Establishment and preliminary application of PRRSV,SS and Pm multiplex Taq-Man real-time PCR detection methods
In order to establish a multiplex TaqMan PCR detection method that can simultaneously detect and identify porcine reproductive and respiratory syndrome virus(PRRSV),Streptococcus suis(SS)and Pasteurella multocida(Pm),the gene sequences of three pathogens registered on NCBI were analyzed by MEGA7.0,and specific primers and probes were designed according to the ORF6 gene of PRRSV,GDH gene of SS and KMT1 gene of Pm,respectively,and the reaction con-ditions were optimized.This method can specifically detect PRRSV,SS and Pm,and has no cross-reactivity against other pathogens.The minimum detection copy numbers for PRRSV,SS and Pm were 9.93×102 copies/μL,1.10 × 102 copies/μL and 1.07×102 copies/μL,respectively.The coeffi-cient of variation was less than 1.85%both within and between groups.The results show that the method has strong specificity,high sensitivity and good stability,and can be applied to clinical de-tection.