Development And Application Of Multi-PCR For Detection Of Actinobacillus Pleuropneumoniae Serotype1,2,6,7

Actinobacillus pleuropneumoniae (APP) is the etiological agent of porcine pleuropneumonia ,which is characterized by fibrinous pleuritis and hemorrhagic,highly contagious pneumonia It caused a lot of economic losses of porcine production. It is difficult in preventation and diagnose because APP have many serotype and each other has nearly no cross-protection. In this study, primers were designed based on Outer membrane lipoprotein(OmlA) and capsular polysaccharide(CPS). Single PCRs were developed and the conditions were optimized. On this base , multi- PCR were developed, specificity and sencetivity of it were tested and it was used in clinic.1 primer designing Primers were designed repectively by Array Designer 2.0 according to the sequences of OlmA and CPS1,2,6,7 of APP in GenBank and were analyzed avoiding the formation of steady dimmers , high homology and complementarity. The same time homology and complementarity of amplified fragments were analyzed. The five specific PCR primers were successfully designed to amplify OmlA 952bp, CPS₁ 630bp, CPS₂ 504bp, CPS₆ 720bp, CPS₇ 389bp, respectively.2 Single PCR Single PCRs of the detection OmlA,CPS1,2,6,7 genes were developed and the conditions of annealing temperature, primer concentration, Mg²⁺ concentration were optimized and specificity and sencetivity of them were tested. Result: on the reaction condition of 10×buffer (Mg²⁺ -Free) 5μl, 25 mM Mg²⁺ 3μl -6μl, 2.5 mM dNTP Mixture 4μl, 25/2pmol/ul primer mixture 1.5μl-3μl, 5u/μl TaKaR Taq 0.4μl, DNA template 5ul in a 50 ul reaction volume;Force-denatured 4 min at 94 ℃, denatured 45 S at 94 ℃, annealed 45 S at 52-58℃, 1 min at 72 ℃,35 cycles, extend at 72 ℃, 4℃ hold, OmlA 952bp, CPS₁ 630bp, CPS₂ 504bp, CPS₆ 720bp, CPS₇ 389bp fragments could be amplified. Specificity test, positive amplifications of OmlA 952bp were obtained from APP reference strains, positive amplifications of CPS₁ 630bp, CPS₂ 504bp, CPS₆ 720bp, CPS7 389bp were obtained with only homologous APP reference strains. negative with...