Fast detection of the V176F mutation of multiresistant Mycobacterium tuberculosis isolates using high-resolution melting curve PCR analysis

Abstract number: P1555

Pietzka A., Stöger A., Zeinzinger J., Allerberger F., Ruppitsch W.

Objective: In tuberculosis (TB), drug resistance to rifampicin, one of the two most potent first-line drugs, is increasing globally. Resistance to rifampicin is mainly associated with single point mutations in the RNA polymerase gene (rpoB) gene. Several point mutations occur in the 81-bp hot-spot region of cluster I (codons 432 to 458) of the rpoB gene. A frequent mutation outside cluster I associated with high-level resistance to rifampicin is the V176F (GTC176TTC) mutation. To improve the detection of multidrugresistant (MDR)-TB isolates with wild type cluster I rpoB sequence, we describe a fast and accurate detection method for the V176F mutation based on high-resolution melting curve PCR analysis.

Methods: Forty-nine rifampicin resistant and nineteen fully susceptible Mycobacterium tuberculosis strains, as determined by conventional drug susceptibility testing, were used to develop a PCR assay to detect the V176F mutation. A 125 bp fragment of the rpoB gene containing the amino acid exchange was amplified for HRM analysis on a LightCycler480 instrument (Roche Diagnostics, Penzberg, Germany).

Results: Three of the forty-nine resistant isolates (6%) showed the single nucleotide polymorphism (SNP) class 2 sequence alteration V176F. The remaining forty-six resistant isolates didn't have any mutation in the 125 bp amplicon as well as the susceptible isolates.

Conclusions: Rifampicin resistance is associated with MDR-TB and is therefore a surrogate marker for genotypic drug susceptibility testing. Molecular detection of mutations within cluster I of the rpoB gene is already well known. In addition we describe the feasibility of a new single-step closed-tube screening method for the occurrence of the frequent V176F mutation in the rpoB gene which allows a broad detection of multi drug resistance and which can easily be combined with an assay for mutation testing in cluster I.

Figure: V176F mutation.