title: Development of a multi-target TaqMan assay to detect eastern equine encephalitis virus variants in mosquitoes.
abstract: Disease outbreaks caused by eastern equine encephalitis virus (EEEV; Togaviridae, Alphavirus) may be prevented by implementing effective surveillance and intervention strategies directed against the mosquito vector. Methods for EEEV detection in mosquitoes include a real-time reverse transcriptase PCR technique (TaqMan assay), but we report its failure to detect variants isolated in Connecticut in 2011, due to a single base-pair mismatch in the probe-binding site. To improve the molecular detection of EEEV, we developed a multi-target TaqMan assay by adding a second primer/probe set to provide redundant targets for EEEV detection. The multi-target TaqMan assay had similar performance characteristics to the conventional assay, but also detected newly-evolving strains of EEEV. The approach described here increases the reliability of the TaqMan assay by creating back-up targets for virus detection without sacrificing sensitivity or specificity.
authors: Center for Vector Biology and Zoonotic Diseases, The Connecticut Agricultural Experiment Station, New Haven, Connecticut 06504, USA. Philip.Armstrong@ct.gov
authors: Philip M
authors: PM
authors: Armstrong
authors: Nicholanna
authors: N
authors: Prince
authors: Theodore G
authors: TG
authors: Andreadis
mesh_terms: D000818:Animals; D009033:Culicidae / Q000821:virology*; D017931:DNA Primers / Q000235:genetics*; D004663:Encephalitis Virus, Eastern Equine / Q000235:genetics / Q000302:isolation & purification*; D004683:Encephalomyelitis, Equine / Q000175:diagnosis* / Q000635:transmission / Q000821:virology; D006801:Humans; D007303:Insect Vectors / Q000821:virology*; D062486:Public Health Surveillance; D012367:RNA, Viral / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D015047:Zoonoses
chemical_list: D017931:DNA Primers; D012367:RNA, Viral