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, SungJae Shin

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)2736-2751
Number of pages16
JournalJournal of Clinical Microbiology
Volume55
Issue number9
DOIs
Publication statusPublished - 2017 Sep 1

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Multiplex Polymerase Chain Reaction
Mycobacterium
Mycobacterium tuberculosis
Genotype
Multilocus Sequence Typing
Computational Biology
Sputum
Real-Time Polymerase Chain Reaction
Tuberculosis
Genome
Polymerase Chain Reaction
DNA
Infection

All Science Journal Classification (ASJC) codes

  • Microbiology (medical)

Cite this

Chae, Hansong ; Han, Seung Jung ; Kim, Su Young ; Ki, Chang Seok ; Huh, Hee Jae ; Yong, DongEun ; Koh, Won Jung ; Shin, SungJae. / Development of a one-step multiplex PCR assay for differential detection of major Mycobacterium species. In: Journal of Clinical Microbiology. 2017 ; Vol. 55, No. 9. pp. 2736-2751.
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Development of a one-step multiplex PCR assay for differential detection of major Mycobacterium species. / Chae, Hansong; Han, Seung Jung; Kim, Su Young; Ki, Chang Seok; Huh, Hee Jae; Yong, DongEun; Koh, Won Jung; Shin, SungJae.

In: Journal of Clinical Microbiology, Vol. 55, No. 9, 01.09.2017, p. 2736-2751.

Research output: Contribution to journalArticle

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