator bacteria are important to ensure safe water for drinking and recreational purposes.A total of 200 water samples obtained from the Three Gorges Reservoir ...Scientifically sound methods to rapidly measure fecal indicator bacteria are important to ensure safe water for drinking and recreational purposes.A total of 200 water samples obtained from the Three Gorges Reservoir during three successive one-year study periods（October 2009 to September 2012） were analyzed using multiple-tube fermentation（MTF）and most probable numbers combined with polymerase chain reaction（MPN-PCR）.The MPN-PCR method was found to be significantly more sensitive than the MTF method for detecting Escherichia coli and Enterococcus spp.,and of equal sensitivity for detecting total coliforms when all surface water samples were grouped together.The two analytical methods had a strong,significant relationship,but MPN-PCR took only 12-18 hr,compared with the 3-8 days needed using the MTF method.Bacterial concentrations varied per sampling site but were significantly lower in the mainstream of the Yangtze River than those in the backwater areas of tributaries.The water quality of 85.8% of water samples from the mainstream was suitable for use as a centralized potable water source,while the water quality of 52.5% of water samples from the backwater areas was unsuitable for recreational activities.Relationships between fecal indicator bacteria showed significant correlation（r = 0.636-0.909,p 〈 0.01,n = 200）,while a weak but significant correlation was found between fecal indicators and water turbidity,water temperature,daily inflow,and total dissolved solids（r = 0.237-0.532,p 〈 0.05,n = 200）.The study indicated that MPN-PCR is a rapid and easily performed deoxyribonucleic acid（DNA）-based method for quantitative detection of viable total coliforms,E.coli,and Enterococcus spp.in surface water.展开更多
目的:建立一种检测低水平JAK2V617F突变的微滴式数字PCR(droplet digital PCR,ddPCR)方法并探讨其在骨髓增殖性肿瘤(myeloproliferative neoplasm,MPN)中的应用价值。方法:利用位点特异性的TaqMan-MGB探针,建立用于检测基因组DNA中JAK2V...目的:建立一种检测低水平JAK2V617F突变的微滴式数字PCR(droplet digital PCR,ddPCR)方法并探讨其在骨髓增殖性肿瘤(myeloproliferative neoplasm,MPN)中的应用价值。方法:利用位点特异性的TaqMan-MGB探针,建立用于检测基因组DNA中JAK2V617F突变的ddPCR方法,应用该方法、实时定量PCR及二代测序方法检测MPN患者JAK2V617F突变,比较不同方法检测结果的一致性。结果:成功建立了一种基于特异性TaqMan-MGB探针检测JAK2V617F突变的ddPCR方法,检测灵敏度至少为0.05%。应用ddPCR方法及实时定量PCR方法对82份MPN患者及8份健康对照者的骨髓或外周血DNA样本同时进行检测,其中87份样本检测结果一致,检出的一致率为96.7%;3份经实时定量PCR方法检测为阴性而ddPCR检测为阳性的样本,ddPCR的定量结果分别为0.015%、0.002%、0.039%,均低于实时定量PCR的检出下限;对于两种方法检测结果均为阳性的52份样本,其定量数据的相关系数为0.9714(P<0.0001)。11例样本同时采用二代测序的方法进行验证,两者定量结果的相关系数为0.9839(P<0.0001)。结论:利用特异性TaqMan-MGB探针检测JAK2V617F突变的微滴式数字PCR方法可便捷、准确地检测JAK2V617F突变,该方法具有高度的灵敏度且可绝对定量,在JAK2V617F突变的筛查及动态监测中具有潜在应用价值。展开更多
基金supported by the Basic and Advanced Research Project of Chongqing CSTC (No.cstc2013jcyj A20011)the Special Foundation for Scientific and Technological Talents of Wanzhou District of Chongqing Province,China
文摘Scientifically sound methods to rapidly measure fecal indicator bacteria are important to ensure safe water for drinking and recreational purposes.A total of 200 water samples obtained from the Three Gorges Reservoir during three successive one-year study periods（October 2009 to September 2012） were analyzed using multiple-tube fermentation（MTF）and most probable numbers combined with polymerase chain reaction（MPN-PCR）.The MPN-PCR method was found to be significantly more sensitive than the MTF method for detecting Escherichia coli and Enterococcus spp.,and of equal sensitivity for detecting total coliforms when all surface water samples were grouped together.The two analytical methods had a strong,significant relationship,but MPN-PCR took only 12-18 hr,compared with the 3-8 days needed using the MTF method.Bacterial concentrations varied per sampling site but were significantly lower in the mainstream of the Yangtze River than those in the backwater areas of tributaries.The water quality of 85.8% of water samples from the mainstream was suitable for use as a centralized potable water source,while the water quality of 52.5% of water samples from the backwater areas was unsuitable for recreational activities.Relationships between fecal indicator bacteria showed significant correlation（r = 0.636-0.909,p 〈 0.01,n = 200）,while a weak but significant correlation was found between fecal indicators and water turbidity,water temperature,daily inflow,and total dissolved solids（r = 0.237-0.532,p 〈 0.05,n = 200）.The study indicated that MPN-PCR is a rapid and easily performed deoxyribonucleic acid（DNA）-based method for quantitative detection of viable total coliforms,E.coli,and Enterococcus spp.in surface water.
文摘目的:建立一种检测低水平JAK2V617F突变的微滴式数字PCR(droplet digital PCR,ddPCR)方法并探讨其在骨髓增殖性肿瘤(myeloproliferative neoplasm,MPN)中的应用价值。方法:利用位点特异性的TaqMan-MGB探针,建立用于检测基因组DNA中JAK2V617F突变的ddPCR方法,应用该方法、实时定量PCR及二代测序方法检测MPN患者JAK2V617F突变,比较不同方法检测结果的一致性。结果:成功建立了一种基于特异性TaqMan-MGB探针检测JAK2V617F突变的ddPCR方法,检测灵敏度至少为0.05%。应用ddPCR方法及实时定量PCR方法对82份MPN患者及8份健康对照者的骨髓或外周血DNA样本同时进行检测,其中87份样本检测结果一致,检出的一致率为96.7%;3份经实时定量PCR方法检测为阴性而ddPCR检测为阳性的样本,ddPCR的定量结果分别为0.015%、0.002%、0.039%,均低于实时定量PCR的检出下限;对于两种方法检测结果均为阳性的52份样本,其定量数据的相关系数为0.9714(P<0.0001)。11例样本同时采用二代测序的方法进行验证,两者定量结果的相关系数为0.9839(P<0.0001)。结论:利用特异性TaqMan-MGB探针检测JAK2V617F突变的微滴式数字PCR方法可便捷、准确地检测JAK2V617F突变,该方法具有高度的灵敏度且可绝对定量,在JAK2V617F突变的筛查及动态监测中具有潜在应用价值。
