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
To improve nitrogen removal performance of wastewater treatment plants （WWTPs）, it is essential to understand the behavior of nitrogen cycling communities, which comprise various microorganisms. This study character...To improve nitrogen removal performance of wastewater treatment plants （WWTPs）, it is essential to understand the behavior of nitrogen cycling communities, which comprise various microorganisms. This study characterized the quantity and diversity of nitrogen cycling genes in various processes of municipal WWTPs by employing two molecular-based methods：most probable number-polymerase chain reaction （MPN-PCR） and DNA microarray. MPN-PCR analysis revealed that gene quantities were not statistically different among processes, suggesting that conventional actwated sludge processes （CAS） are similar to nitrogen removal processes in their ability to retain an adequate population of nitrogen cycling microorganisms. Furthermore, most processes in the WWTPs that were researched shared a pattern：the nitS and the bacterial amoA genes were more abundant than the nirK and archaeal amoA genes, respectivelv. DNA microarray analysis revealed that several kinds of nitrification and denitrification genes were detected in both CAS and anaerobic-oxic processes （AO）, whereas limited genes were detected in nitrogen removal processes. Results of this study suggest that CAS maintains a diverse community of nitrogen cycling microorganisms; moreover, the microbial communities in nitrogen removal processes may be specific.展开更多
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。
文摘To improve nitrogen removal performance of wastewater treatment plants （WWTPs）, it is essential to understand the behavior of nitrogen cycling communities, which comprise various microorganisms. This study characterized the quantity and diversity of nitrogen cycling genes in various processes of municipal WWTPs by employing two molecular-based methods：most probable number-polymerase chain reaction （MPN-PCR） and DNA microarray. MPN-PCR analysis revealed that gene quantities were not statistically different among processes, suggesting that conventional actwated sludge processes （CAS） are similar to nitrogen removal processes in their ability to retain an adequate population of nitrogen cycling microorganisms. Furthermore, most processes in the WWTPs that were researched shared a pattern：the nitS and the bacterial amoA genes were more abundant than the nirK and archaeal amoA genes, respectivelv. DNA microarray analysis revealed that several kinds of nitrification and denitrification genes were detected in both CAS and anaerobic-oxic processes （AO）, whereas limited genes were detected in nitrogen removal processes. Results of this study suggest that CAS maintains a diverse community of nitrogen cycling microorganisms; moreover, the microbial communities in nitrogen removal processes may be specific.
