TaqMan 实时 RT-PCR 检测法用于检测和区分日本脑炎病毒。
TaqMan Real-time RT-PCR Assay for Detecting and Differentiating Japanese Encephalitis Virus.
机构信息
Department of Viral Encephalitis, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; State Key Laboratory of Infectious Disease Prevention and Control, Beijing 102206, China.
State key laboratory for genetic engineering and molecular virology, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
出版信息
Biomed Environ Sci. 2018 Mar;31(3):208-214. doi: 10.3967/bes2018.026.
OBJECTIVE
To detect Japanese encephalitis virus (JEV) rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction (RT-PCR) detection system was developed.
METHODS
By aligning the full-length sequences of JEV (G1-G5), six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay.
RESULTS
With the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/µL. The coefficients of variation of this real-time RT-PCR were all < 2.8%. The amplification efficiency of this method was between 90% and 103%.
CONCLUSION
A TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.
目的
为了快速检测日本脑炎病毒（JEV）并区分其基因型，开发了一种基于 TaqMan 的逆转录定量聚合酶链反应（RT-PCR）检测系统。
方法
通过对齐 JEV（G1-G5）的全长序列，基于 JEV 的高度保守的 NS1、NS2 和 M 基因设计了六组高度特异性的 TaqMan 实时 RT-PCR 引物和探针，其中包括一组用于非特异性 JEV 检测和五组用于检测特定 JEV 基因型。使用 20 批蚊子样本评估我们的定量 PCR 检测方法。
结果
使用特异性检测方法，未检测到其他黄病毒。该系统的检测下限为 JEV 滴度的 1 pfu/mL 和 100 RNA 拷贝/µL。该实时 RT-PCR 的变异系数均<2.8%。该方法的扩增效率在 90%至 103%之间。
结论
成功建立了 TaqMan 实时 RT-PCR 检测系统，可用于检测和区分所有五种 JEV 基因型。
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