Comparison of biotinylated DNA and RNA probes for rapid detection of varicella-zoster virus genome by in situ hybridization.
We describe a general method for the production of nonisotopic DNA and RNA probes for the detection of the varicella-zoster virus (VZV) genome by in situ hybridization. VZV DNA was extracted from purified viral nucleocapsids, cleaved with restriction enzyme (RE) BamHI, and cloned into plasmid pBR322 by the standard vector insert procedure. We cloned over 85% of the VZV genome and obtained 18 recombinants. Plasmids containing the B, F, G, H, and J fragments of VZV DNA were labeled by the nick translation method with biotin-11-dUTP as the dTTP analog. Additionally, the B fragment was cleaved with RE AvaI, subcloned into the plasmid pGEM-4 transcription vector, and subsequently linearized with REs PstI and EcoRI. RNA was transcribed with T7 or SP6 polymerase, with a substitution of allylamine-UTP as the UTP analog, and labeled with epsilon-caproylamidobiotin-N-hydroxysuccinimide ester. The DNA and RNA probes were used under full-stringency conditions for in situ hybridization with alkaline phosphatase as the detector and 5-bromo-4-chloro-3-indolyl phosphate-Nitro Blue Tetrazolium as the substrate. When tested under comparable conditions, the RNA probe was slightly more sensitive than was the DNA probe: both probes showed homology only with VZV-infected cells and clinical tissues and not with the other herpesviruses. Probes prepared from variable regions of the genome (fragments F and J) performed as well as did those from conserved regions (fragments B. G. and H). Biotinylated probes have distinct advantages over isotopic probes and retain their full potency for more than 2 years when stored properly.
Forghani
B
B
Division of Laboratories, California State Department of Health Services, Berkeley 94704.
Yu
G J
GJ
Hurst
J W
JW
D001710:Biotin; D003001:Cloning, Molecular; D015342:DNA Probes; D004279:DNA, Viral / Q000235:genetics; D005814:Genes, Viral; D005822:Genetic Vectors; D014645:Herpesvirus 3, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D015336:Molecular Probe Techniques; D009693:Nucleic Acid Hybridization; D015347:RNA Probes
D015342:DNA Probes; D004279:DNA, Viral; D015347:RNA Probes; D001710:Biotin