Two New Rapid SNP-Typing Methods for Classifying Mycobacterium tuberculosis Complex into the Main Phylogenetic Lineages

Abstract

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the “Beijing” sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae . Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive.

Novel Single Nucleotide Polymorphism-Based Assay for Genotyping Mycobacterium avium subsp. paratuberculosis

“…Although WGS is becoming cheaper, it is still too expensive to be used for routine genotyping of M. avium subspecies paratuberculosis isolates and requires robust data handling and analysis processes. Single nucleotide polymorphisms (SNPs) have been used successfully to type several genetically monomorphic pathogens, including M. tuberculosis (13), Mycobacterium bovis (14), Salmonella enteritica serovar Typhi (15), and Yersinia pestis (16). SNP assays have been used to discriminate between M. avium subspecies paratuberculosis strain types I, II, and III (17,18) and between strains derived from animal and human hosts (19).…”

Simple Multiplex PCR Assay for Identification of Beijing Family Mycobacterium tuberculosis Isolates with a Lineage-Specific Mutation in Rv0679c

“…Since this typical spoligotype pattern has become a specific marker of the Beijing genotype, some PCR methods to detect this specific deletion, named region of difference 207 (RD207), have been developed (20)(21)(22). In addition to RD207, another deleted region named RD105 was also shown to be a good marker for discrimination of the Beijing genotype, although this deletion is common for all the east Asian lineages, including the non-Beijing strains (10,23); however, most of these published detection methods require expensive real-time PCR equipment and high-cost reagents (24). The conventional PCR assay targeting RD207 still seems to be at a disadvantage, since it relies on an unstable inser-tion sequence that is likely to be a target of homologous recombination.…”

“…The conventional PCR assay targeting RD207 still seems to be at a disadvantage, since it relies on an unstable inser-tion sequence that is likely to be a target of homologous recombination. Instead of unstable repetitive structures, single nucleotide polymorphisms (SNPs) were recently considered to be a robust target for defining the accurate position of a strain on the phylogenetic tree, since horizontal gene transfer or gene recombination between different strains is rare in the M. tuberculosis complex (MTC) (12,24,25). Filliol et al (26) drew phylogenetic trees of the MTC using several typing methods and showed that the dendrogram drawn with SNPs most accurately reflected the true evolution of the MTC.…”

Mycobacterial Typing Systems: The Good, the Bad and the Unattainable

“…Where there is a low IS6110 copy number, other typing methods must be employed to facilitate sufficient discrimination. Typing methods that rely on single nucleotide polymorphisms (SNPs) and polymorphic GC-rich repetitive sequences (PGRS) have proven to be effective [8][9][10].…”