A real-time PCR assay for the detection of varicella-zoster virus DNA and differentiation of vaccine, wild-type and control strains

Abstract

Varicella-zoster vaccine is a live attenuated virus. It is, therefore, necessary to have a test to differentiate vaccine from wild-type varicella-zoster virus (VZV) strains for the investigation of varicella or zoster-like rash illness in individuals vaccinated previously. In addition, it is necessary to have a rapid VZV assay for use in the context of smallpox bioterrorism laboratory testing. Using specific primers and hybridization probes, a rapid method to differentiate vaccine strain VZV from wild-type VZV was developed based on the presence or absence of a Pst I restriction site within open reading frame (ORF) 38. Using this ORF 38 assay in conjunction with a similar previously described ORF 62 assay allows for further differentiation of vaccine strain, wild-type and a laboratory control strain (Ellen) VZV. This is accomplished because Ellen VZV is similar to wild-type VZV with respect to the ORF 38 assay but is similar to vaccine strain VZV with respect to the ORF 62 assay. The hybridization probes for each ORF are labeled with different fluorescent tags thus allowing both assays to be run simultaneously in a single tube. Both assays demonstrate a high degree of specificity for VZV and can reliably detect between 10 and 100 copies of VZV DNA. Thus, the real-time polymerase chain reaction (PCR) assay for VZV described below provides a rapid assay allowing the simultaneous differentiation of vaccine, wild-type and laboratory control strains of VZV.