Development Of Multiplex PCR For Detection Of Pathogenic Bacteria

The majority of pathogenic bacteria by damaging the mucous membrane of gastrointestinaland respiratory tract, breaking through the immunologic barrier, into the blood circulatory system,which is causing the bacteriosis. Animal husbandry status shows us, the drug resistant strains aremore, because of the forage full of antibiotics.The ecumenic antibiotics can’t stop the disoperationof bacteriosis, additionally deficiency of the new tape and high performance antibacterials, lots ofpathogenic bacteria initiate similar clinical symptoms, the veterinary surgeon should use moreantibiotics and broad spectrum antibiotic to cure the disease, these all give the chance todeteriorate the problem. So a rapid differential diagnosis was needed to direct medication.Thisstudy was face to the current situation, aimed to develop a high performance and specific methodof differential diagnosis of aetiology to detect and different the conditional pathogenic bacteria ofgastrointestinal and respiratory tract diseases.Based on the corresponding sequences of the ureR, invA, α-toxin, kmt, the lktA-artJ intergenicregion and plo, six pairs of primers were designed. Two multiplex PCR methods were establishedand we got the optimal conditions. Amplification of the fragments and sequence analysis showtarget gene and designed primers were specificity.The ideal minimum detection limit of multiplexPCR(Proteus mirabilis, Salmonella and Clostridium perfringens)was4pg/μL lower than PCR ofthese (by turns0.4pg/μL,0.4pg/μL and4×10-4pg/μL). The ideal minimum detection limit ofmultiplex PCR(Pasteurella multocida, Mannheimia haemolytica and Trueperella pyogenes)was40pg/μL which is accordant with PCR of these (by turns4pg/μL,40pg/μL and40pg/μL).There were17bacteria stains to use to detect the specificity of multiplex PCR.Different stains, positivesamples and negative samples were detected for many times.The result were accordant.It provesthese methods have stability and repeatability. This method was preliminarily used to detect thepathological tissue of challenged mice and intentionaly contaminated tissue, which indicated themultiplex PCR was also valuable. The isolation were accordant with multiplex PCR. Our workhere will be helpful for the differential diagnosis of these conditional pathogenic bacteria.