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. 2000 Nov;38(11):4066-71.
doi: 10.1128/JCM.38.11.4066-4071.2000.

Rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay

R S Lanciotti  1 , A J Kerst, R S Nasci, M S Godsey, C J Mitchell, H M Savage, N Komar, N A Panella, B C Allen, K E Volpe, B S Davis, J T Roehrig
Affiliations

Rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay

R S Lanciotti et al. J Clin Microbiol. 2000 Nov.

Abstract

The authors report on the development and application of a rapid TaqMan assay for the detection of West Nile (WN) virus in a variety of human clinical specimens and field-collected specimens. Oligonucleotide primers and FAM- and TAMRA-labeled WN virus-specific probes were designed by using the nucleotide sequence of the New York 1999 WN virus isolate. The TaqMan assay was compared to a traditional reverse transcriptase (RT)-PCR assay and to virus isolation in Vero cells with a large number ( approximately 500) of specimens obtained from humans (serum, cerebrospinal fluid, and brain tissue), field-collected mosquitoes, and avian tissue samples. The TaqMan assay was specific for WN virus and demonstrated a greater sensitivity than the traditional RT-PCR method and correctly identified WN virus in 100% of the culture-positive mosquito pools and 98% of the culture-positive avian tissue samples. The assay should be of utility in the diagnostic laboratory to complement existing human diagnostic testing and as a tool to conduct WN virus surveillance in the United States.

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Figures

FIG. 1
FIG. 1
Sensitivity comparison of the TaqMan and RT-PCR assays for WN virus. The amplification plot was obtained from TaqMan assay testing of previously titrated WN virus dilutions (10,000 to 0.1 PFU; tests with 1 and 0.1 PFU were performed in duplicate) with the 3′NC primer-probe set. The inset in the upper portion depicts agarose gel electrophoresis of the RT-PCR products obtained from the same dilution series of WN virus with the 233-640c primer pair. ΔRn, change above threshold fluorescence.
FIG. 2
FIG. 2
Estimated quantity of WN virus detected in CSF samples from patients serologically confirmed to have WN virus encephalitis. The viral titers were calculated by generating a standard curve (correlation coefficient, 0.996) with a previously titrated WN virus seed and using the PE 7700 Sequence Detection System software. The results for day 3 represent data from two different specimens, and the results for day 17 represent data for three specimens.
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