摘要
为建立直接检测可疑病料中胸膜肺炎放线杆菌(APP)的PCR诊断方法,对用以扩增apxⅣA毒力基因3′端长约442 bp的核苷酸片段的引物、模板、TaqDNA聚合酶、dNTPs的最适剂量及退火温度进行了优化筛选,并进行了特异性及重复性试验;以筛选出的优化条件,对临床可疑病料进行了PCR检测。结果,在25μL体系中,以5 pmol/μL引物、0.25μLTaqDNA聚合酶、2 mmol/L dNTPs、56℃退火温度对APP标准菌株apxⅣA毒力基因的扩增效果最好,重复性和稳定性高;而对类症菌株的扩增结果则为阴性,表明其特异性强。对分离到APP的病料PCR阳性率为100%(5/5);未分离到APP的纤维渗出性病料中,新鲜与陈旧病料PCR阳性率分别为70.59%(12/17)、50.00%(4/8),而非纤维渗出性病料的PCR结果均为阴性(0/45、0/9)。结果表明,建立的PCR方法特异、快速、稳定、敏感,优于细菌学方法,它可作为APP可疑病料的理想诊断方法。
To establish a diagnostic PCR assay for the detection of Actinobacillus pleuropneumoniae （APP） in the suspected samples, concentrations of primers,template, Taq DNA polymerase or dNTPs, and denaturing temperatures were optimized and screened for the amplification of 442 bp fragment of 3＇end of apxⅣ A gene of APP, and the specificity and repeatability of the PCR assay were evaluated. 5 pmol/μL primers,0.25 U Taq DNA polymerase,2 mmol/L dNTPs,56 ℃ for denaturing were recommended for amplification of the fragment. The positive rate of the PCR amplification was 100% （5/5） in accordance with the APP isolation. For fresh and dated pig samples with appearance of fibrous effusion, from which no APP not isolated,the positive rates were 70.59% （12/17） and 50.00% （4/8）, respectively. The positive rates of case tissues without appearance of fibrous effusion, from which APP were not isolated too, were . negative （0/45, 0/9）. The results suggested that the established PCR assay was more specific, rapid, stable and sensitive in comparison with the bacteriological method, and can be used as the most satisfactory method for diagnoses of suspected samples with APP.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2007年第2期117-120,共4页
Chinese Veterinary Science
基金
广东省自然科学基金项目(034064)
佛山市科技发展专项基金项目(03020031)
关键词
猪
胸膜肺炎放线杆菌
PCR
临床应用
swine
Actinobacillus pleuropneumoniae
PCR
clinical application
