猪胸膜肺炎放线杆菌半套式PCR检测方法的建立及应用

Development and application of a semi-nested PCR assay for detection of porcine Actinobacillus pleuropneumoniae

根据1型胸膜肺炎放线杆菌apxⅣA基因3′端序列设计3条引物,建立了检测猪胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)的半套式PCR方法。筛选了该方法的最佳反应条件,并采用该方法进行临诊检测,比较了不同处理临床样品的检测结果。结果表明,该方法能检测出所有供试血清型APP,不能从猪上呼吸道常见细菌基因组DNA中扩增出条带,特异性好;最低DNA检出量为16.5 fg,其灵敏性是常规PCR方法的1 000倍;重复性试验结果表明,该方法稳定可靠。应用半套式PCR方法检测临床样品,检出率显著高于细菌分离鉴定;样品保存方法和核酸抽提方法能影响阳性样品的检出率。

Three primers were designed according to the 3＇ terminal sequence of apxⅣA gene of Actinobacillus pleuropneumoniae （APP） serotype 1 to develop a semi-nested PCR assay for detection of APP. Reaction conditions of the semi-nested PCR were optimized. The efficacy of detection was compared by using different sample preservation and DNA extraction methods. The developed semi-nested PCR could amplify unique band of approximately 250 bp in size from genomic DNA of all tested samples of APP strains,while no other band was observed from bacteria other than APP in porcine upper respiratory tract, indicating that the semi-nested PCR possessed good specificity. 16.5 fg of genomic DNA was detected by the semi-nested PCR,and the sensitivity of the semi-nested PCR was 1 000 fold higher than that of the onestep PCR method. The products by the semi-nested PCR were always consistent for 5 times in reproducibility. The positive rate of clinical samples detected by the semi-nested PCR was higher than that by the bacterial isolation method. The preservation conditions and the extracting methods for DNA significantly affected the positive rate by the semi-nested PCR.