Newly Developed Primers for Comprehensive Amplification of the rpoB Gene and Detection of Rifampin Resistance in Mycobacterium tuberculosis

Abstract

New rpoB gene primers for detecting Rif r in Mycobacterium tuberculosis complex bacteria achieved 100% specificity and 88% (fresh sputa) and 92% (ethanol-preserved sputa) diagnostic sensitivity and detected up to 4 CFU/sample. Of the 99 Rif r isolates examined, 97% had mutations within cluster I, 2% at codon 176, and 1% at codon 497.Molecular detection of rifampin-resistant (Rif r ) Mycobacterium tuberculosis usually relies on amplification of the hotspot zone for resistance-conferring mutations (cluster I, covering codons 432 to 458 according to the M. tuberculosis nomenclature) (7) of the rpoB gene (4,8,10,12,13,17). Previous studies have shown that 94 to 98% of Rif r M. tuberculosis strains show a mutation in cluster I (10,11,14,15). Resistance-associated mutations have also been described for cluster II (codons 496 and 497) and for codons 176, 486, 558, and 598 (1, 6, 7).We describe and evaluate new primers, covering the entire region with all currently known significant mutations in a single assay.Oligonucleotides were designed on the basis of an alignment of the rpoB gene sequence from the H37Rv M. tuberculosis reference strain (NC 000962; NCBI bank), some relevant nontuberculous mycobacteria (NTM), and nonmycobacterial species using ClustalX (version 1.83.1) software. Amplify software (version 1.2; University of Wisconsin-Madison) was used to estimate the stabilities and binding capacities of the selected oligonucleotides and to simulate PCRs. Figure 1 shows the relative locations of the selected primers.A single PCR with primers rpoBgeneSA (5Ј-GGTTCGCCGC GCTGGCGCGAAT-3Ј) and rpoBgeneRB (5Ј-GACCTCCTCG ATGACGCCGCTTTCT-3Ј) was used for bacterial suspensions, whereas a nested PCR with primers rpoBgeneSAnew (5Ј-GCAA AACAGCCGCTAGTCCTAGTCCGA-3Ј) and rpoBgeneRA (5Ј-GCGCCATCTCGCCGTCGTCAGTACAG-3Ј) for the first run, and rpoBgeneSA and rpoBgeneRB as inner primers, was used to amplify clinical specimens.The first run of the nested PCR was performed with a final volume of 50 l containing 10 mM Tris-HCl (pH 8.6), 50 mM KCl, 1.65 mM MgCl 2 , 200 M of each deoxynucleoside triphosphate, 12.5 pmol of each primer, 1.5 U Taq polymerase (Promega, Madison, WI), and 5 l of DNA extract from clinical specimens. PCR was performed using a PTC 100 MJResearch thermocycler (Whaltham, MA) as follows: a hot start (90°C) followed by 5 min at 94°C; 45 cycles of 45 s at 94°C, 1 min 30 s at 66°C, and 45 s at 72°C; and a final extension of 10 min at 72°C. The second run was performed with a final volume of 25 l enzyme mixture with 0.5 U Taq polymerase and 0.25 l of the first PCR amplicon as follows: a hot start (90°C) followed by 5 min at 94°C, 29 cycles of 45 s at 94°C, 1 min 45 s at 72°C (annealing and extension), and a final extension of 10 min at 72°C. For bacterial suspensions, a single PCR was run under similar conditions but using a 50-l enzyme mixture and 45 cycles. Amplicons were analyzed with a 2% (wt/vol) agarose gel.DNA was extracted from sputum by an adapted Boom extraction method (16) and from bacterial suspensions in 1ϫ TE bu...

Rifampin Resistance Missed in Automated Liquid Culture System for Mycobacterium tuberculosis Isolates with Specific rpoB Mutations

“…Detection of rpoB mutations, targeting a 1,674-bp region from codon 176 to 672, from which RMP resistance-conferring mutations have been described, was performed as described before (10). DNA extracts from clinical specimens were prepared using an adapted Maxwell extraction method (Promega).…”

Evaluation of Direct Detection of Mycobacterium tuberculosis Rifampin Resistance by a Nitrate Reductase Assay Applied to Sputum Samples in Cotonou, Benin

“…On the isolate with discordant result, rpoB gene sequencing was performed at the ITM as described previously (16).…”

Detection of Mycobacterium tuberculosis and Drug Resistance: Opportunies and Challenges in Morocco

“…Rare loci found outside the hotspot region of rpoB are associated with resistance without associated mutation known for conferring resistance (Taniguchi, 1996;Fang, 1999;Schilke, 1999;Yuen, 1999;Heep, 2001;Zhang, 2005;Rigouts, 2007;Prammananan, 2008).…”

“…Mutated strains in Val146Phe (Heep, 2001;Rigouts, 2007;Prammananan, 2008) show a lowlevel resistance (MIC 4 µg/ml) (Rigouts, 2007). The Ala381Val mutation (Taniguchi, 1996) is described on a strain of MIC 200, with no other mutation on the rpoB gene.…”