Nghiem Minh Ngoc, Nguyen Bac Van, Nguyen Son Thai, Vo Thuy Thi Bich, Nong Hai Van
Institute of Genome Research, Vietnam Academy of Science and Technology, Cau Giay, Hanoi, Vietnam.
Institute of Biotechnology, Vietnam Academy of Science and Technology, Cau Giay, Hanoi, Vietnam.
J Microbiol Biotechnol. 2015 May;25(5):745-52. doi: 10.4014/jmb.1409.09089.
Tuberculosis (TB) is the most common mycobacterial infection in developing countries, requiring a rapid, accurate, and well-differentiated detection/diagnosis. For the rapid detection and discrimination of Mycobacterium tuberculosis complex (MTC) from nontuberculous mycobacteria (NTM), a novel, simple, and primer-combined single-step multiplex PCR using three primer pairs (6110F-6110R, 1081F-1081R, and 23SF-23SR; annealing on each of IS6110, IS1081, and 23S rDNA targets), hereafter referred to as a triplex PCR, has been developed and evaluated. The expected product for IS6110 is 416 bp, for IS1081 is 300 bp, and for 23S rDNA is 206 bp by single PCR, which was used to verify the specificity of primers and the identity of MTC using DNA extracted from the M. tuberculosis H37Rv reference strain (ATCC, USA) and other mycobacteria other than tuberculosis (MOTT) templates. The triplex PCR assay showed 100% specificity and 96% sensitivity; the limit of detection for mycobacteria was ~100 fg; and it failed to amplify any target from DNA of MOTT (50 samples tested). Of 307 blinded clinical samples, overall 205 positive M. tuberculosis samples were detected by single PCR, 142 by conventional culture, and 90 by AFB smear methods. Remarkably, the triplex PCR could subsequently detect 55 positive M. tuberculosis from 165 culture-negative and 115 from 217 AFB smear-negative samples. The triplex PCR, targeting three regions in the M. tuberculosis genome, has proved to be an efficient tool for increasing positive detection/ discrimination of this bacterium from clinical samples.
结核病（TB）是发展中国家最常见的分枝杆菌感染，需要快速、准确且鉴别性良好的检测/诊断方法。为了从非结核分枝杆菌（NTM）中快速检测和鉴别结核分枝杆菌复合群（MTC），已开发并评估了一种新型、简单且引物组合的单步多重PCR方法，该方法使用三对引物（6110F - 6110R、1081F - 1081R和23SF - 23SR；分别在IS6110、IS1081和23S rDNA靶标上退火），以下简称三重PCR。通过单重PCR，IS6110的预期产物为416 bp，IS1081为300 bp，23S rDNA为206 bp，用于验证引物特异性以及使用从结核分枝杆菌H37Rv参考菌株（美国ATCC）和其他非结核分枝杆菌（MOTT）模板中提取的DNA来鉴定MTC。三重PCR检测显示特异性为100%，灵敏度为96%；分枝杆菌的检测限约为100 fg；并且未能从MOTT的DNA（测试50个样本）中扩增出任何靶标。在307份盲法临床样本中，通过单重PCR共检测到205份结核分枝杆菌阳性样本，通过传统培养法检测到142份，通过抗酸杆菌涂片法检测到90份。值得注意的是，三重PCR随后可从165份培养阴性样本中检测到55份结核分枝杆菌阳性样本，从217份抗酸杆菌涂片阴性样本中检测到115份。靶向结核分枝杆菌基因组三个区域的三重PCR已被证明是一种有效的工具，可提高从临床样本中阳性检测/鉴别该细菌的能力。
