Whelen A C, Felmlee T A, Hunt J M, Williams D L, Roberts G D, Stockman L, Persing D H
Division of Clinical Microbiology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
J Clin Microbiol. 1995 Mar;33(3):556-61. doi: 10.1128/jcm.33.3.556-561.1995.
Recent analysis of the gene encoding the beta subunit of Mycobacterium tuberculosis RNA polymerase (rpoB) has demonstrated a small region that harbors the mutations most frequently associated with rifampin resistance. Earlier reports have described a high degree of sequence conservation of rpoB among mycobacteria other than M. tuberculosis and other GC-rich bacteria that can lead to false-positive amplification when applied directly to clinical specimens. We developed reagents for PCR amplification that are based on signature nucleotides discovered by comparative sequence analysis of the rpoB genes of organisms phylogenetically related to M. tuberculosis. The specificities of the reagents were challenged with 20 isolates of multiple-drug-resistant M. tuberculosis and more than 20 species of mycobacteria other than M. tuberculosis and other GC-rich organisms. A single-tube heminested PCR protocol was devised to obtain sensitivity equal to those of an IS6110-based PCR assay and culture in spiked sputum experiments. The assay correctly identified 21 of 24 (87.5%) culture-positive specimens, 13 of which were acid-fast smear-negative, in a panel of 51 clinical specimens. Three specimens that were false-positive initially were negative upon repeat testing when the assay was modified to eliminate the potential for aerosol carryover of the first-round amplification product during the open-tube addition of the second set of reaction reagents. This assay is the most sensitive and specific test to date for the direct detection of M. tuberculosis rpoB in clinical specimens. This rapid PCR-based assay can be used for the simultaneous identification of M. tuberculosis and its rifampin susceptibility genotype.
最近对结核分枝杆菌RNA聚合酶β亚基（rpoB）编码基因的分析表明，有一个小区域存在与利福平耐药性最常相关的突变。早期报告描述了结核分枝杆菌以外的分枝杆菌以及其他富含GC的细菌中rpoB的高度序列保守性，当直接应用于临床标本时可能导致假阳性扩增。我们基于对与结核分枝杆菌系统发育相关的生物体rpoB基因进行比较序列分析发现的特征性核苷酸，开发了用于PCR扩增的试剂。用20株耐多药结核分枝杆菌以及20多种结核分枝杆菌以外的分枝杆菌和其他富含GC的生物体对这些试剂的特异性进行了验证。设计了一种单管半巢式PCR方案，以在加标的痰液实验中获得与基于IS6110的PCR检测和培养相当的灵敏度。在一组51份临床标本中，该检测正确鉴定出24份培养阳性标本中的21份（87.5%），其中13份抗酸涂片阴性。最初为假阳性的3份标本在修改检测方法以消除在开放管中添加第二组反应试剂期间第一轮扩增产物气溶胶携带可能性后，重复检测时变为阴性。该检测是迄今为止用于临床标本中直接检测结核分枝杆菌rpoB最灵敏和特异的检测方法。这种基于PCR的快速检测可用于同时鉴定结核分枝杆菌及其利福平药敏基因型。
