Establishment and application of multiplex RT-PCR procedure for detection of three virus from swine

ABSTRACT

Based on the M gene sequence of TGEV and PEDV and VP2 gene sequence of PoRV, the optimal reaction system and amplification procedure were established by optimizing primer, probe concentration and annealing temperature, and the Quantitative PCR method of TaqMan probes for three viruses is successfully established. On this basis, after further optimization of conditions, a triple real-time fluorescent quantitative PCR method for detecting TGEV, PEDV, and PoRV was established. The detection sensitivity of this method for TGEV, PEDV, and PoRV were 2.49 copies/ L, 4.36 copies/ L, and 4.96 copies/ L respectively. The maximum value of CV in repeated trials detected by TGEV, PEDV and PoRV were 2.5%, 3.8%, 4.3%, and the maximum value of CV in repeated trials between groups were 3.7%, 3.4%, 3.2%, which are no more than 5%.indicating that the established method has good reproducibility. Using this method to detect PRV, PCV1, and PRRSV virus samples, there is no cross-reaction, indicating that the method is specific. Using the established method to detect 40 clinical diseases, the samples were tested, and the positive rates of TGEV, PEDV, and PoRV were 5%, 30%, and 12.5%respectively. The mixed infection rate of TGEV and PEDV was 2.5%, the mixed infection rate of PEDV and PoRV was 5%. The results of the multiple fluorescence quantitative PCR method are consistent with those of the detection of a single fluorescent RT-PCR method, indicating that the established method has good clinical application value.