Isolation And Identification Of Haemophilus Parasuis And Multiple PCR Methods Detecting Hps And APP

Haemophilus parasuis(Hps) is a causative agent of swine polyserositis,polyarthritis and meningitis.Actinobacillus pleuropneumoniae(APP) cause a highly contagious swine respiratory disease which is characterized by acute hemorrhagic fibrinous pleuropneumonia and chronic necrotizing fibrinous pleuropneumonia.with the development of pig industry,Hps and APP infection a marked increase in the trend,has become one of the important pathogen for the pig farming industry and causing significant economic losses.The study reported here on the isolation and identification of Haemophilus parasuis and developed a multiple PCR method for Hps and APP.Study were as follows:1.Haemophilus parasuis isolation and identificationWith the NAD and calf serum medium of TSB and TSA inoculation training,a suspected of Haemophilus parasuis isolated from pericardial effusion of sick pigs in Sichuan.On the separation of the identification of bacterial strains:growth characteristics of their colony cultured in TSA for 24～48h smooth,transparent gray;to their level of blood agar plate line,and then choose Staphylococcus aureus perpendicular to the horizontal line designated line,37℃cultured 24～48h,a typical "satellite growth" phenomenon,and there was no hemolysis.Bacterial morphology with pleomorphic small balls of gram-negative bacilli,a diameter of about 0.5-1mm.Biochemical identification with negative oxidase test,catalase test positive,test negative for urease,indole test negative,nitrate reduction test positive,can ferment glucose,sucrose,fructose,etc.,do not ferment xylose,galactose,mannose etc..Test Agar diffusion test and molecular biology typing methods for identification of its serotype 12 serum.Pathogenicity in mice experiments to determine the LD₅₀ for the 1.43×109CFU/mL.At the same time test their characterization of the molecular biology,the design of Haemophilus parasuis 16S rRNA primers to isolate the DNA as a template for PCR amplification of the isolated strains of 16S rRNA,and PCR products were cloned,sequencing and homology comparison,the comparative analysis of the isolates from reference strains with foreign genes homology reached 98%.Experiments show that:the success of this study was isolated from pig farms in Sichuan and identification of a Haemophilus parasuis and named HS SC-1.2.establishment of multiple PCR methods detected Hps and APPAPP apxIV and Hps 16S rRNA gene sequence reference genes in GenBank,analysis of their homology with Primer premier 5.0 software for the design of apxIV and 16S rRNA conservative gene,two pairs of designed primers and primers designed for dimerization body,homology,complementarity analysis,experimental design gene fragments amplified APP apxIV for 442bp,amplified Hps 16S rRNA gene fragment was 1090bp.The use of primers designed,respectively,and APP apxIVA of lips 16S rRNA gene PCR amplification,According to single PCR,by optimizing the annealing temperature, primer concentration,magnesium ion concentration,established the multi-PCR method to detect Hps and APP.Multiple PCR amplification of the best system:10×buffer(Mg2 +-Free) 2.5μl,25mM Mg2+1μl,2.5mM dNTP 2μl,10pmol of upstream and downstream primer 0.8μl,2.5U/μl Taq DNA polymerase 0.5μl,DNA template 2μl,the last with sterile deionized water up to 25μl.PCR reaction program predegeneration 94℃for 3 min,then 94℃denaturation 1min,55-57℃renaturation between the 45s,72℃extension of 1min,a total of 30 cycles,final extension of 72℃in 10min,4℃preservation.Multiple PCR-specific detection of common pathogens of Mycoplasma hyopneumoniae,Pasteurella multocida,Salmonella enteritidis,Streptococcus suis,swine erysipelas bacteria E.coli, were not amplified in any fragment.It could detected the volume of 10pg of DNA for susceptibility testing.Experiments show that:In this study,the initial establishment of a multiple PCR method of detection of Hps and APP,the detection method has the advantages of good specificity,high sensitivity detection and simultaneous detection of APP and Hps.