Development of a DNA probe for identification of bovine herpesvirus 4.
A sensitive and specific DNA probe for detection and identification of bovine herpesvirus 4 (BHV-4) was developed. Cloned fragments from a library of HindIII fragments of the BHV-4 (DN-599) genome were labeled with 32P or digoxigenin and were tested for sensitivity and specificity in detecting viral DNA by dot-blot hybridization. Two probes were identified that detected 10 pg of purified viral DNA, and detected viral DNA in 0.001 micrograms of total DNA extracted from BHV-4-infected cells. Both probes labeled with 32P and 1 labeled with digoxigenin detected viral DNA in samples prepared from cells infected with 2 prototype strains (DN-599 and Movar 33/63) and 4 field isolates of BHV-4. The DNA probes did not hybridize to total DNA prepared from uninfected bovine cells or from cells infected with BHV-1, BHV-2, alcelaphine herpesvirus 1, pseudorabies virus, or equine herpesvirus 1. One probe, labeled with digoxigenin, was tested further by dot-blot hybridization with infected cell lysates that were simply treated with sodium dodecyl sulfate and proteinase K prior to application to the membrane, avoiding extensive DNA purification procedures. This simplified procedure also resulted in specific detection of field isolates of BHV-4 and prototype strains of BHV-4.
Galik
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PK
Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL 36849-5519.
van Santen
V L
VL
Stringfellow
D A
DA
Bird
R C
RC
Wright
J C
JC
Smith
P C
PC
D000818:Animals; D015139:Blotting, Southern / Q000379:methods; D002417:Cattle; D002418:Cattle Diseases; D002460:Cell Line; D003001:Cloning, Molecular; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D016679:Genome, Viral; D006564:Herpesviridae / Q000235:genetics / Q000302:isolation & purification*; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D007668:Kidney; D015183:Restriction Mapping
D015342:DNA Probes; D004279:DNA, Viral