Study On Genechip For Detecting Of Actinobacillus Pleuropneumoniae, Mycoplasma Hyopneumoniae And Pasteurella Multocida And Genotyping Of Actinobacillus Pleuropneumoniae

Microarray technology can simultaneously identify multi-pathogens at the species, subspecies, type, and subtype level in a single assay, which offers greater powerful and attractive method for detection and typing of multi-pathogens. Microarray technology possess highthrongh, parallelism, hight sensitivity and specificity. In this study, detecting and/or typing target genes of Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae, Pasteurella multocida were designed and amplified by PCR. APP-Mhp-PM detecting genechip and APP typing genechip developed with amplified target genes are a new detection technology, which will produce a marked effect on monitoring, association diagnosis, precaution and treatment of three bacterial diseases.23 primer pairs were designed and adapted froml6SrDNA, apxIVA, dsbE, omlA, apx toxin and capsular genes of A. pleuropneumoniae, 16SrDNA and P36 genes of M. hyopneumoniae, psl and KMT1 genes of P. multocida. Other primer pairs were designed and selected by using Array Designer 2.0 except for cited primers. 25 target genes were amplified by 23 pair primers and cloned into vector pMD18-T. 7 detecting target genes of them were A16S, dsbE, apxIV of A. pleuropneumoniae, M16S and P36 of M hyopneumoniae, psl and KMT1 of P. multocida, respectviely; 17 typing target genes of them were omlAl, omlA2, omlA3, omlA4, cpsl, cps2, cps6, cps7, cps8, cpx3, cpx6, cpx9, apxIA, apxID, apxIIA, apxIIIA and apxIIID of A. pleuropneumoniae. Alignment of all 25 target genes sequenced showed A16S target sequence had above 95% homology with 16S r RNA sequences of Actinoballus species, M16S target sequence had above 88%, 95%, 100% homology with 16S rRNA sequences of M. hyorhinitis, M.flocculare and M hyopneumoniae respectively, other detecting target sequences had above 92% homology with the same gene of respective different serotypes or strains bacteria, and it had below 67% homology among typing target sequences except that typing target gene apxID had 75% homology with apxIIID.7 detecting target genes were used to construct APP-Mhp-PM detecting genechip. 15 typing target genes of omlAl, omlA2, omlA3, omlA4, cpsl, cps2, cps6, cps7, cps8, cpx6,...