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

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 complex (MTBC) and NTM using or RD750, (ii) differentiate () from MTBC using RD9, (iii) selectively identify the widespread Beijing genotype by targeting, and (iv) simultaneously detect five clinically important NTM (, ,, , and) by targeting IS, DT1, , and An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 10 and 10 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 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,, Beijing genotype, and major NTM species.

Antibiograms and Molecular Characterization of Drug Resistance ofMycobacterium abscessuscomplex from Patients with Multidrug-Resistant Pulmonary Tuberculosis (MDR TB) Infection

“…M. abscessus isolates showed the expected 310bp band and M. massiliense showed the 1145bp band using the primers for mass _3210. The mass_ 3210 is a specific gene previously identified and used to discriminate between M. abscessus complex species (Chae et al , 2017). Further analysis of the mass_ 3210 aligned sequences of representative isolates showed two clustered groups: MAB01-MAB05 and MAS01-MAS05 (Figure 4).…”

“…It was centrifuged for 15 minutes at 12,000 x g. The supernatant was transferred to a sterile Eppendorf® tube and was used as the source of DNA for the one-step multiplex PCR assay. The one-step multiplex PCR assay designed by Chae et al (2017) was used (1) to discriminate pan-mycobacterial from non-mycobacterial species by amplifying the16S rRNA gene, (2) to distinguish between MTB complex and NTM species in mycobacteria by amplifying the rv0577 , (3) to identify M. tuberculosis by amplifying the RD9, (4) to identify M. tuberculosis Beijing family by amplifying the mtbk_20680 , and (5) to identify the five major NTM species and subspecies by amplifying IS 1311 ( M. avium ), DT1 ( M. intracellulare ), mass_3210 ( M. abscessus and M. massiliense ), mkan_rs12360 ( M. kansasii ).…”

Development of a cost-effective line probe assay for rapid detection and differentiation of Mycobacterium species

“…Most of the PCR assays which are used for detecting mycobacteria, identify a single or limited number of Mycobacteriums species [24]. Therefore, these methods seem to be inefficient for identifying Mycobacteria to the species level.…”

Twenty-four hour face mask sampling in pulmonary tuberculosis reveals three distinct patterns of bacterial aerosol production dissociated from conventional markers of transmission risk

“…As differences in IS6110 copy number between bacterial strains limits confidence in comparative analysis, a subset of samples for each participant was further analysed using the Mtb-specific RD9 DNA sequence, single copy gene 9 . Comparative statistical analyses have been assessed using both IS6110 copy numbers and genome copy number derived using the ratio between IS6110 and RD9 (see supplemental data).…”