Development of a rapid and sensitive polymerase chain reaction assay for detection of bovine herpesvirus type 1 in bovine semen.
We developed a polymerase chain reaction (PCR) assay to detect bovine herpesvirus type 1 (BHV-1) in bovine semen. Since bovine semen contains components that inhibit PCR amplification, a protocol was developed to purify BHV-1 DNA from bovine semen. To identify failures of PCR amplification, we used an internal control template that was coamplified by the same PCR primers. When separated fractions of BHV-1-contaminated semen were analyzed by the PCR, we found that more than 90% of the BHV-1 DNA was present in a pooled fraction consisting of seminal fluid, nonsperm cells, and virus adsorbed to spermatozoa. By using this fraction, three to five molecules of BHV-1 DNA in 50 microliters of bovine semen could be detected. A pilot study to compare this PCR assay with the routinely used virus isolation method showed that this PCR assay is 2- to 100-fold more sensitive. In addition, the results of the PCR assay are available in 1 day, whereas the virus isolation method takes 7 days. Therefore, the PCR assay may be a good alternative to the virus isolation method.
van Engelenburg
F A
FA
Department of Virology, Central Veterinary Institute, Lelystad, The Netherlands.
Maes
R K
RK
van Oirschot
J T
JT
Rijsewijk
F A
FA
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005069:Evaluation Studies as Topic; D005814:Genes, Viral; D007242:Herpesvirus 1, Bovine / Q000235:genetics* / Q000302:isolation & purification*; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D011786:Quality Control; D012015:Reference Standards; D012091:Repetitive Sequences, Nucleic Acid; D012661:Semen / Q000382:microbiology*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014759:Viral Envelope Proteins / Q000235:genetics
D017931:DNA Primers; D004279:DNA, Viral; D014759:Viral Envelope Proteins; C038535:glycoprotein gC, herpes simplex virus type 1