Rapid and high sensitivity test for direct detection of bovine herpesvirus-1 genome in clinical samples.
A procedure for direct detection of the BHV-1 genome in clinical samples, including semen, was developed. The method is based on the PCR amplification of a highly conserved DNA fragment within the glycoprotein gI sequence of the virus (323 bp between nt 1491 to nt 1814). The method is rapid and highly specific for all 27 subtypes assayed, which are included in the clinical and genetically different groups of BHV-1. The viral origin of the PCR product was assessed by Southern hybridization, with an internal probe. The method for DNA isolation from clinical samples included a fast extraction procedure with Chelex 100 resin allowing the loading of larger amounts of DNA in the PCR and in turn increasing the sensitivity of the method of detection. The level of sensitivity achieved by PCR was in the range of 1 TCID(50). This PCR assay may be an useful tool for BHV-1 monitoring in semen banks at low cost.
Santurde
G
G
Departamento de Patologia Animal L Universidad Complutense de Madrid. Facultad de Veterinaria, Madrid, Spain.
Da Silva
N
N
Villares
R
R
Tabares
E
E
Solana
A
A
Bautista
J M
JM
Castro
J M
JM
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002418:Cattle Diseases / Q000821:virology*; D002460:Cell Line; D017124:Conserved Sequence; D004279:DNA, Viral / Q000032:analysis; D016679:Genome, Viral; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D007242:Herpesvirus 1, Bovine / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000235:genetics
D004279:DNA, Viral; D014764:Viral Proteins; C046998:bovine herpesvirus type-1 glycoproteins