Sensitive and specific detection of proviral bovine leukemia virus by 5' Taq nuclease PCR using a 3' minor groove binder fluorogenic probe.
Sensitive assays are required to detect proviral bovine leukemia virus (BLV) in donor cattle used for the in vivo preparation of Australian tick fever vaccines. 5' Taq nuclease assays using 3' minor groove binder DNA probes (TaqManMGB) were developed and compared to conventional PCR assays for sensitive detection of Australian BLV. Seven beef and dairy herds were screened using DNA prepared by a variety of protocols to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log(10) dilutions of plasmids with inserted BLV sequences. Animals were also screened by the BLV standard agar-gel immunodiffusion test (AGID) and commercial enzyme linked immunosorbent assays (ELISA) for antibodies, and an ELISA for detecting viral antigens expressed (VAE) in lymphocyte cultures. The TaqMan MGB assay based on the pol region was the most sensitive and specific for the detection of BLV. This is the first report of a sensitive BLV 5' Taq nuclease assay.
Lew
Ala E
AE
Agency for Food and Fibre Sciences, Queensland Department of Primary Industries, c/o Locked Mail Bag No. 4, 4105, QLD, Moorooka, Australia. ala.lew@dpi.qld.gov.au
Bock
Russell E
RE
Molloy
John B
JB
Minchin
Catherine M
CM
Robinson
Susan J
SJ
Steer
Penelope
P
D042322:Anaplasma marginale / Q000276:immunology; D000818:Animals; D001315:Australia; D001403:Babesia / Q000276:immunology; D016793:Babesia bovis / Q000276:immunology; D001428:Bacterial Vaccines / Q000302:isolation & purification; D001483:Base Sequence; D002417:Cattle; D004279:DNA, Viral / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific; D004340:Drug Contamination; D005456:Fluorescent Dyes; D001909:Leukemia Virus, Bovine / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D016052:Protozoan Vaccines / Q000302:isolation & purification; D011533:Proviruses / Q000235:genetics* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods* / Q000706:statistics & numerical data
D001428:Bacterial Vaccines; D004279:DNA, Viral; D005456:Fluorescent Dyes; D016052:Protozoan Vaccines; D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases