多重PCR快速鉴定分枝杆菌方法的建立

Establishment of a multiplex PCR for rapid identification of Mycobacterium species

目的建立并评估一种快速鉴定分枝杆菌的多重PCR法,为结核病的快速病原诊断提供技术手段.方法选取16S rRNA、Rv0577、RD9和mtbk_20680基因,设计特异性引物,经条件优化,建立多重PCR方法.对非洲分枝杆菌、牛分枝杆菌、卡介苗(BCG)菌株、7种常见的非结核分枝杆菌(NTM)、7种常见的呼吸道病原菌和甘肃省93株结核分枝杆菌(MTB)临床分离株进行检测鉴定,评价其敏感性和特异性.结果16S rRNA、Rv0577、RD9和mtbk_20680基因产物长度分别为543 bp、786 bp、369 bp和231 bp.MTB北京基因型的4个基因均为阳性,而非北京基因型mtbk_20680基因为阴性;非洲分枝杆菌、牛分枝杆菌、BCG菌株均为16S rRNA和Rv0577阳性;7种NTM仅16S rRNA为阳性;7种呼吸道菌检测结果均为阴性.93株MTB临床分离株中鉴定为北京基因型菌株的有80株,占86.02%,非北京型菌株有13株,占13.98%.该方法的特异性为100%.结论该多重PCR法可以准确鉴别分枝杆菌、结核分枝杆菌复合群(MTBC)、NTM、MTB、北京基因型与非北京基因型MTB,具有高度的敏感性和特异性.

Objective To establish and evaluate a multiplex PCR method for rapid identification of Mycobacterium species in order to provide an approach for rapid diagnosis of pathogens causing tuberculo-sis.Methods Four genes including 16S rRNA,Rv0577,RD9 and mtbk_20680 were selected to establish the multiplex PCR method.Specific primers were designed and the reaction system and conditions were opti-mized.The sensitivity and specificity of the multiplex PCR method were evaluated through identifying Myco-bacterium africanum(M.africanum),Mycobacterium bovis(M.bovis),M.bovis BCG,seven common non-tuberculosis Mycobacterium(NTM),seven reference species of common respiratory bacteria and 93 clinical strains of Mycobacterium tuberculosis(MTB)isolated from patients with tuberculosis in Gansu Province of China.Results The fragments of 16S rRNA,Rv0577,RD9 and mtbk_20680 genes were 543 bp,786 bp,369 bp and 231 bp in length,respectively.MTB strains of the Beijing genotype were positive for all of the four genes,while the non-Beijing genotype strains were negative for mtbk_20680.M.africanum,M.bovis and M.bovis BCG strains were negative for 16S rRNA and Rv0577.NTM strains only carried 16S rRNA and none of four genes were detected in the seven species of respiratory bacteria.Among the 93 clinical MTB strains,80(86.02%)belonged to the Beijing genotype and the other 13(13.98%)were non-Beijing gen-otype strains.The specificity of the multiplex PCR method was 100%.Conclusions The established multi-plex PCR method could accurately distinguish Mycobacterium,Mycobacterium tuberculosis complex(MTBC),NTM,MTB,and Beijing and non-Beijing genotype MTB with high sensitivity and specificity.