Sensitive and specific detection of strains of Japanese encephalitis virus using a one-step TaqMan RT-PCR technique. Export

@article{citeulike:672790, abstract = {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.}, address = {Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan, Republic of China. JauLingH@mail.cju.edu.tw}, author = {Huang, J. L. and Lin, H. T. and Wang, Y. M. and Weng, M. H. and Ji, D. D. and Kuo, M. D. and Liu, H. W. and Lin, C. S.}, citeulike-article-id = {672790}, citeulike-linkout-0 = {http://dx.doi.org/10.1002/jmv.20218}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/15484282}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=15484282}, doi = {10.1002/jmv.20218}, issn = {0146-6615}, journal = {J Med Virol}, keywords = {load, mosquito, viral}, month = {December}, number = {4}, pages = {589--596}, posted-at = {2006-05-28 06:58:03}, priority = {2}, title = {Sensitive and specific detection of strains of Japanese encephalitis virus using a one-step TaqMan RT-PCR technique.}, url = {http://dx.doi.org/10.1002/jmv.20218}, volume = {74}, year = {2004} }

TY - JOUR ID - citeulike:672790 L3 - citeulike-article-id:672790 N2 - 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. IS - 4 JF - J Med Virol SN - 0146-6615 AD - Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan, Republic of China. JauLingH@mail.cju.edu.tw EP - 596 TI - Sensitive and specific detection of strains of Japanese encephalitis virus using a one-step TaqMan RT-PCR technique. VL - 74 SP - 589 KW - load KW - mosquito KW - viral AU - Huang, JL AU - Lin, HT AU - Wang, YM AU - Weng, MH AU - Ji, DD AU - Kuo, MD AU - Liu, HW AU - Lin, CS PY - 2004/12// UR - http://dx.doi.org/10.1002/jmv.20218 ER -