Detection and genotyping of varicella-zoster virus by TaqMan allelic discrimination real-time PCR

Abstract

A proportion of individuals vaccinated with live attenuated Oka varicella-zoster virus (VZV) vaccine subsequently develop attenuated chicken pox and/or herpes zoster. To determine whether postvaccination varicella infections are caused by vaccine or wild-type virus, a simple method for distinguishing the vaccine strain from wild-type virus is required. We have developed a TaqMan real-time PCR assay to detect and differentiate wild-type virus from Oka vaccine strains of VZV. The assay utilized two fluorogenic, minor groove binding probes targeted to a single nucleotide polymorphism in open reading frame 62 that distinguishes the Oka vaccine from wild-type strains. VZV DNA could be genotyped and quantified within minutes of thermocycling completion due to real-time monitoring of PCR product formation and allelic discrimination analysis. The allelic discrimination assay was performed in parallel with two standard PCR-restriction fragment length polymorphism (RFLP) methods on 136 clinical and laboratory VZV strains from Canada, Australia, and Japan. The TaqMan assay exhibited a genotyping accuracy of 100% and, when compared to both PCR-RFLP methods, was 100 times more sensitive. In addition, the method was technically simpler and more rapid. The TaqMan assay also allows for high-throughput genotyping, making it ideal for epidemiologic study of the live attenuated varicella vaccine.

FIG. 1.

Real-time PCR plasmid standard curve for WT and V-Oka assays. Dilutions of both WT (♦) and V-Oka (▪) plasmid standards ranging from 10⁷ to 1 copy/reaction were amplified by the TaqMan method. The resulting C_T values were plotted as a function of the log plasmid copy number. Each data point is the average of the results for 5 replicate reactions performed simultaneously. The linear ranges of detection for the WT and V-Oka probes were 10² to 10⁷ and 10³ to 10⁷ plasmid copies/reaction, respectively.

FIG. 2.

Allelic discrimination plot of clinical isolates and V-Oka strain. DNA from clinical isolates (♦), V-Oka DNA (▵), WT DNA (○), and controls containing no template (□) were amplified in duplicate by the TaqMan method. Data were plotted by using the absolute fluorescence of each reporter dye probe. In the depicted run, all samples except one which failed to amplify were identified as WT virus, and the V-Oka DNA was identified as vaccine DNA.

FIG. 3.

Allelic discrimination plot of VZV DNA isolated from zoster lesions in a vaccinated individual. DNA was extracted from a vesicle scraping obtained from a vaccinated individual with HZ. Specimen DNA from two DNA extractions (♦), V-Oka DNA (▵), WT DNA controls (○), and controls containing no template (□) were amplified in duplicate by the TaqMan method. Data were plotted by using the absolute fluorescence of each reporter dye probe. The clinical VZV DNA specimen clustered with the V-Oka DNA and was identified as vaccine DNA.