Abstract:

The aim of this essay was to establish a specific and sensitive novel detection method for porcine rotavirus A (PoRVA) using droplet digital PCR (ddPCR). This study designed specific primers and probes for PoRVA based on the conserved sequences of the VP6 gene from GenBank, and successfully constructed a ddPCR detection method for PoRVA by optimizing the reaction conditions and determined the specificity, sensitivity, repeatability, and made some clinical sample testing. The optimization of the reaction conditions showed that the optimal concentrations of upstream and downstream primers and the corresponding probe were 600 nmol·L^-1and 300 nmol·L^-1, respectively, with the highest copy number of virus-positive droplets and the best effect at an annealing temperature of 56 ℃ The specificity results show that the self-established detection method can only detect PoRVA, while the nucleic acid detection results for seven control viruses such as porcine deltacorona virus and porcine epidemic diarrhea virus are all negative. Sensitivity results showed that the detection limit of this method was 3.97 copies·μL^-1, which is 1 000 times and 10 times higher than that of conventional PCR (3 970 copies·μL^-1) and quantitative fluorescent PCR (39.7 copies·μL^-1), respectively. Repeatability results showed that the intra- and inter-group coefficients of variation were less than 10%. Testing of 142 clinical samples showed that the positive detection rate by ddPCR was higher than that of qPCR and PCR. The results indicate that the novel ddPCR detection method for PoRV established in this study has strong specificity, high sensitivity, and good repeatability. It is suitable for early infection diagnosis and epidemiological investigation of PoRV, and can provide a reliable means for continuous monitoring of porcine rotavirus epidemics in the future.

Key words: porcine rotavirus A, droplet digital PCR, detection method