RpoB gene

Some strains of Mycobacterium tuberculosis, the causative agent of tuberculosis, are resistant to rifampicin. These strains become resistant through mutations that alter the rpoB gene, which encodes the /3 subunit of the RNA polymerase. Rifampicin cannot bind to the mutant RNA polymerase and so is unable to block the initiation of transcription. DNA sequences from a large number of rifampicin-resistant M. tuberculosis strains have been found to have mutations in a specific 69 bp region of rpoB. One well-characterized strain with rifampicin resistance has a single base pair alteration in rpoB that results in a single amino acid substitution in the J3 subunit a His residue is replaced by an Asp residue. 

In combination with an alkaline phosphatase reaction-linked assay, these two schemes have been used successfully for the identification of mutations in the rpoB gene of Mycobacterium tuberculosis from clinical isolates that show rifampin resistance (Rifr). The advantages and disadvantages of the new approach are discussed. 

Step 2. Amplify a 130 bp segment of the rpoB gene that contains the RRDR region by PCR and purify the product. 

Table 4 Probes designed to detect mutations in the rpoB gene ...

The scheme was first used to scan the 130 bp rpoB gene PCR products and to detect mutations occurring in codon 531, which arise frequently in Rifr M. tuberculosis. In this scheme, only one common probe is needed in the reaction to detect four alleles located at different positions of codon 531. Two substitutions (CGA > CAA and CGA > CCA) were correctly identified by this scheme [26]. 

Table 5 Detection of rpoB gene mutations of M. tuberculosis in Wuhan by SOLAC compared with DNA sequencing results...

The RRDR of the rpoB gene was subsequently sequenced to analyze the mutations associated with rifampin resistance and to verify the detection results of the SOLAC-GOS assay. DNA sequencing analysis of the 55 Rifr isolates showed that 50 strains had point mutations, one strain had a double mutation, and one strain had a one-base deletion in the 81-bp RRDR of the rpoB gene. Three strains were found to have no mutation. A total of 52 mutations of 12 different types including 11 point mutations and one deletion were identified (Table 5). The most frequent mutations were found at codons 531, 526, and 516, with frequencies of 52.7,23.6, and 9.1%, respectively. Similar results have been reported by other investigators [36-38]. 

Renouf, V., Claisse, O., Miot-Sertier, C, Lonvaud-Funel, A. (2006). LAB evolution during winemaking use of the rpoB gene as target for PCR-DGGE analysis. Food Microbiol., 23,136-145. 

Drancourt M, Raoult D (2002) rpoB gene sequence-based identification of Staphylococcus species. J Clin Microbiol 40 1333-1338... 

Rantsiou, K., Comi, G., Cocolin, L. (2004). The rpoB gene as a target for PCR-DGGE analysis to follow lactic acid bacterial population d5mamics during food fermentations. Food Microbiology, 21, 481 87. 

Case RJ, Boucher Y, DahUdf I, Hoknstrom C, Doolittle WF, Kjelleberg S. Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology smdies. Appl Environ Microbiol 2007 73(l) 278-288. 

Qaisse, O., Renouf, V., and Lonvaud-Funel, A. (2007) Differentiation of wine lactic add baclaia spedes based on RFLP analysis of a partial sequence of rpoB gene. J Microbiol Met 69, 387-390. 

RIF is bactericidal against M. tuberculosis at 0.005-0.2 pg/mL and works by inhibiting RNA synthesis by binding to the bacterial DNA-dependent RNA polymerase P-subunit encoded by the rpoB gene. Resistance occurs rapidly when used alone and arises because of a mutation in a defined 81-bp region of the rpoB gene and can cause cross-resistance to other rifamycins such as rifabutin and... 

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