Study On Rapid Detection Technique Of Actinobacillus Pleuropneumoniae

1 Cloning, sequencing and expression of one section of ApxIV gene from A ctinobacillus pleuropneumoniaeA section of ApxIV gene of Actinobacillus pleuropneumoniae amplified by PCR was cloned into pMD18-T vector, and transformed into host strain E.coli DH_5a The plasmid of positive clone was enzymolysized and sequenced. The result of sequencing showed that the consistency was 100% compared with the refernce sequence from GenBank. Then the target DNA was cloned into pET-32a vector. The recombinant plasmid was transformed into E.coli BL21. The target protein with about 60kD was induced by IPTG and detected by western blotting.2 Application and development of indirect ELISAs based on Cps and rApxlVThe recombinant prote(rApxIV)was purified and used as antigen for developing a rApx IV-based indirect enzyme-linked immunosorbent assay(rApxIV-ELlSA). The conditions of rApxIV-ELISA were determined as follows: 0.625 μg/ml of rApxIV used to coat ELISA plate, 1:100 of sera as detecting samples and serum sample being incubatedat 37°Cfor 90 minutes. The ELISA was used to detect 18 sera from pigs artificially infected with App and 54 sera from pigs vaccinated with killed App.The results indicated that this protein is a promising candidate for distinguishing vaccination from infection.The capsular polysaccharide ( Cps ) of App was extracted and used as antigen for developing a Cps-based indirect enzyme-linked immunosorbent assay ( Cps-ELISA ) . The conditions of Cps-ELISA were determined as follows: 3μg/ml of CPS used to coat ELISA plate, 1:100 of sera as detecting samples and serum sample being incubatedat 37℃ for 90 minutes. A total of 250 sera were detected in parallel by the Cps-ELISA and indirect hemagglutination test kit for App. 88% of them were detected positively by Cps-ELISA, while 81.2% of them were positive by indirect hemagglutination test kit.The developed indirect ELISAs based on recombinant ApxIV and Cps were used to sera of pig after artificial infection,vaccination and natural infection, Theresults showed that the Cps-ELISA can surveil antibody changes agaist App after vaccination, and the rApxIV-ELISA can detect the natural infection of App. 3 PCR Assay for rapid detection of Actinobacillus pleuropneumoniaeA pair of primers from the 5' and 3' termini of outer membrane lipoprotein (OmlA) genes of Actinobacillus pleuropeumoniae (App) were selected to be the basis for development of a specific PCR assay. A DNA fragment of 1,010bp was amplified from lysed App serotypes 1,2,3,4,5a,5b,6,7,9 of biovar 1. None of PCR products was detected when chromosomal DNAs from bacteria of S. equisimilis, P.pnenmotropica, S.typhimurium, H.parasuis and E.coli were used as target DNAs. Digested with appropriate restriction endonuclease, the PCR products could produce the expected target DNA fragments. The PCR assay developed was very sensitive, with lower detection limits of 10 CFU of App cells. The PCR were used to detect the artificially infected and clinical specimens.