Hunt J M, Roberts G D, Stockman L, Felmlee T A, Persing D H
Division of Clinical Microbiology, Mayo Clinic, Rochester, MN 55905.
Diagn Microbiol Infect Dis. 1994 Apr;18(4):219-27. doi: 10.1016/0732-8893(94)90024-8.
The polymerase chain reaction (PCR) and automated DNA sequencing were used to detect a genetic locus, rpoB, associated with rifampin resistance in Mycobacterium tuberculosis (TB) in clinical isolates and directly in clinical specimens. Primers derived from the sequence of a TB rpoB gene fragment were used to amplify DNA from bacterial and mycobacterial isolates. An rpoB-specific PCR product was obtained for five of five TB, seven of eight other mycobacterial species, Nocardia sp., Corynebacterium sp., Streptomyces sp., Actinomyces sp., and Rhodococcus sp., but not for 15 isolates (eight genera) representing usual bacterial flora. Sequence comparison of the amplified rpoB region revealed the occurrence of TB-specific "signature nucleotides" at three positions. PCR yielded amplification products for seven of 16 clinical specimens. Five of the seven contained TB-specific DNA, as well as sequences that predicted rifampin susceptibility in accord with agar dilution results. None of ten specimens that were culture negative for TB yielded TB-specific PCR products. These results with a limited number of clinical specimens demonstrate the feasibility of direct detection by PCR of rifampin-resistant TB in clinical specimens. Such testing may serve as a rapid surrogate test for multidrug-resistant TB in laboratories with PCR and automated sequencing capability.
采用聚合酶链反应（PCR）和自动DNA测序技术，检测临床分离株及直接从临床标本中结核分枝杆菌（TB）与利福平耐药相关的遗传位点rpoB。源自结核分枝杆菌rpoB基因片段序列的引物用于扩增细菌和分枝杆菌分离株的DNA。从5株结核分枝杆菌、8株其他分枝杆菌属（包括诺卡菌属、棒状杆菌属、链霉菌属、放线菌属和红球菌属）中的7株获得了rpoB特异性PCR产物，但代表常见细菌菌群的15株分离株（8个属）未获得该产物。扩增的rpoB区域的序列比较显示在三个位置出现了结核分枝杆菌特异性的“特征性核苷酸”。PCR从16份临床标本中的7份产生了扩增产物。7份中的5份含有结核分枝杆菌特异性DNA，以及与琼脂稀释结果一致的预测利福平敏感性的序列。10份结核培养阴性的标本均未产生结核分枝杆菌特异性PCR产物。这些对有限数量临床标本的研究结果证明了通过PCR直接检测临床标本中耐利福平结核分枝杆菌的可行性。这种检测可作为具有PCR和自动测序能力的实验室中耐多药结核分枝杆菌的快速替代检测方法。
