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首页> 外文期刊>Journal of Clinical Microbiology >A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples.
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A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples.

机译：用于检测临床样品中结核分枝杆菌的更可靠的PCR。

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Diagnostic techniques based on PCR have two major problems: false-positive reactions due to contamination with DNA fragments from previous PCRs (amplicons) and false-negative reactions caused by inhibitors that interfere with the PCR. We have improved our previously reported PCR based on the amplification of a fragment of the Mycobacterium tuberculosis complex-specific insertion element IS6110 with respect to both problems. False-positive reactions caused by amplicon contamination were prevented by the use of uracil-N-glycosylase and dUTP instead of dTTP. We selected a new set of primers outside the region spanned by the formerly used primers to avoid false-positive reactions caused by dTTP-containing amplicons still present in the laboratory. With this new primer set, 16 copies of the IS6110 insertion element, the equivalent of two bacteria, could be amplified 10(10) times in 40 cycles, resulting in a mean efficiency of 77% per cycle. To detect the presence of inhibitors of the Taq polymerase, which may cause false-negative reactions, part of each sample was spiked with M. tuberculosis DNA. The DNA purification method using guanidinium thiocyanate and diatoms effectively removed most or all inhibitors of the PCR. However, this was not suitable for blood samples, for which we developed a proteinase K treatment followed by phenol-chloroform extraction. This method permitted detection of 20 M. tuberculosis bacteria per ml of whole blood. Various laboratory procedures were introduced to reduce failure or inhibition of PCR and avoid DNA cross contamination. We have tested 218 different clinical specimens obtained from patients suspected of having tuberculosis. The samples included sputum (n=145), tissue biopsy samples (n=25), cerebrospinal fluid (n=15), blood (n=14), pleural fluid (n=9), feces, (n=7), fluid from fistulae (n=2), and pus from a wound (n=1). The results obtained by PCR were consistent with those obtained with culture, which is the "gold standard." We demonstrate that PCR is a useful technique for the rapid diagnosis of tuberculosis at various sites.
机译：基于PCR的诊断技术有两个主要问题：由于先前PCR（扩增子）的DNA片段污染导致的假阳性反应，以及干扰PCR的抑制剂引起的假阴性反应。针对这两个问题，我们基于结核分枝杆菌复合物特异性插入元件IS6110片段的扩增，改进了先前报道的PCR。通过使用尿嘧啶-N-糖基化酶和dUTP代替dTTP可以防止扩增子污染引起的假阳性反应。我们选择了一组新的引物，该区域位于以前使用的引物所跨越的区域之外，以避免实验室中仍然存在的含dTTP的扩增子引起的假阳性反应。使用这种新的引物组，可以在40个循环中将16个拷贝的IS6110插入元件（相当于两种细菌）扩增10（10）次，平均每个循环效率为77％。为了检测可能引起假阴性反应的Taq聚合酶抑制剂的存在，将每个样品的一部分掺入结核分枝杆菌DNA。使用硫氰酸胍和硅藻的DNA纯化方法有效去除了大多数或所有PCR抑制剂。但是，这不适用于血液样本，为此我们开发了蛋白酶K处理方法，然后进行苯酚-氯仿提取。该方法允许每毫升全血检测20株结核分枝杆菌。引入了各种实验室程序以减少PCR的失败或抑制并避免DNA交叉污染。我们测试了从怀疑患有肺结核的患者获得的218种不同的临床标本。样本包括痰液（n = 145），组织活检样本（n = 25），脑脊液（n = 15），血液（n = 14），胸膜液（n = 9），粪便（n = 7），来自瘘管的液体（n = 2）和来自伤口的脓液（n = 1）。通过PCR获得的结果与通过培养获得的结果一致，这是“黄金标准”。我们证明，PCR是在各个部位快速诊断结核的有用技术。

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