Comparison ofBiotinylated DNA andRNA Probes forRapid Detection ofVaricella-Zoster VirusGenomeby InSituHybridization

We describe a general methodfortheproduction ofnonisotopic DNA andRNA probes forthedetection of thevaricella-zoster virus (VZV)genome byinsitu hybridization. VZVDNA was extracted frompurified viral nucleocapsids, cleaved withrestriction enzyme (RE)BamHI,andcloned into plasmid pBR322bythestandard vector insert procedure. We cloned over85%oftheVZV genome andobtained 18recombinants. Plasmids containing theB,F,G,H,andJfragments ofVZV DNA were labeled bythenicktranslation methodwith biotin-11-dUTP asthedTTPanalog. Additionally, theBfragment was cleaved withREAvaI,subcloned into theplasmid pGEM-4transcription vector, andsubsequently linearized withREsPstIandEcoRI.RNA was transcribed withT7orSP6polymerase, with asubstitution ofallylamine-UTP astheUTPanalog, andlabeled withr-caproylamidobiotin-N-hydroxysuccinimide ester. TheDNA andRNA probes were usedunderfullstringency conditions forinsitu hybridization withalkaline phosphatase asthedetector and5-bromo-4-chloro3-indolyl phosphate-Nitro BlueTetrazolium asthesubstrate. Whentested undercomparable conditions, the RNA probe was slightly more sensitive thanwas theDNA probe; bothprobes showedhomology onlywith VZV-infected cells andclinical tissues andnotwiththeother herpesviruses. Probes prepared fromvariable regions ofthegenome (fragments FandJ)performed aswell asdidthose fromconserved regions (fragments B,G,andH).Biotinylated probes havedistinct advantages overisotopic probes andretain their full potency formore than2yearswhenstored properly.