A型牛轮状病毒(BRV-A)是引起牛腹泻的重要病原之一,对养牛业危害最大。为建立一种针对BRV-A的快速、高通量、低成本检测方法,根据BRV-A的VP6基因保守区设计特异性引物,建立了检测BRV-A的SYBR Green I RT-qPCR方法。该方法对浓度梯度为4....A型牛轮状病毒(BRV-A)是引起牛腹泻的重要病原之一,对养牛业危害最大。为建立一种针对BRV-A的快速、高通量、低成本检测方法,根据BRV-A的VP6基因保守区设计特异性引物,建立了检测BRV-A的SYBR Green I RT-qPCR方法。该方法对浓度梯度为4.41×10^(8)~4.41×10^(2) copies/μL质粒标准品的扩增Ct值与拷贝数浓度呈良好的线性关系,熔解曲线为单峰;与其他引起牛腹泻的常见病原无交叉反应,最低检测限为4.41×10^(1) copies/μL,批内、批间重复性试验Ct值变异系数均低于1%;对临床粪便样品的阳性检出率高于地方标准中的PCR方法,且检测结果符合性较好。综上,本研究建立的BRV-A SYBR Green I RT-qPCR检测方法灵敏、特异、稳定,且操作简单、成本低,为临床样品的大规模BRV-A检测及其感染的早期诊断提供了技术支撑。展开更多
Objective:To develop a rapid,cost effective RT-PCR method for the mass scale diagnosis of such diseases at the vireraia stage to find out the actual disease burden in that area.Methods:For this purpose,cases with the ...Objective:To develop a rapid,cost effective RT-PCR method for the mass scale diagnosis of such diseases at the vireraia stage to find out the actual disease burden in that area.Methods:For this purpose,cases with the history of only short febrile illness were considered.Thus 157 samples with the history of dengue/chikungunya like illness and only 58 samples with a history of acute encephalitis syndrome(AES)were selected.Results:Out of 157 samples,42 and 74 were detected as dengue and chikungunya,respectively and out of 58 AES cases only 23 could be detected as Japanese encephalitis by this RT-PCR method.Conclusions:This cost effective RT-PCR method can detect the total positive cases that remain undetected by EL1SA method.Moreover,this method is capable to detect the viral RNA from patients'sera even after the appearance of IgM antibody at one fifth costs as compared with the other commercially available kits.展开更多
文摘A型牛轮状病毒(BRV-A)是引起牛腹泻的重要病原之一,对养牛业危害最大。为建立一种针对BRV-A的快速、高通量、低成本检测方法,根据BRV-A的VP6基因保守区设计特异性引物,建立了检测BRV-A的SYBR Green I RT-qPCR方法。该方法对浓度梯度为4.41×10^(8)~4.41×10^(2) copies/μL质粒标准品的扩增Ct值与拷贝数浓度呈良好的线性关系,熔解曲线为单峰;与其他引起牛腹泻的常见病原无交叉反应,最低检测限为4.41×10^(1) copies/μL,批内、批间重复性试验Ct值变异系数均低于1%;对临床粪便样品的阳性检出率高于地方标准中的PCR方法,且检测结果符合性较好。综上,本研究建立的BRV-A SYBR Green I RT-qPCR检测方法灵敏、特异、稳定,且操作简单、成本低,为临床样品的大规模BRV-A检测及其感染的早期诊断提供了技术支撑。
基金supported by the Department of Science and Technology,Goverment of West Bengal.India[grant No.705(Sanc.)ST/P/S&T/9G-27/2007]
文摘Objective:To develop a rapid,cost effective RT-PCR method for the mass scale diagnosis of such diseases at the vireraia stage to find out the actual disease burden in that area.Methods:For this purpose,cases with the history of only short febrile illness were considered.Thus 157 samples with the history of dengue/chikungunya like illness and only 58 samples with a history of acute encephalitis syndrome(AES)were selected.Results:Out of 157 samples,42 and 74 were detected as dengue and chikungunya,respectively and out of 58 AES cases only 23 could be detected as Japanese encephalitis by this RT-PCR method.Conclusions:This cost effective RT-PCR method can detect the total positive cases that remain undetected by EL1SA method.Moreover,this method is capable to detect the viral RNA from patients'sera even after the appearance of IgM antibody at one fifth costs as compared with the other commercially available kits.
