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Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species

 Hansong Chae  ;  Seung Jung Han  ;  Su-Young Kim  ;  Chang-Seok Ki  ;  Hee Jae Huh  ;  Dongeun Yong  ;  Won-Jung Koh  ;  Sung Jae Shin 
 Journal of Clinical Microbiology, Vol.55(9) : 2736-2751, 2017 
Journal Title
 Journal of Clinical Microbiology 
Issue Date
Humans ; Multilocus Sequence Typing/methods ; Multiplex Polymerase Chain Reaction/methods* ; Mycobacterium Infections, Nontuberculous/diagnosis* ; Mycobacterium Infections, Nontuberculous/microbiology ; Mycobacterium tuberculosis/genetics ; Mycobacterium tuberculosis/isolation & purification* ; Nontuberculous Mycobacteria/genetics ; Nontuberculous Mycobacteria/isolation & purification* ; RNA, Ribosomal, 16S/genetics ; Tuberculosis, Pulmonary/diagnosis* ; Tuberculosis, Pulmonary/microbiology
Beijing genotype ; Mycobacterium abscessus complex ; Mycobacterium avium complex ; Mycobacterium kansasii ; Mycobacterium tuberculosis ; multiplex PCR ; nontuberculous mycobacteria
The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis (M. tuberculosis) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680, and (iv) simultaneously detect five clinically important NTM (M. avium, M. intracellulare, M. abscessus, M. massiliense, and M. kansasii) by targeting IS1311, DT1, mass_3210, and mkan_rs12360 An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 103 and 104 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis, M. tuberculosis Beijing genotype, and major NTM species.
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1. College of Medicine (의과대학) > Dept. of Microbiology (미생물학교실) > 1. Journal Papers
1. College of Medicine (의과대학) > Dept. of Laboratory Medicine (진단검사의학교실) > 1. Journal Papers
Yonsei Authors
신성재(Shin, Sung Jae) ORCID logo https://orcid.org/0000-0003-0854-4582
용동은(Yong, Dong Eun) ORCID logo https://orcid.org/0000-0002-1225-8477
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