A novel method for the rapid and prospective identification of Beijing Mycobacterium tuberculosis strains by high-resolution melting analysis

Abstract

Genotypic analysis of Mycobacterium tuberculosis (MTB) has enabled the definition of several lineages. The Beijing family, which is considered highly virulent and transmissible, has been associated with resistance in certain settings and involved in severe outbreaks, making it one of the most closely-monitored lineages. Therefore, rapid prospective identification of Beijing MTB strains could be relevant. In the present study, we evaluate a real-time PCR followed by high-resolution melting (HRM) based on the identification of a single nucleotide polymorphism (SNP) in the Rv2629 gene which defines Beijing lineage (A191C for Beijing genotype and A191A for non-Beijing genotype). This combined methodology efficiently differentiated Beijing and non-Beijing strains in 100% of the isolates from a collection of reference strains without requiring specific DNA probes. Additionally, HRM was able to assign a Beijing/non-Beijing genotype in 90.9% of the respiratory specimens assayed. Its applicability was tested on a Peruvian sample of circulating MTB strains, in which it identified 10.7% as belonging to the Beijing genotype; this proportion reached 20% in the North Lima area. HRM analysis of the A191C SNP is a rapid, reliable, and sensitive method for the efficient prospective survey of high-risk Beijing MTB strains, even in developing settings where MTB culture is often not available.

Rapid Test for Identification of a Highly Transmissible Mycobacterium tuberculosis Beijing Strain of Sub-Saharan Origin

“…MIRU loci, specific deletions, or single-nucleotide polymorphisms (SNPs). However, most of these methods for identification are not specific, expensive, or difficult to perform (4,7,8,17). The results obtained in this work show that our multiplex PCR has demonstrated the specificity to identify the GC1237 genotype isolates.…”

Importance of the Genetic Diversity within the Mycobacterium tuberculosis Complex for the Development of Novel Antibiotics and Diagnostic Tests of Drug Resistance

“…In addition, phylogenetic analyses showed that the D64A mutation is not associated with RIF resistance but instead constitutes a marker for the Beijing lineage (29,74,97,115). As a result, an assay using high-resolution melting analysis that relies on this change has been developed to allow for the rapid identification of Beijing strains (8). The usefulness of this assay has recently been called into question by a report that identifies this mutation in non-Beijing strains as well (174).…”

Analysis of Mutations in Streptomycin-Resistant Strains Reveals a Simple and Reliable Genetic Marker for Identification of the Mycobacterium tuberculosis Beijing Genotype

“…Other methods are based on the detection of specific genomic deletions, such as region of difference 105 (RD105) or an intact pks15/1 gene (8). Recently, the detection of a single-nucleotide polymorphism (SNP) in the Rv2629 gene by using real-time PCR followed by high-resolution melting has also been described (3).…”

“…The isolates of the Beijing family of M. tuberculosis are characterized by their increased ability to spread and cause disease and their increased association with drug resistance in comparison with the levels for non-Beijing strains (2). Because of this, many methods have been developed that are aimed at identifying and differentiating M. tuberculosis Beijing isolates (3)(4)(5)(6). There are several methods for identifying Beijing isolates, including spoligotyping, restriction fragment length polymorphism (RFLP) using IS6110 as a probe, or the presence of a copy of IS6110 in the dnaA region (7).…”