Sensitive and specific detection of strains of Japanese encephalitis virus using a one-step TaqMan RT-PCR technique
| Autor: | Huan Wun Liu, Chang-Shen Lin, Yu Ming Wang, Jau Ling Huang, Ming Hui Weng, Hui Tsu Lin, Da Der Ji, Ming Der Kuo |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
NS3
encephalitis viruses Biology Sensitivity and Specificity Article chemistry.chemical_compound flavivirus Virology TaqMan medicine Taq Polymerase Encephalitis Japanese Plaque-forming unit Encephalitis Virus Japanese Reverse Transcriptase Polymerase Chain Reaction Japanese encephalitis medicine.disease biology.organism_classification Molecular biology Reverse transcriptase Reverse transcription polymerase chain reaction Flavivirus Infectious Diseases Real-time polymerase chain reaction chemistry TaqMan assay JEV RNA Viral real‐time PCR Taq polymerase Research Article |
| Zdroj: | Journal of Medical Virology |
| ISSN: | 1096-9071 0146-6615 |
| DOI: | 10.1002/jmv.20218 |
| Popis: | 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‐rTtH 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. |
| Databáze: | OpenAIRE |
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