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首页> 外文期刊>Journal of Clinical Microbiology >Direct detection of Mycobacterium tuberculosis in respiratory specimens in a clinical laboratory by polymerase chain reaction.
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Direct detection of Mycobacterium tuberculosis in respiratory specimens in a clinical laboratory by polymerase chain reaction.

机译：通过聚合酶链反应在临床实验室中直接检测呼吸道标本中的结核分枝杆菌。

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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 microliters) was added to an amplification mixture containing 12 pmol of IS6110 primers, 20 pmol of PAB primers, 2 mM MgCl2, 200 microM 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.
机译：结核分枝杆菌流行性多药耐药性（MDR）菌株的出现与已报告的结核病（TB）病例数的增加是一个主要的公共卫生问题。鉴于最近在我们中心爆发的耐多药结核分枝杆菌，我们开始开发一种聚合酶链反应（PCR）分析法，用于快速诊断肺结核，它使用了两组引物，其中一组基于IS6110重复的M序列结核病和其他基于蛋白质的抗原b（PAB）。首先针对适当的提取方案以及引物对，核苷酸和MgCl2的浓度优化反应条件。在对临床标本进行初步评估后，对提取和扩增步骤进行了进一步修改。通过我们的优化PCR分析，在合并的结核分枝杆菌阴性菌落中，PAB和IS6110引物分别在结核分枝杆菌的2至23和0.023至0.23 CFU之间检测到。在常规处理分枝杆菌后，随后扩增了734个标本。通过用Tween 20沸腾并搅打沉淀物来获得用于扩增的DNA。对于每个反应，将DNA（10微升）添加到含有12 pmol IS6110引物，20 pmol PAB引物，2 mM MgCl2、200 microM核苷酸的扩增混合物中，和2.5 U Taq聚合酶，然后将混合物扩增40个循环。 PCR检测的灵敏度和特异性分别为87.2和97.7％。我们无法解释7个样本（1％）的结果。根据我们的经验，PCR被证明是在临床环境中对结核病有用的快速诊断测试，并且是确定医院环境中暴露人群的有价值的流行病学工具。我们的发现还强调了对PCR测定条件进行系统优化的必要性。

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