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首页> 外文期刊>Journal of Clinical Microbiology >Detection of Mycobacteria, Mycobacterium avium Subspecies, and Mycobacterium tuberculosis Complex by a Novel Tetraplex Real-Time PCR Assay
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Detection of Mycobacteria, Mycobacterium avium Subspecies, and Mycobacterium tuberculosis Complex by a Novel Tetraplex Real-Time PCR Assay

机译：新型四联体实时荧光定量PCR检测分枝杆菌，鸟分枝杆菌亚种和结核分枝杆菌复合体

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Mycobacterium tuberculosis complex, Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n = 38) and nonmycobacterial (n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n = 57), archival clinical samples (n = 233), and strains isolated from various hosts (n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses.
机译：结核分枝杆菌复合物，鸟分枝杆菌和许多其他非结核分枝杆菌是分布广泛的微生物，具有重要的医学和兽医学意义。考虑到野生生物-畜禽-人与人之间的相互关系在流行病学方面的重要性日益提高，开发具有高特异性和敏感性的快速检测工具对于评估它们的存在并加快诊断分枝杆菌病的过程至关重要。在这里，我们描述了一种新型的四重实时荧光定量PCR的开发和评估，用于在内部监控的单一测定中同时检测分枝杆菌属，鸟分枝杆菌亚种和结核分枝杆菌复合物。使用分枝杆菌属（ n = 38）和非分枝杆菌属（ n = 28）菌株的DNA评估了该方法，这些组织掺入了不同CFU量的三种分枝杆菌属物种（ n = 57），档案临床样本（ n = 233）和从各种宿主分离的菌株（ n = 147）。每个反应的最小可检测DNA量对于牛分枝杆菌BCG和堪萨斯分枝杆菌为50 fg，对于鸟分枝杆菌亚种为5 fg。人参。在分析加标样品时，该方法始终可检测到每克低至100至1,000的分枝杆菌CFU。使用经过验证的基于培养物的方法作为参考方法，一组临床样品的敏感性和特异性值为97.5和100％。对临床分离株进行的测定证实了这些结果。该PCR能够在一个反应​​中鉴定同一样品中的鸟分枝杆菌和结核分枝杆菌复合体。总之，我们设计的四重实时荧光定量PCR代表了一种高度特异性和灵敏的工具，可用于常规实验室诊断中分枝杆菌的检测和鉴定，并可能具有其他用途。

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