Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantitation of Japanese encephalitis virus
Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantitation of Japanese encephalitis virus
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DOI：
10.1016/j.jviromet.2007.02.011
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发表时间：
2007-07-01
影响因子：
3.1
通讯作者：
Rao, P. V. Lakshmana
中科院分区：
文献类型：
--
作者：
Santhosh, S. R.;Parida, M. M.;Rao, P. V. Lakshmana
以NS3基因为靶点，建立了一种基于SYBR Green i的急性期乙型脑炎病毒（JEV）快速检测和定量的实时RT-PCR方法。与传统的RT-PCR相比，本研究中开发的测定方法更为敏感。通过熔化曲线分析和与黄病毒相关成员的交叉反应性研究，确定了该检测系统的特异性。利用2005年印度Gorakhpur流行的32例疑似急性期脑脊液样本验证了实时荧光定量PCR法在临床诊断中的适用性。与传统RT-PCR相比，实时RT-PCR的灵敏度提高体现在多提取10个模板拷贝数低的样本上。通过绘制周期阈值（C-t）值与RNA模板拷贝数的关系获得标准曲线，对急性期脑脊液样本中的病毒载量进行定量。这是应用实时RT-PCR检测和定量患者脑脊液样本中乙脑病毒的首次报道。这些发现表明，所报道的检测方法具有潜在的临床应用潜力，可作为快速实时检测和定量急性期临床样本中乙脑病毒的灵敏诊断试验。(C) 2007 Elsevier B.V.版权所有
One-step SYBR Green I-based real-time RT-PCR assay for rapid detection as well as quantitation of Japanese encephalitis virus (JEV) in acute-phase patient CSF samples by targeting the NS3 gene was developed. The assay developed in this study was found to be more sensitive as compared to conventional RT-PCR. The specificity of the reported assay system was established through melting curve analysis as well as by cross-reactivity studies with related members of Flavivirus. The applicability of Real-time PCR assay for clinical diagnosis was validated with 32 suspected acute-phase CSF samples of Gorakhpur epidemic, India, 2005. The improved sensitivity of real-time RT-PCR was reflected by picking up 10 additional samples with low copy number of template in comparison to conventional RT-PCR. The quantitation of the viral load in acute-phase CSF samples was done using a standard curve obtained by plotting cycle threshold (C-t) values versus copy numbers of the RNA template. This is the first report on the application of real-time RT-PCR for detection as well as quantitation of JEV from patient CSF samples. These findings demonstrate the potential clinical application of the reported assay as a sensitive diagnostic test for rapid and real-time detection and quantitation of JEV in acute-phase clinical samples. (C) 2007 Elsevier B.V. All rights reserved.