Establishment and application of multiplex PCR method for Haemophilus parasuis,Pasteurella multocida and Actinobacillus pleuropneumoniae

The multiplex PCR method for Haemophilus parasuis(HPS),Pasteurella multocida(Pm) and Actinobacillus pleuropneumoniae(APP) has been developed based on the established single PCR for these pathogens.The multiplex PCR procedure was further optimized.A 256 bp fragment of APP,a 457 bp fragment of Pm and a 822 bp fragment of HPS could be simultaneously amplified in a PCR reaction.The test sensitivity showed that the PCR method could detect 60 pg of HPS,120 pg of Pm and 50 pg of APP,respectively.Thirty-nine strains of bacteria isolated from the pigs infected with porcine respiratory and reproductive syndrome virus(PRRSV) were tested by the multiplex PCR.The results showed 12 strains of HPS,16 of Pm strains and 2 of APP,which is consistent with conventional bacterial isolation and identification results.So the multiplex PCR developed in this study is suitable for the clinical detection of HPS,Pm and APP.