LAUSR

ABSTRACT Aims To evaluate the fitness of three PCR assays for the detection of Mycoplasma bovis in dilute (extended) bovine semen, and a reverse transcriptase-PCR (RT-PCR) adaptation as a proxy for viability. Materials and methods Four commercial kit-based methods for nucleic acid extraction were compared to test for the presence of PCR inhibitors in nucleic acid extracted from undiluted and diluted semen. Then, analytical sensitivity, analytical specificity, and diagnostic specificity of two real-time PCR and one conventional PCR were evaluated for the detection of M. bovis DNA in semen and compared against microbial culture. Furthermore, an RT-PCR was adapted to detect RNA only and tested on viable and non-viable M. bovis to establish its ability to discriminate between the two. Results No significant PCR inhibition was detected from the dilute semen. All DNA extraction methods except one were equivalent, regardless of semen dilution. The analytical sensitivity of the real-time PCR assays was estimated as 45.6 cfu per 200 µL semen straw (2.2 × 102 cfu/mL). The conventional PCR was 10 times less sensitive. No cross-reactivity was observed for the real-time PCR for any of the bacteria tested and the diagnostic specificity was estimated as 100 (95% CI = 94.04–100) %. The RT-PCR was poor in distinguishing between viable and non-viable M. bovis. The mean quantification cycle (Cq) values for RNA extracted from different treatments to kill M. bovis remained unchanged 0–48 hours after inactivation. Conclusion and clinical relevance The real-time PCR were fit for the purpose of screening dilute semen for the detection of M. bovis to prevent incursion via importation of infected semen. The real-time PCR assays can be used interchangeably. The RT-PCR could not reliably indicate the viability of M. bovis. Based on the results from this study, a protocol and guidelines have been produced for laboratories elsewhere that wish to test bovine semen for M. bovis.