Long PCR Amplification of Varicella-zoster Virus DNA in Clinical Specimens from the Patients with Varicella and Herpes Zoster

Abstract

Long PCR amplification of the 7.7 to 33.5Kbp regions of varicella-zoster virus (VZV) genomic DNA was performed using template DNAs extracted from clinical specimens such as vesicle fluid and crusts which had been obtained from varicella or herpes zoster patients. PCR products of 7.7-14.4Kbp in length were efficiently amplified from all of the 14 template DNAs of crust specimens. Targets of 18.6-20.0Kbp DNA could be also amplified from 14 crust samples except one. From all of the 7 samples derived from infected cells, the DNA targets up to 27.2 Kbp in length could be amplified. Whereas, the efficiency of amplification of 27.2Kbp DNAs from crust samples was somewhat lower (9/14, 64%) than that of DNAs from infected cells. In 83% (5/6) of target DNAs from infected cells, amplification of DNA as long as 33.5Kbp was possible, while only in 40% (2/5) of these from crust specimens. From crust samples, the efficiency of amplification of DNA longer than 20 Kbp tended to decline. We also confirmed that long target DNA was amplifiable directly from vesicle fluid specimens as effective as from crust specimens. Restriction fragment length polymorphism (RFLP) analyses combined with R2-nested PCR of the long PCR products allowed classification of the 14 clinical specimens into 9 groups.
Long PCR derived from clinical specimens was demonstrated to be applicable to RFLP analyses and sequencing without laborious test of virus isolation. Furthermore, the long PCR method described here will be useful for studies of the molecular epidemiology of VZV and for investigating variations among VZV isolates.