title: Detection and differentiation of genotype I and III Japanese encephalitis virus in mosquitoes by multiplex reverse transcriptase-polymerase chain reaction.
abstract: Japanese encephalitis (JE) is a disease that threatens both human and animal populations in Asian countries, and the causative agent of JE, Japanese encephalitis virus (JEV), has recently changed from genotype III (GIII) to genotype I (GI). However, a test for the rapid differentiation of GI and GIII JEV is still unavailable, especially one that can be used for mosquito-based surveillance. We have designed GI- and GIII-specific primer sets for the rapid detection and differentiation of GI and GIII JEV by multiplex reverse transcriptase-polymerase chain reaction (multiplex RT-PCR). The GI-specific and GIII-specific primer sets were able to specifically amplify the target gene from GI and GIII JEV, respectively. The limitations of detection were 0.00225 and 0.225 pfu for the GI-specific and GIII-specific primers, respectively. Using a mixture of GI-specific and GIII-specific primers, the multiplex RT-PCR was able to specifically detect and differentiate GI and GIII JEV. The multiplex RT-PCR was able to successfully differentiate GI and GIII virus in JEV-infected mosquitoes. Thus, a sensitive and specific multiplex RT-PCR system for the rapid detection and differentiation of GI and GIII JEV has been developed, and this test is likely to be valuable when carrying out mosquito-based JEV surveillance.
authors.affiliation: Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Y Y
authors.initials: YY
authors.lastname: Chen
authors.forename: J W
authors.initials: JW
authors.lastname: Lin
authors.forename: Y C
authors.initials: YC
authors.lastname: Fan
authors.forename: S S
authors.initials: SS
authors.lastname: Chiou
mesh_terms: D000818:Animals; D009033:Culicidae / Q000821:virology*; D017931:DNA Primers; D004664:Encephalitis Virus, Japanese / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004672:Encephalitis, Japanese / Q000635:transmission / Q000821:virology*; D005838:Genotype; D006801:Humans; D007564:Japan; D008969:Molecular Sequence Data; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D012194:RNA-Directed DNA Polymerase