Identification of non-tuberculous mycobacteria by real-time PCR coupled with a high-resolution melting system

Abstract

Non-tuberculous mycobacteria (NTM) are increasingly important opportunistic pathogens responsible for a variety of clinical diseases. The aim of this study was to evaluate a novel technique, real-time PCR coupled with high-resolution melting analysis (real-time PCR-HRMA), for NTM identification. Two pairs of unique primers targeted to the 16S rRNA gene and the 16S-23S internal transcribed spacer region were selected for further evaluation. A total of 149 mycobacterial clinical isolates were subjected to analysis using the real-time PCR-HRMA system. Overall, 134 NTM identified by the 16S rRNA full-gene sequencing method were categorized into four major groups: Mycobacterium avium complex, Mycobacterium chelonae group, Mycobacterium gordonae and Mycobacterium fortuitum group. Of the 134 prevalent mycobacterial isolates, 101 mycobacteria (75.4 %) could be identified correctly by the real-time PCR-HRMA system. The individual sensitivities for the M. avium complex, M. chelonae group, M. gordonae and M. fortuitum groups were 90.9, 89.1, 100 and 36.8 %, respectively. The specificity of identifying these groups varied from 96.4 to 100 %. When identification failed, mostly it was attributable to various species in the M. fortuitum group. The real-time PCR-HRMA system is therefore a rapid and sensitive method for identifying prevalent NTM in a clinical laboratory.

High-Resolution Melting Curve Analysis for Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis: a Meta-Analysis

“…HRMA is a rapid, simple, cost-effective, and closed-tube method, and it has been applied to a variety of diseases, such as inherited, infectious, and oncological diseases (14). This PCR-based method was successfully used for rapid identification and susceptibility testing of Mycobacterium tuberculosis in previous studies (2,4,15,16). On the other hand, previous studies have demonstrated that RIF resistance is a good surrogate marker for MDR-TB, especially when molecular methods are used for rapid detection of RIF-resistant M. tuberculosis isolates, as 90% of RIF-resistant M. tuberculosis isolates, or an even greater proportion, are also resistant to INH (17)(18)(19)(20).…”

Rapid differentiation of mycobacteria by simplex real-time PCR with melting temperature calling analysis

“…The accuracy of our method was lower than that of a two-tube real-time PCR which was coupled with HRMA and did not use oligonucleotide probe (Perng et al 2012).…”

“…The accuracy of our method was lower than that of a two‐tube real‐time PCR which was coupled with HRMA and did not use oligonucleotide probe (Perng et al . ). This largely attributed to relatively higher conservation of 16S rRNA gene and 16S‐23S ITS region, whereas the two‐tube real‐time PCR was only used for differentiation and identification of NTM species; so it did not show the ability to differentiate MTC from NTM.…”

Ultraviolet Light Enhances the Bovine Serum Albumin Fixation for Acid Fast Bacilli Stain

“…A mycobacteria growth indicator tube (MGIT) culture medium that yielded a bacterial growth signal using the MGIT 960 system (Becton Dickinson, Franklin Lakes, NJ, USA) was to be further confirmed for AFB by microscopic observation. All of the AFB positive MGIT broths in this study were identified as MTB complex using the MGIT TBc Identification Test (TBc ID; Becton Dickinson), as described by the manufacturer [14]. An aliquot of the positive MGIT culture was also inoculated onto Lowenstein–Jensen (LJ) slants (Becton Dickinson) for further identification.…”

“…An aliquot of the positive MGIT culture was also inoculated onto Lowenstein–Jensen (LJ) slants (Becton Dickinson) for further identification. The clinical NTM isolates were identified to the species level by DNA sequencing of the partial- length 16S rRNA gene [13], [14].…”