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

ABSTRACT Molecular surveillance of rifampin-resistant Mycobacterium tuberculosis can help to monitor the transmission of the disease. The Xpert MTB/RIF Ultra assay detects mutations in the rifampin resistance-determining region (RRDR) of the rpoB gene by the use of melting temperature (Tm) information from 4 rpoB probes which can fall in one of the 9 different assay-specified Tm windows. The large amount of Tm data generated by the assay offers the possibility of an RRDR genotyping approach more accessible than whole-genome sequencing. In this study, we developed an automated algorithm to specifically identify a wide range of mutations in the rpoB RRDR by utilizing the pattern of the Tm of the 4 probes within the 9 windows generated by the Ultra assay. The algorithm builds a RRDR mutation-specific “Tm signature” reference library from a set of known mutations and then identifies the RRDR genotype of an unknown sample by measuring the Tm distances between the test sample and the reference Tm values. Validated using a set of clinical isolates, the algorithm correctly identified RRDR genotypes of 93% samples with a wide range of rpoB single and double mutations. Our analytical approach showed a great potential for fast RRDR mutation identification and may also be used as a stand-alone method for ruling out relapse or transmission between patients. The algorithm can be further modified and optimized for higher accuracy as more Ultra data become available.

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Comparative antimycobacterial activities of rifampin, rifapentine, and KRM-1648 against a collection of rifampin-resistant Mycobacterium tuberculosis isolates with known rpoB mutations.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.11.2655 ◽

1996 ◽

Vol 40 (11) ◽

pp. 2655-2657 ◽

Cited By ~ 69

Author(s):

S L Moghazeh ◽

X Pan ◽

T Arain ◽

C K Stover ◽

J M Musser ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Rna Polymerase ◽

Gene Mutations ◽

Rpob Gene ◽

Genetic Alterations ◽

Beta Subunit ◽

Polymerase Beta ◽

Antimycobacterial Agents ◽

Rifampin Resistance

A collection of 24 rifampin-resistant clinical isolates of Mycobacterium tuberculosis with characterized RNA polymerase beta-subunit (rpoB) gene mutations was tested against the antimycobacterial agents rifampin, rifapentine, and KRM-1648 to correlate levels of resistance with specific rpoB genotypes. The results indicate that KRM-1648 is more active in vitro than rifampin and rifapentine, and its ability to overcome rifampin resistance in strains with four different genetic alterations may prove to be useful in understanding structure-function relationships.

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Amplification Refractory Mutation System – Polymerase Chain Reaction for Rapid Detection of rpoB Gene Mutations in Mycobacterium tuberculosis

International Journal of Applied Sciences and Biotechnology ◽

10.3126/ijasbt.v5i1.17020 ◽

2017 ◽

Vol 5 (1) ◽

pp. 81-85 ◽

Cited By ~ 1

Author(s):

Hemanta Kumari Chaudhary ◽

Mitesh Shrestha ◽

Prakash Chaudhary ◽

Bal Hari Poudel

Keyword(s):

Mycobacterium Tuberculosis ◽

Rpob Gene ◽

Amplification Refractory Mutation System ◽

Chain Reaction ◽

Arms Pcr ◽

Mutation System ◽

Mdr Tb ◽

Total Dna ◽

Polymerase Chain ◽

Rifampin Resistance

Multidrug-resistant tuberculosis (MDR-TB) has become a serious worldwide threat including in Nepal. MDR-TB refers to the pathological condition whereby Mycobacterium tuberculosis becomes resistant to the first line of drug treatment i.e. rifampin and isoniazid. Resistance to rifampin (RIF) is mainly caused by the mutations in the rpoB gene which codes for the β-subunit of RNA polymerase. In this study, Amplification Refractory Mutation System – Polymerase Chain Reaction (ARMS – PCR) technique has been used to detect mutations in the rpoB gene of Mycobacterium tuberculosis. Total DNA samples of 34 phenotypic MDR-TB were subjected to ARMS – PCR using three different codon specific primers (516, 526 and 531). These three codons occupy large portion of total mutation responsible for rifampin resistance. Out of the total DNA samples, all were bearing mutation in at least one of the three codons mentioned. Of those bearing mutation, the highest number had mutation in codon 531 (97.05 %) followed by codon 516 (17.64 %) and finally in codon 526 (11.76%) respectively. Hence, ARMS – PCR may be used as an alternative diagnostic technique for detection of rifampin resistance in Mycobacterium tuberculosis strains, especially for a developing country like Nepal.Int. J. Appl. Sci. Biotechnol. Vol 5(1): 81-85

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