Establishment And Application Of Multiple PCR Diagnosis Methods For Major Bacterial Pathogens In Sheep Respiratory Tract Infection

In order to determine the bacterial pathogens of dead sheep with symptoms of respiratory disease in sheep farms of scale in Shihezi,a strain of D-type Pasteurella multocida was isolated used conventional bacterial isolation and identification method,16 S rRNA sequence analysis,Pasteurella multocida specific gene identification method.The tissue came from sheep with respiratory symptoms.The tissue was derived from respiratory symptoms.5 capsular serotype specific genes were used to determine their serotypes and16 virulence related genes of the isolates were amplified.Then detected of pathogenicity and drug sensitivity of isolates.A multiplex PCR diagnostic method that can identify Pasteurella multocida,Klebsiella pneumoniae,Arcanobacterium pyogenes was successfully established.Based on kmt gene of Pasteurella multocida,dcbF gene of D-type Pasteurella multocida,khe gene of Klebsiella pneumoniae,and plo gene of Arcanobacterium pyogenes,optimization on final primer concentration,template ratio,annealing temperature,annealing time and cycletimes,appraisal on the performance of this method through interprime specificity,interspecies specificity,sensitivity and repeatability through.55 cases of artificial infections and 11 cases of clinical respiratory diseases,were tested with multiplex PCR method and bacterial isolation and identification at the same time.The findings of this study are as follows:1.A D-type Pasteurella multocida was isolated from diseased sheep,the sheep with respiratory symptoms from sheep farm in Shihezi,Xinjiang.The isolate have strong pathogenicity and founded that isolate carries 8 virulence among the 16 major virulence related genes currently found.The homology of the amplified fragment is more than 99% compared with the reference strain of Pasteurella multocida that has been published on NCBI.The isolated strain is resistant to penicillin,lincomycin,gentamicin and SMZ-TMP,and presents sensitivity of varying degrees to other 19 types of drugs.2.Multiplex PCR method capable of detecting 3 kinds of bacteria was successfully established.The optimal final primer concentrations were 0.16 μmol/L,0.16 μmol/L,0.48 μmol/L and 0.48 μmol/L,optimal template ratio was 1 μL,0.5 μL and 2 μL,optimal annealing temperature was 55.1 °C,annealing time is 30 s and the optimal number of cycles is 35 times,in this multiplex PCR method.The established method has good specificity and sensitivity to the differential diagnosis of Pasteurella multocida,Klebsiella pneumoniae,Arcanobacterium pyogenes.3.55 cases of artificial infections and 11 cases of clinical respiratory diseases,were detected with multiplex PCR method and bacterial isolation and identification at the same time.The coincidence rate of multiplex PCR test results and separation and identification test results is 100%...