Rapid Detection of Multidrug-Resistant Mycobacterium tube rculosis by Use of Real-Time PCR and High-Resolution Melt Analysis

Abstract

The current study describes the development of a unique real-time PCR assay for the detection of mutations conferring drug resistance in Mycobacterium tuberculosis. The rifampicin resistance determinant region (RRDR) of rpoB and specific regions of katG and the inhA promoter were targeted for the detection of rifampin (RIF) and isoniazid (INH) resistance, respectively. Additionally, this assay was multiplexed to discriminate Mycobacterium tuberculosis complex (MTC) strains from nontuberculous Mycobacteria (NTM) strains by targeting the IS6110 insertion element. High-resolution melting (HRM) analysis following real-time PCR was used to identify M. tuberculosis strains containing mutations at the targeted loci, and locked nucleic acid (LNA) probes were used to enhance the detection of strains containing specific single-nucleotide polymorphism (SNP) transversion mutations. This method was used to screen 252 M. tuberculosis clinical isolates, including 154 RIF-resistant strains and 174 INH-resistant strains based on the agar proportion method of drug susceptibility testing (DST). Of the 154 RIF-resistant strains, 148 were also resistant to INH and therefore classified as multidrug resistant (MDR). The assay demonstrated sensitivity and specificity of 91% and 98%, respectively, for the detection of RIF resistance and 87% and 100% for the detection of INH resistance. Overall, this assay showed a sensitivity of 85% and a specificity of 98% for the detection of MDR strains. This method provides a rapid, robust, and inexpensive way to detect the dominant mutations known to confer MDR in M. tuberculosis strains and offers several advantages over current molecular and culture-based techniques.

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

“…Previous studies have combined HRMA with real-time PCR assays for rapid detection and identification of clinically important bacteria (Ajitkumar et al, 2012;Chen et al, 2011;Cheng et al, 2006, Ricchi et al, 2011Choi et al, 2010;Douarre et al, 2012;Pang et al, 2011;Pietzka et al, 2009;Tortoli, 2009;Won et al, 2010;Yang et al, 2009). The literature suggests that a combined real-time PCR-HRMA assay is a novel and rapid technique suitable for the genotyping of drug-resistant isolates of M. tuberculosis (Chen et al, 2011;Pietzka et al, 2009;Ramirez et al, 2010). Accordingly, the objective of this study was to establish a combined real-time PCR-HRMA assay for common clinical NTM identification.…”

Role of Real Time PCR in Detection of MTB, MDR and XDR TB Directly from Sputum Specimens of Clinical Suspects of PTB

“…14 It was also reported that real time PCR has a sensitivity of 85% and 98% for the detection of MDR TB strains. 15 The result for the determination of clinical efficacy of the real time PCR in the detection of MDR TB was found to be 100% concordant with Gene Xpert. The present study also aimed at the diagnosis of XDR TB among the smear positive as well as smear negative cases, with the diagnosis of one XDR TB case.…”

Validitas Metode Polymerase Chain Reaction GeneXpert MTB/RIF pada Bahan Pemeriksaan Sputum untuk Mendiagnosis Multidrug Resistant Tuberculosis

“…4,5 Beberapa penelitian menyatakan lebih dari 90% penderita TB yang resisten rifampisin, juga mengalami resistensi terhadap isoniazid, sehingga resistensi terhadap rifampisin merupakan penanda pengganti (surrogate marker) yang mewakili suatu MDR-TB. [6][7][8][9] Rifampisin bekerja dengan berikatan terhadap subunit-β ribonucleic acid (RNA) polimerase yang dikode oleh gen rpoB, suatu komponen penting dalam proses transkripsi. Terhambatnya transkripsi RNA ini menyebabkan terhambatnya sintesis protein.…”