Abstract
The aim of this study was to improve molecular methods for the detection of bovine viral diarrhoea virus (BVDV). A single-tube nested reverse-transcriptase polymerase chain reaction (nRT-PCR) employing the 5′–3′-exonuclease assay (TaqMan ®) system was optimised for use with bulk milk, semen and whole blood samples. An artificial template (mimic) was engineered to provide in-tube validation of negative samples by demonstrating the absence of substances inhibitory to RT or PCR. This mimic was constructed by disrupting the BVDV amplicon at the TaqMan ® probe site by inserting a 295 bp fragment of human genomic DNA. The mimic amplicon was discriminated from the BVDV RT-PCR products using a second TaqMan ® probe, with a different fluorochrome specific for the inserted DNA. This new method was more sensitive than BVDV antigen ELISA methods and the existing RT-PCR method used in the laboratory for detection of BVDV in bulk milk. Furthermore, RNA extracted by robotic methods has proved suitable for use in this assay. This TaqMan ® nRT-PCR will be a valuable method for the detection of BVDV in a variety of biological matrices including milk and semen.
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