Development and application of reverse transcriptase droplet digital PCR technology for sensitive detection of BVDV-1 and BTV in bovine semen.

BACKGROUND

Bovine viral diarrhoea virus genotype 1 (BVDV-1) and bluetongue virus (BTV) are potent viral pathogens that may be transmitted through semen, resulting in the spread of diseases via artificial insemination. Thus, establishing an early detection method for BVDV-1 and BTV infection is important for the trading of semen. In this study, we developed two RT‒ddPCR methods to detect BVDV-1 and BTV, and each method was evaluated for repeatability, limit of detection and specificity. The sensitivity of these methods was compared with that of RT‒qPCR (WOAH) by analysing clinical samples.

RESULTS

The RT‒ddPCR results revealed that both methods exhibited good repeatability at low concentrations, with detection limits of 1.05 copies/µL and 0.662 copies/µL per reaction for BVDV-1 and BTV, respectively; additionally, both methods exhibited high specificity and did not exhibit cross-reaction with other important semen-transmitted pathogens. Eighty bovine semen samples and twenty mixed semen samples were tested. The results revealed that the positivity rates of BVDV-1 and BTV RT‒ddPCR (25% and 23%, respectively) were greater than those of RT‒qPCR (19% and 18%, respectively).

CONCLUSIONS

RT‒ddPCR was highly sensitive for detecting low concentrations of BVDV-1 and BTV in clinical samples and could be a good supplement for qPCR testing.