DIRECT-DETECTION OF MYCOBACTERIUM-TUBERCULOSIS IN RESPIRATORY SPECIMENS IN A CLINICAL LABORATORY BY POLYMERASE CHAIN-REACTION
DIRECT-DETECTION OF MYCOBACTERIUM-TUBERCULOSIS IN RESPIRATORY SPECIMENS IN A CLINICAL LABORATORY BY POLYMERASE CHAIN-REACTION
复制标题
DOI：
10.1128/jcm.31.7.1688-1694.1993
复制
发表时间：
1993-07-01
影响因子：
9.4
通讯作者：
HICKS, KES
中科院分区：
文献类型：
--
作者：
FORBES, BA;HICKS, KES
结核分枝杆菌（Mycobacterium tuberculosis，TB）的流行性多重耐药（multiple-drug-resistant，MDR）菌株的出现以及报告的结核病（TB）病例数量的增加代表了一个主要的公共卫生问题。鉴于最近爆发的MDR M。在本中心的结核病研究中，我们开始使用两组引物开发用于肺结核快速诊断的聚合酶链反应（PCR）测定法，一组引物基于M.结核病和基于蛋白抗原B（PA B）的另一种。反应条件首先优化为适当的提取方案和引物对，核苷酸和MgCl 2的浓度。在对临床标本的检测进行初步评价后，进一步修改了提取和扩增程序。PAB和IS 6110引物检测到的M菌数量在2至23之间，在0.023至0.23之间。tuberculosis，M.结核病阴性结核分枝杆菌通过我们优化的PCR检测。在常规处理分枝杆菌后，随后扩增了734份标本。将沉淀物用吐温20煮沸、打浆，得到扩增用DNA。对于每个反应，将DNA（10穆尔）加入到含有12 pmol IS 6110引物、20 pmol PAB引物、2 mM MgCl 2、200 μ M核苷酸和2.5 U Taq聚合酶的扩增混合物中，然后将混合物扩增40个循环。本实验的敏感性和特异性分别为87.2%和97.7%。我们无法解释7份标本（1%）的结果。根据我们的经验，PCR被证明是一种有用的结核病临床快速诊断检测方法，也是一种有价值的流行病学工具，可用于确定医院环境中的暴露人群。我们的研究结果还强调了系统优化PCR检测条件的必要性。
The emergence of epidemic multiple-drug-resistant (MDR) strains of Mycobacterium tuberculosis in conjunction with an increase in the number of reported cases of tuberculosis (TB) represents a major public health problem. In light of a recent outbreak of MDR M. tuberculosis at our center, we began the development of a polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary TB using two sets of primers, one based on the IS6110 repeated sequence of M. tuberculosis and the other based on the protein antigen b (PAB). Reaction conditions were first optimized as to the appropriate extraction protocol and the concentrations of primer pairs, nucleotides, and MgCl2. Following a preliminary evaluation of the assay with clinical specimens, extraction and amplification procedures were further modified. PAB and IS6110 primers detected between 2 and 23 and 0.023 and 0.23 CFU of M. tuberculosis, respectively, in pooled, M. tuberculosis-negative sputa by our optimized PCR assay. After routine processing for mycobacteria, 734 specimens were subsequently amplified. DNA for amplification was obtained by boiling and beating the sediments with Tween 20. For each reaction, DNA (10 mul) was added to an amplification mixture containing 12 pmol of IS6110 primers, 20 pmol of PAB primers, 2 mM MgCl2, 200 muM nucleotides, and 2.5 U of Taq polymerase and the mixture was then amplified for 40 cycles. The sensitivity and specificity of our PCR assay were 87.2 and 97.7%, respectively. We were unable to interpret the results for seven specimens (1%). In our experience, PCR proved to be a useful rapid diagnostic test for TB in a clinical setting and a valuable epidemiological tool for determining exposure groups in the hospital setting. Our findings also underscore the need for the systematic optimization of PCR assay conditions.