Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

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

Spoligotyping, single nucleotide polymorphism (SNP) and region of difference (RD) PCR results from the three <i>Mycobacterium tuberculosis</i> isolates belonging to Lineage 3.

1<p>Spoligotype International Type (SIT) according to the definition in SpolDB4 database using SITVIT2 (<a href="http://www.pasteur-guadeloupe.fr:8081/SITVITDemo/index.jsp" target="_blank">http://www.pasteur-guadeloupe.fr:8081/SITVITDemo/index.jsp</a>).</p><p>+, present; −, absent; RD, region of difference; SNP, single nucleotide polymorphism.</p

Sequence information of probes and primers used in this study to detect main phylogenetic lineages of <i>Mycobacterium tuberculosis</i> complex isolates by single nucleotide polymorphisms genotyping.

<p>*as reported in Ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024737#pone.0024737-Comas1" target="_blank">[8]</a>.</p><p>F: forward; R: reverse; SNP, single nucleotide polymorphisms.</p><p>Probes are minor groove binder probes.</p

Primer and probe sequences for TaqMan assays.

1<p>Nomenclature according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041253#pone.0041253-Coscolla1" target="_blank">[2]</a>.</p>2<p>Nomenclature according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041253#pone.0041253-Gagneux1" target="_blank">[20]</a>.</p>3<p>Nomenclature according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041253#pone.0041253-Filliol2" target="_blank">[72]</a>.</p>4<p>Position of SNP in reference to the H37Rv genome.</p>5<p>6FAM and VIC, fluorescent dyes at the 5′-end of probes; MGBNFQ, MinorGrooveBinder-NonFluorescentQuencher at the 3′-end.</p

Lineage assignments of MOL-PCR, TaqMan and spoligotyping for the 46 MTBC strains as in Table S1.

1<p>Lineage-classification using spoligotype prototypes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041253#pone.0041253-Comas1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041253#pone.0041253-KatoMaeda2" target="_blank">[63]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041253#pone.0041253-Demay1" target="_blank">[64]</a>.</p>2<p>As described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041253#pone.0041253-Demay1" target="_blank">[64]</a>.</p>3<p>No lineage-classification possible based on spoligotyping.</p>4<p>“Pseudo-Beijing” as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041253#pone.0041253-Fenner1" target="_blank">[14]</a>.</p

Schematic illustration of MOL-PCR.

<p>A. Three oligonucleotides and two fluorescently labelled beads are used for interrogation of one SNP. B. MOL-PCR consists of three steps. 1. One of two competitive allele-specific left-hand probe oligonucleotides (LPO) and one universal right-hand probe oligonucleotide (RPO) are hybridized to the template DNA and ligated. 2. With PCR and a reporter-labelled forward primer, the ligated oligonucleotides (LPO+RPO) are amplified. 3. After denaturation of the PCR product, allele-specific fluorescent beads carrying an allele-specific <i>antiTag</i> sequence are hybridized to the amplicons. This will result in beads carrying reporter fluorescence (bead 1 in example) and beads not carrying fluorescence (bead 2). Reporter fluorescence (Alexa532) per bead is measured with a flow cytometric device (Luminex). For the 8-plex assay, a total of 24 oligonucleotides and 16 beads are used in the same reaction.</p

Oligonucleotides used for Luminex MOL-PCR.

1<p>Nomenclature according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041253#pone.0041253-Coscolla1" target="_blank">[2]</a>.</p>2<p>Position of SNP in reference to H37Rv.</p>3<p>SNP in an essential gene (“ess”); synonymous SNP (“syn”).</p>4<p>Strand orientation of oligonucleotides.</p>5<p>LPO, left probe oligonucleotide, annealing upstream of SNP of interest and including it; RPO, right probe oligonucleotide, annealing downstream of SNP of interest.</p>6<p>x<i>Tag</i> bead with coupled <i>antiTag</i>-sequence.</p>7<p>Universal primer sequences (upper case, not underlined), allele-specific <i>tag</i>-sequences (lower case), and sequences hybridizing to the template genomic sequence (underlined). RPO are 5′-phosphorylated.</p

Comparison of MOL-PCR and TaqMan for allele calling and lineage assignment.

1<p>excluding N/A as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041253#pone.0041253.s009" target="_blank">Table S1</a>.</p>2<p>excluding M. microti, M. pinnipedi.</p>3<p>excluding <i>M. bovis</i>, <i>M. caprae</i>, <i>M. microti</i>, <i>M. pinnipedii</i>.</p

"Pseudo-Beijing" : evidence for convergent evolution in the direct repeat region of Mycobacterium tuberculosis

Mycobacterium tuberculosis has a global population structure consisting of six main phylogenetic lineages associated with specific geographic regions and human populations. One particular M. tuberculosis genotype known as "Beijing" has repeatedly been associated with drug resistance and has been emerging in some parts of the world. "Beijing" strains are traditionally defined based on a characteristic spoligotyping pattern. We used three alternative genotyping techniques to revisit the phylogenetic classification of M. tuberculosis complex (MTBC) strains exhibiting the typical "Beijing" spoligotyping pattern.; MTBC strains were obtained from an ongoing molecular epidemiological study in Switzerland and Nepal. MTBC genotyping was performed based on SNPs, genomic deletions, and 24-loci MIRU-VNTR. We identified three MTBC strains from patients originating from Tibet, Portugal and Nepal which exhibited a spoligotyping patterns identical to the classical Beijing signature. However, based on three alternative molecular markers, these strains were assigned to Lineage 3 (also known as Delhi/CAS) rather than to Lineage 2 (also known as East-Asian lineage). Sequencing of the RD207 in one of these strains showed that the deletion responsible for this "Pseudo-Beijing" spoligotype was about 1,000 base pairs smaller than the usual deletion of RD207 in classical "Beijing" strains, which is consistent with an evolutionarily independent deletion event in the direct repeat (DR) region of MTBC.; We provide an example of convergent evolution in the DR locus of MTBC, and highlight the limitation of using spoligotypes for strain classification. Our results indicate that a proportion of "Beijing" strains may have been misclassified in the past. Markers that are more phylogenetically robust should be used when exploring strain-specific differences in experimental or clinical phenotypes