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Sensitive and specific detection of strains of Japanese encephalitis virus using a one‐step TaqMan RT‐PCR technique
Author(s) -
Huang JauLing,
Lin HuiTsu,
Wang YuMing,
Weng MingHui,
Ji DaDer,
Kuo MingDer,
Liu HuanWun,
Lin ChangShen
Publication year - 2004
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.20218
Subject(s) - taqman , japanese encephalitis , virology , virus , biology , real time polymerase chain reaction , reverse transcription polymerase chain reaction , polymerase chain reaction , plaque forming unit , microbiology and biotechnology , encephalitis , gene , messenger rna , genetics
A rapid, sensitive, and accurate laboratory diagnostic test is needed for distinguishing Japanese encephalitis virus (JEV) from other diseases featuring similar clinical symptoms and also for preventing potential outbreaks. In this study, a TaqMan reverse transcription (RT)‐polymerase chain reaction (PCR) assay was developed for rapid detection and quantification of the viral RNA of various JEV strains. A consensus JEV NS3 region was chosen to design the primers and the TaqMan probe. The JEV TaqMan assay used the EZ‐r TtH RT‐PCR system featuring advantages such as a one‐step, high‐temperature RT reaction modality and preventing carry‐over contamination. The sensitivity of the JEV TaqMan assay for detecting in vitro‐transcribed JEV NS3 RNA was estimated to be one to five copies of RNA per reaction. For cultured JE virions, less than 40 plaque forming unit (PFU)/ml of virus load (corresponding to 0.07 PFU/test) could be detected. In addition, the JEV TaqMan assay could detect all seven strains of JEV tested, but provided negative results for nine other flaviviruses and encephalitis viruses tested. The JEV TaqMan assay demonstrated greater sensitivity and specificity than traditional RT‐PCR methods as has been previously reported. The application of the JEV TaqMan assay herein has been shown to the sensitive detection of the JEV from both mosquito pools and also JEV‐spiking human blood. The assay should be of use in diagnostic laboratory conduct and could be used to replace or complement time‐consuming viral‐culture methods, thus achieving more rapid, sensitive, and highly specific identification of JEV infection. J. Med. Virol. 74:589–596, 2004. © 2004 Wiley‐Liss, Inc.