Rapid genotyping of varicella-zoster virus vaccine and wild-type strains with fluorophore-labeled hybridization probes

We developed a single-tube rapid method for the detection and differentiati on of varicella-zoster virus (VZV) vaccine and wild-type strains that combi nes rapid-cycle PCR with wild-type-specific fluorescent probe melting profi les for product genotyping. A region including the polymorphic site in VZV open reading frame (ORF) 62 was amplified in the presence of two fluorescen ce-labeled hybridization probes. During the annealing step of the thermal c ycling, both probes bound to their complementary sequences in the amplicon, resulting in resonance energy transfer, thus providing real-time fluoresce nce monitoring of PCR. Continuous acquisition of fluorescence data during a melting curve analysis at the completion of PCR revealed that loss of fluo rescence occurred in a strain-specific manner as the detection probe, which was fully complementary to the wild-type VZV ORF 62 region, melted off the template. Use of this method allowed genotyping of samples within minutes after the completion of PCR, eliminating the need for post-PCR sample manip ulation. In addition to reducing the time required to produce a result, thi s method substantially reduces the risk of contamination of the final produ ct as well as the risk of sample tracking errors. The genotypes of 79 VZV-p ositive samples determined by this fluorescent resonance energy transfer (F RET) method were identical to the genotypes obtained by conventional PCR an d restriction fragment length polymorphism analysis. The genotyping of VZV strains by the FRET method is a rapid and reliable method that is suitable fbr typing and that is also practical for use for the processing of large n umbers of specimens.