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. 2015 May;52(3):491-9.
doi: 10.1093/jme/tjv021. Epub 2015 Mar 18.

Multiplex qRT-PCR for the Detection of Western Equine Encephalomyelitis, St. Louis Encephalitis, and West Nile Viral RNA in Mosquito Pools (Diptera: Culicidae)

Aaron C Brault  1 , Ying Fang  2 , William K Reisen  3
Affiliations

Multiplex qRT-PCR for the Detection of Western Equine Encephalomyelitis, St. Louis Encephalitis, and West Nile Viral RNA in Mosquito Pools (Diptera: Culicidae)

Aaron C Brault et al. J Med Entomol. 2015 May.

Abstract

Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer-probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk.

Keywords: St. Louis encephalitis virus; West Nile virus; multiplex; surveillance; western equine encephalitis virus.

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Figures

Fig. 1.
Fig. 1.
Mosquito pools tested in California from 2000 to 2013. Shown are the annual number of pools tested and the number positive for WEEV, SLEV, and WNV viruses each year. Arrow shows the start of testing using only qRT-PCR.
Fig. 2.
Fig. 2.
Sensitivity of the primer–probe sets run in duplicate as single or multiplex. Plotted are mean qRT-PCR Ct scores as a function of virus titer assayed by Vero cell plaque titration as log10 PFU per 0.1 ml. RNA extraction by MagMax, RT-PCR by ABI7900.
Fig. 3.
Fig. 3.
Results of blind proficiency panel testing for WNV RNA by nine local agencies and CVEC (fitted line) in 2013. Shown are the RT-PCR Ct score plotted as a function of WNV titer estimated by plaque assay on Vero cell culture. Note variation in sensitivity. At CVEC, RNA was extracted by MagMax Express and RT-PCR run using a one-step fast kit on an ABI Vii7a platform.
Fig. 4.
Fig. 4.
Average processing time in days for mosquito pools after receipt at CVEC during 2006.
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