Improved diagnostic PCR assay for Actinobacillus pleuropneumoniae based on the nucleotide sequence of an outer membrane lipoprotein
Improved diagnostic PCR assay for Actinobacillus pleuropneumoniae based on the nucleotide sequence of an outer membrane lipoprotein
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DOI：
10.1128/jcm.36.2.443-448.1998
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发表时间：
1998-02-01
影响因子：
9.4
通讯作者：
Ahrens, P
中科院分区：
文献类型：
--
作者：
Gram, T;Ahrens, P
编码胸膜肺炎放线杆菌血清型1和5的外膜蛋白的基因（omlA）已经在前面描述，并且已经形成了开发特异性PCR测定的基础。对胸膜肺炎生物变型1的参考菌株进行了序列测定，序列比对显示其保守的末端和可变的中间区，将参考菌株分为4个不同的组。从该基因保守的5 '端和3'端选择引物，从102个供试的A.从肺获得的胸膜肺炎。通过对来自50个A.为了检测该反应的特异性，还对23种与A.胸膜肺炎或分离自猪的病毒。PCR检测结果均为阴性。胸膜肺炎阴性畜群。通过PCR产物的琼脂糖凝胶分析评估的灵敏度为10（2）CFU/PCR试管。与培养法相比，该PCR的特异性和敏感性表明，该PCR可用于A.胸膜肺炎
The gene (omlA) coding for an outer membrane protein of Actinobacillus pleuropneumoniae serotypes 1 and 5 has been described earlier and has formed the basis for development of a specific PCR assay, The corresponding regions of all 12 A. pleuropneumoniae reference strains of biovar 1 were sequenced, Alignment of the sequences revealed conserved terminal and variable middle regions, which divided the reference strains into four distinct groups. Primers were selected from the conserved 5' and 3' termini of the gene, A 950-bp amplicon was obtained from each of 102 tested field isolates of A. pleuropneumoniae obtained from lungs. Their identity was verified by sequencing approximately 500 bp of the amplification product from 50 of the A. pleuropneumoniae isolates, which all showed the expected DNA sequence characteristic of the serotype, To test the specificity of the reaction, 23 other bacterial species related to A. pleuropneumoniae or isolated from pigs were assayed. They were all found negative in the PCR, as were tonsil cultures from 50 pigs of an A. pleuropneumoniae-negative herd. The sensitivity assessed by agarose gel analysis of the PCR product was 10(2) CFU/PCR test tube. The specificity and sensitivity of this PCR compared to those of culture suggest the use of this PCR for routine identification of A. pleuropneumoniae.