title: Allele-specific qRT-PCR demonstrates superior detection of single nucleotide polymorphisms as genetic markers for West Nile virus compared to Luminex® and quantitative sequencing.
abstract: To enable in vivo and in vitro competitive fitness comparisons among West Nile viruses (WNV), three reference viruses were marked genetically by site-directed mutagenesis with five synonymous nucleotide substitutions in the envelope gene region of the genome. Phenotypic neutrality of the mutants was assessed experimentally by competitive replication in cell culture and genetic stability of the substituted nucleotides was confirmed by direct sequencing. Luminex(®) technology, quantitative sequencing and quantitative RT-PCR (qRT-PCR) were compared in regard to specificity, sensitivity and accuracy for quantitation of wildtype and genetically marked viruses in mixed samples based on RNA obtained from samples of known viral titers. Although Luminex(®) technology and quantitative sequencing provided semi-quantitative or qualitative measurements, a sequence-specific primer extension approach using a specific reverse primer set in singleplex qRT-PCR demonstrated the best quantitation and specificity in the detection of RNA from wildtype and mutant viruses.
authors.affiliation: Center for Vectorborne Diseases, Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Worwa
authors.forename: Christy C
authors.initials: CC
authors.lastname: Andrade
authors.forename: Tara C
authors.initials: TC
authors.lastname: Thiemann
authors.forename: Bborie
authors.initials: B
authors.lastname: Park
authors.forename: Payal D
authors.initials: PD
authors.lastname: Maharaj
authors.forename: Michael
authors.initials: M
authors.lastname: Anishchenko
authors.forename: Aaron C
authors.initials: AC
authors.lastname: Brault
authors.forename: William K
authors.initials: WK
authors.lastname: Reisen
mesh_terms: D000483:Alleles; D000818:Animals; D005819:Genetic Markers; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017421:Sequence Analysis / Q000379:methods; D014773:Virology / Q000379:methods*; D014902:West Nile virus / Q000235:genetics*
chemical_list: D005819:Genetic Markers
keywords: Fitness competition; Genetic marker; Luminex(®) technology; Quantitative sequencing; West Nile virus; qRT-PCR