Abstract: The multiple real-time RT-PCR method was used to improve the speed and sensitivity of multiple pathogen detection, which can promote rapid diagnosis and prompt treatment of calf diarrhea. Four pairs of effective primers and probes were designed,synthesized and experimentally screened targeting the conserved region of ORF2 Gene from bovine astroviruses (BAstV), 5'-UTR from bovine viral diarrhea virus 1 (BVDV-1), N pro gene from bovine coronavirus (BCV) and VP6 gene from bovine rotavirus (BRV), respectively. The concentrations of the primers, probes and the reaction parameters and conditions of the real-time RT-PCR reaction system were further optimized with four recombinant plasmids containing the target fragments of BAstV, BVDV-1, BCV and BRV respectively. The standard curves were drawn to assess the specificity, sensitivity and reproducibility of the real-time RT-PCR reaction system. The results showed that the optimum concentrations of the primers were 300, 300, 400 and 500 nmol·L^-1, and the optimum concentrations of the probes were 250, 150, 100 and 300 nmol·L^-1 for BAstV, BVDV-1, BCV and BRV respectively, and the annealing temperature and time were 50.0℃ and 45 s. The minimum detectable limits of BAstV, BVDV-1, BCV and BRV were 1 000, 100, 100 and 100 copies·μL^-1 respectively, and the quadruple real-time RT-PCR has good specificity and repeatability and the positive detection rate of clinical collection of fecal samples were higher than that of the PCR method. In conclusion, the results showed that the quadruple real-time RT-PCR method established in this paper can be used for the rapid differential diagnosis of BAstV, BVDV-1, BCV and BRV which are common causes of diarrhea in calves.

Key words: bovine astroviruses, bovine viral diarrhea virus, bovine coronavirus, bovine rotavirus, Real-time PCR