A sensitive and efficient detection method for bovine viral diarrhea virus (BVDV) in single preimplantation bovine embryos.

The objective was to develop a method to accurately and efficiently detect minute amounts of bovine viral diarrhea virus (BVDV) associated with a single embryo. There are two major challenges for BVDV detection in a single embryo: the test sensitivity and the efficiency of viral molecule recovery. These become even more critical when attempts are made to detect BVDV infections that occurred naturally, not through artificial exposure of the embryos to high affinity BVDV strains. We have developed a one-step sample preparation method that has increased the viral molecule recovery rate compared to the standard RNA isolation procedure by 7-100-fold. Instead of using the traditional virus exposure approach, we generated BVDV positive embryos via somatic cell nuclear transfer (SCNT) technology using BVDV positive donor cells. By combining the highly efficient sample preparation procedure with a sensitive one-step, real-time PCR system, we have developed a sensitive test that allows detection of as low as two copies of BVDV in a single embryo. This method will allow systematic risk assessment for BVDV transmission during in vitro embryo production via IVF or SCNT procedures.