Rapid Detection of rpoB Gene Mutations Conferring Rifampin Resistance in Mycobacterium tuberculosis

Abstract

b Multidrug-resistant Mycobacterium tuberculosis strains are widespread and present a challenge to effective treatment of this infection. The need for a low-cost and rapid detection method for clinically relevant mutations in Mycobacterium tuberculosis that confer multidrug resistance is urgent, particularly for developing countries. We report here a novel test that detects the majority of clinically relevant mutations in the beta subunit of the RNA polymerase (rpoB) gene that confer resistance to rifampin (RIF), the treatment of choice for tuberculosis (TB). The test, termed TB ID/R, combines a novel target and temperature-dependent RNase H2-mediated cleavage of blocked DNA primers to initiate isothermal helicase-dependent amplification of a rpoB gene target sequence. Amplified products are detected by probes arrayed on a modified silicon chip that permits visible detection of both RIF-sensitive and RIF-resistant strains of M. tuberculosis. DNA templates of clinically relevant single-nucleotide mutations in the rpoB gene were created to validate the performance of the TB ID/R test. Except for one rare mutation, all mutations were unambiguously detected. Additionally, 11 RIF-sensitive and 25 RIF-resistant clinical isolates were tested by the TB ID/R test, and 35/36 samples were classified correctly (96.2%). This test is being configured in a low-cost test platform to provide rapid diagnosis and drug susceptibility information for TB in the point-of-care setting in the developing world, where the need is acute.

Attomolar quantitation of Mycobacterium tuberculosis by asymmetric helicase-dependent isothermal DNA-amplification and electrochemical detection

“…Among them, helicase dependent amplification (HDA) is one of the simplest approaches that closely resembles PCR reaction scheme but at a constant temperature, in the presence of helicase for DNA unwinding. Since its first description (Vincent et al, 2004), it has been applied to the detection of several pathogen organisms mainly using electrophoresis (Barbieri et al, 2014), colorimetric (Ao et al, 2012) or fluorimetric (Tong et al, 2012) detection of amplicons. It has also been carried out directly on the surface of microarrays (Andresen et al, 2009) or integrated with microfluidic (Mahalanabis et al, 2010;Ramalingam et al, 2009) and vertical flow strip systems (Jordan et al, 2012) for developing portable devices that can be used in point-of-care testing.…”

Isothermal Nucleic Acid Amplification Techniques and Their Use in Bioanalysis

“…The level of the off target background amplification in HDA is higher than in PCR [29]. In order to minimize the primer/primer duplex formation, primer modifica tion have been suggested [30]. Another approach to min imizing the background amplification involves the use of dimethyl sulfoxide, betaine, and sorbitol, which, unfortu nately, could also inhibit DNA polymerase.…”

MTB-DR-RIF 9G test: Detection and discrimination of tuberculosis and multi-drug resistant tuberculosis strains

“…However, rifampicin-resistant M. tuberculosis (MTB-DR-RIF) strains have mutations in the rpoB gene which make them resistant to rifampicin [7]. Reported studies indicate that there are at least 87 point mutations, short insertions, and deletions in the 81-bp core RRDR of rpoB codons 507 (c507) to 533 (c533) [8].…”