Rapid Detection of West Nile Virus from Human Clinical Specimens, Field-Collected Mosquitoes, and Avian Samples by a TaqMan Reverse Transcriptase-PCR Assay

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 (≈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.

Real-time Fluorescent PCR Techniques to Study Microbial–Host Interactions

“…We and others have found the consistently low inter-assay and intra-assay variability especially useful compared to other assays (Schutten et al, 2000;Nitsche et al, 2000;Locatelli et al, 2000). Real-time PCR also has equivalent or improved sensitivity compared to microbial culture, or conventional single-round and nested PCR (Kearns et al, 2001c;Clarke et al, 2002;Kupferschmidt et al, 2001;Kennedy et al, 1997b;Locatelli et al, 2000;Capone et al, 2001;Leutenegger et al, 1999;Smith et al, 2001;Monopoeho et al, 2000;van Elden et al, 2001;Lanciotti et al, 2000). It has been reported to be at least as sensitive as Southern blot, still considered by some as the gold standard for probe-based hybridisation assays (Capone et al, 2001).…”

Ribosomal RNA depletion or exclusion has negligible effect on the detection of viruses in a pan viral microarray

“…The virus specific real time PCR mix for all viruses except rabies virus (RV) was composed of 1× QuantiTect Virus + ROX Vial Kit (Qiagen, Manchester, UK), forward and reverse primers at a final concentration of 0.4 M and virus specific TaqMan probe at a final concentration of 0.2 M, 1× ROX, 3 l of template DNA and water to total a volume of 20 l (Mcgoldrick et al, 1998;Lanciotti et al, 2000;Marriott et al, 2006;Bilk et al, 2012) The thermal profile used was 95 • C for 5 min and 45 cycles of 95 • C for 15 s, 60 • C for 45 s. The 18S rRNA real time PCR was performed using 0.6 l 18S rRNA primers/probe mix (Life Technologies, Paisley, UK), the Quan-tiTect Virus + ROX Vial Kit as described above and 2 l template DNA. For RV, 10 l Brilliant ® II SYBR ® Green QPCR with low ROX master mix (Agilent Technologies, Cheshire, UK) was used with JW12 and N165-146 primers, each totalling a final concentration of 1 M, 3 l template DNA and water to a final volume of 20 l (Wakeley et al, 2005).…”

Advances in Real‐Time PCR: Application to Clinical Laboratory Diagnostics

“…A significant number of real-time PCR assays have been developed and applied for detection, quantification, and diVerentiation of viruses inducing a wide range of human and animal diseases such as coronavirus [139], flaviviruses [140][141][142], gyrovirus [21], hepadnaviruses [143,144], herpesviruses [145][146][147], orthomyxoviruses [148], parvoviruses [149], papovaviruses [150], paramyxoviruses [8], picornaviruses [151,152], poxviruses [153], retroviruses [154,155], and rhabdoviruses [156].…”