Kim J-U, Cha C H, An H K
Department of Laboratory Medicine, Gangneung Asan Hospital, University of Ulsan College of Medicine, Gangneung, Korea.
J Appl Microbiol. 2015 Jun;118(6):1498-506. doi: 10.1111/jam.12780. Epub 2015 Mar 19.
To directly identify clinically relevant mycobacteria from clinical specimens, we have developed a multiplex real-time PCR assay with hydrolysis probes that can identify 20 mycobacterial species.
The assay was initially evaluated using 248 strains, including both reference strains and clinical isolates. Then, the assay was implemented according to a scheme in our laboratory. The scheme based on the clinical differences between the Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) consisted of three stepwise PCRs. MTC and NTM were differentially detected in the step 1 PCR, and the NTM species were identified in the step 2 and step 3 PCRs. During a 2·5-year period, 1877 isolates of MTC (1142 directly recovered from clinical specimens) and 596 isolates of NTM (143 directly recovered from clinical specimens) were detected, and the species of 590 (99·0%) of the 596 NTM isolates were identified.
Our experience shows that this is a new paradigm for rapidly and accurately identifying clinically relevant mycobacteria, in which a multiplex real-time PCR assay is directly applied to clinical specimens in a stepwise fashion.
This study is the first report of a multiplex real-time PCR assay for identifying clinically important mycobacterial species directly from clinical specimens and its application in a clinical microbiology laboratory.
为了直接从临床标本中鉴定出具有临床相关性的分枝杆菌，我们开发了一种带有水解探针的多重实时PCR检测方法，该方法可鉴定20种分枝杆菌。
该检测方法最初使用包括参考菌株和临床分离株在内的248株菌株进行评估。然后，按照我们实验室的方案实施该检测方法。该方案基于结核分枝杆菌复合群（MTC）和非结核分枝杆菌（NTM）之间的临床差异，由三步PCR组成。在第一步PCR中对MTC和NTM进行差异检测，在第二步和第三步PCR中鉴定NTM的种类。在2.5年的时间里，共检测到1877株MTC分离株（其中1142株直接从临床标本中获得）和596株NTM分离株（其中143株直接从临床标本中获得），并鉴定出了596株NTM分离株中590株（99.0%）的种类。
我们的经验表明，这是一种快速准确鉴定具有临床相关性分枝杆菌的新范式，即直接将多重实时PCR检测方法逐步应用于临床标本。
本研究首次报道了一种直接从临床标本中鉴定临床上重要分枝杆菌种类的多重实时PCR检测方法及其在临床微生物实验室中的应用。
