RESUMO
Sensitive assays are required to detect bovine retroviruses 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 the sensitive detection of bovine syncytial virus (BSV) and bovine immunodeficiency virus (BIV). Seven beef and dairy herds were screened to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log(10) dilutions of plasmids with inserts containing corresponding provirus sequences. Published PCR assays targeting BIV env sequences did not adequately amplify Australian BIV sequences. Pol sequences from Australian strains of BIV and BSV were used to design TaqMan MGB assays, which improved sensitivity 10-fold (BIV) and 100-fold (BSV), respectively, over conventional PCR tests. This is the first report of Australian sequences of BIV and BSV and the first 5' Taq nuclease assays described to detect these viruses. These methods could be applied to future studies requiring sensitive detection of these two bovine retroviruses.
Assuntos
Doenças dos Bovinos/diagnóstico , Vírus da Imunodeficiência Bovina/isolamento & purificação , Infecções por Lentivirus/veterinária , Reação em Cadeia da Polimerase/métodos , Infecções por Retroviridae/veterinária , Spumavirus/isolamento & purificação , Animais , Sequência de Bases , Bovinos , Doenças dos Bovinos/virologia , Sondas de DNA , Corantes Fluorescentes , Genes Virais/genética , Genes env/genética , Genes pol/genética , Infecções por Lentivirus/diagnóstico , Infecções por Lentivirus/virologia , Sondas Moleculares , Dados de Sequência Molecular , Provírus/genética , Provírus/isolamento & purificação , Infecções por Retroviridae/diagnóstico , Infecções por Retroviridae/virologia , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
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.