Development of a double-nest droplet digital PCR technology for detection of BVDV and BTV

Bovine viral diarrhea virus (BVDV) and bluetongue virus (BTV) are important pathogens that affect ruminants, they have an overlapping host range, and mixed infections are easily caused in animals under mixed breeding or grazing together. It has always been a hot issue of concern in the field of livestock veterinary, causing significant economic losses to the global animal husbandry industry. Thus, it is crucial to establish a highly sensitive and specific detection method that can detect BVDV and BTV synchronously. In this study, a double-nest droplet digital PCR(ddPCR) detection method was established basing on the conserved regions of NS3 gene of BVDV and VP7 gene of BTV by optimizing the reaction system and droplet generation conditions. The double-nest ddPCR results revealed that this method exhibited highly sensitive with the lowest detection limits of BVDV and BTV were 4.8 × 10 − 3 copies/µL and 4.7 × 10 − 2 copies/µL, respectively. Meanwhile, the method had stable repeatability for samples with low-concentrations of for BVDV and BTV with a coefficient of variation of less than 20% both within and between batches. Additionally, the method was high specificity and did not have cross-reaction with other important ruminant pathogens.32 samples of ram semen were tested, the results showed that the both positive rate of BVDV and BTV was 6.25%(2/32). The double-nest ddPCR method is highly sensitive and specific, allowing for the precise quantitative detection of both viruses synchronously. It can provide reliable technical support for clinical diagnosis and epidemiological investigation.