Sensitive and specific detection of proviral bovine leukemia virus by 5′ Taq nuclease PCR using a 3′ minor groove binder fluorogenic probe

• Published in: Journal of virological methods 2004-02, Vol.115 (2), p.167-175
• Main Authors: Lew, Ala E., Bock, Russell E., Molloy, John B., Minchin, Catherine M., Robinson, Susan J., Steer, Penelope
• Format: Article
• Language: English
• Subjects: 5′ Taq nuclease; Anaplasma marginale - immunology; Animals; Australia; Babesia - immunology; Babesia bovis - immunology; Bacterial Vaccines - isolation & purification; Base Sequence; Biological and medical sciences; Bovine leukemia virus; Cattle; Deoxyribonucleases, Type II Site-Specific; DNA, Viral - genetics; Drug Contamination; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Leukemia Virus, Bovine - genetics; Leukemia Virus, Bovine - isolation & purification; Microbiology; Minor groove binder; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - statistics & numerical data; Protozoan Vaccines - isolation & purification; Proviruses - genetics; Proviruses - isolation & purification; Sensitivity and Specificity; TaqMan; Techniques used in virology; Virology; Virology - methods; Virology - statistics & numerical data
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2003.09.029

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 (TaqMan ®MGB) 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.