Development of a multiplex real-time PCR for the detection of BoHV-1 and BVDV in cryopreserved bovine semen
Keywords:
artificial insemination, TaqMan, biosafetyAbstract
In Mexico, artificial insemination constitutes one of the main reproductive biotechnologies applied in cattle farming, aimed at increasing herd productivity and genetic improvement. However, a large proportion of the semen used in this technique is processed in the field without strict sanitary control, which increases the risk of spreading infectious agents such as bovine viral diarrhea virus (BVDV) and bovine herpesvirus type 1 (BoHV-1). To strengthen the detection of these pathogens, a multiplex real-time PCR assay with TaqMan probes was developed to simultaneously identify both viruses in cryopreserved bovine semen. Positive controls consisted of reference viruses replicated in MDBK (Madin Darby Bovine Kidney) cells, and negative controls included supernatants of non-infected cells and semen free of both viruses. Primers and probes were designed from sequences deposited in GenBank: the E2 gene for BVDV and the gB glycoprotein for BoHV-1; additionally, the bovine ?-actin gene of Bos taurus was used as a constitutive control. The primers showed amplification in endpoint assays and satisfactory results in real-time assays with positive controls. These findings establish a multiplex detection assay that will contribute to biosafety in artificial insemination programs, reducing the risk of BVDV and BoHV-1 transmission through semen.
https://doi.org/10.21929/abanicomicrobiano/2025.6
2025-6
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