TaqMan Real-time RT-PCR Assay for Detecting and Differentiating Japanese Encephalitis Virus.

Nan Shao; Fan LI; Kai Nie; Shi Hong FU; Wei Jia Zhang; Ying HE; Wen Wen Lei; Qian Ying Wang; Guo Dong Liang; Yu Xi Cao; Huan Yu Wang

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TaqMan Real-time RT-PCR Assay for Detecting and Differentiating Japanese Encephalitis Virus.

Author: Nan SHAO ^{1
,

2} ; Fan LI ^{1
,

2} ; Kai NIE ³ ; Shi Hong FU ^{1
,

2} ; Wei Jia ZHANG ^{1
,

4
,

5} ; Ying HE ^{1
,

2} ; Wen Wen LEI ^{1
,

2} ; Qian Ying WANG ^{1
,

2} ; Guo Dong LIANG ^{1
,

2} ; Yu Xi CAO ⁶ ; Huan Yu WANG ^{1
,

2}
Author Information

1. Department of Viral Encephalitis, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
2. State Key Laboratory of Infectious Disease Prevention and Control, Beijing 102206, China.
3. 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.
4. State Key Laboratory of Infectious Disease Prevention and Control, Beijing 102206, China
5. School of Public Health, Shandong University, Jinan 250012, Shandong, China.
6. Office of laboratory management, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
Publication Type:Journal Article
Keywords: Genotype; Japanese encephalitis virus; TaqMan real-time RT-PCR
MeSH: Animals; Culicidae; virology; Encephalitis Virus, Japanese; genetics; isolation & purification; Polymerase Chain Reaction; methods; Reproducibility of Results; Sensitivity and Specificity
From: Biomedical and Environmental Sciences 2018;31(3):208-214
CountryChina
Language:English
Abstract:
OBJECTIVETo 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.
METHODSBy 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.
RESULTSWith 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%.
CONCLUSIONA TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.