Allele-specific qRT-PCR demonstrates superior detection of single nucleotide polymorphisms as genetic markers for West Nile virus compared to Luminex® and quantitative sequencing.

•Development of a fitness-neutral genetic marker in the E gene of West Nile virus.•Comparison of methods used to quantitatively detect marked strain and wildtype virus.•Allele-specific qRT-PCR using unique reverse primers quantified both strains.•qRT-PCR was superior to Luminex® and quantitative sequencing due to detection range.•qRT-PCR is now used in fitness competitions in cell culture, birds and mosquitoes.