四种血清型胸膜肺炎放线杆菌多重PCR检测方法的建立

Development of a multiplex PCR assay for detection of Actinobacillus pleuropneumoniae serotypes 1,2,6 and 7

根据胸膜肺炎放线杆菌（APP）外膜蛋白OmlA序列设计种特异性引物，根据血清型1、2、6、7荚膜多糖（CPS）序列分别设计型特异性引物，通过优化PCR扩增条件，分别扩增出OralA（952bp）、CPS1（630bp）、CPS2（504bp）、CPS6（720bp）、CPS7（389bp）特异性片段，建立了既可对APP进行定性检测又可对APP1、2、6、7进行定型检测的多重PCR。特异性试验表明，此多重PCR能从APP1～4、6～10标准株中扩增出与引物相应的特异性片段；对猪副嗜血杆菌、猪肺炎霉形体、多杀性巴氏杆菌、肠炎沙门菌、猪链球菌、大肠杆菌和猪丹毒杆菌进行扩增，均未扩增出片段。敏感性试验表明，此多重PCR能够检测到DNA的量为10pg。45头疑似病猪病料的细菌分离鉴定结果与此多重PCR的鉴定结果一致。上述结果表明，建立的多重PCR适合于APP1、2、6、7的鉴别诊断及流行病学调查。

Specific primers for OmlA gene of Actinobacillus pleuropneumoniae （APP） and CPS gene of serotypes 1,2,6 and 7 were designed and synthesized. Through optimizing conditions of PCR,OmlA（952 bp） ,CPS1（630 bp）,CPS2（504 bp）,CPS6（720 bp） and CPS7（389 bp） genes were amplified by PCR,respectively. Thus, a multiplex PCR assay for detection of APP serotypes 1,2,6 and 7 was developed. The specific test for the multiplex PCR showed that positive amplifications were obtained with homologous APP reference strains, but not with Haemoph ilus suis , Mycoplasma h yopneumonia , Pasteurella multocida , Salmonella choleraesuis, Streptococcus suis, Escherichia coli and Erysipelothrix rhusiopathiae. In a sensitivity test for the multiplex PCR, 10 pg of genomic DNA could be detected. The bacterium isolation results of 45 pigs infected suspiciously with APP were in coincidence with the results of the multiplex PCR. These re sults showed that this multiplex PCR was an effective assay for rapid diagnosis,identification and epidemiological investigation of APP serotypes 1,2,6 and 7.