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

David Stucki,Sonia Borrell,Sebastien Gagneux,Bijaya Malla,Mireia Coscollà,Dorothy Yeboah-Manu,Thembela Huna,Iñaki Comas,Julia Feldmann,Lukas Fenner,Simon Hostettler,Igor Mokrousov

doi:10.1371/journal.pone.0041253

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.

Highlights

Genotyping of human-associated Mycobacterium tuberculosis complex (MTBC) plays an increasing role for understanding the epidemiology and biology of tuberculosis (TB) [1,2]
Depending on the particular technique used, different, but phylogenetically equivalent single nucleotide polymorphisms (SNP) sets will be required. These typing methods vary largely in their throughput, cost, and flexibility. With respect to the latter, we identified a lack of methods, which are at the same time flexible and rapid (,1 day/ 96 samples), and amendable to a variable number of strains and a limited number of SNPs (5–50)
Thousands of informative SNPs have become available from whole genome sequences, which can be used for SNP-typing

Summary

Introduction

Genotyping of human-associated Mycobacterium tuberculosis complex (MTBC) plays an increasing role for understanding the epidemiology and biology of tuberculosis (TB) [1,2]. On the one hand, genotyping techniques are key for standard molecular epidemiological investigations of TB such as defining chains of ongoing transmission and differentiating patient relapse from exogenous re-infection [1]. Strain genotyping in MTBC is important for defining the evolutionary background of clinical isolates. There is mounting evidence that the strain genetic background might influence the outcome of TB infection and disease. To study the effect of strain variation and detect relevant genotypephenotype associations, suitable phylogenetic markers and appropriate genotyping methods are required [10]

Methods

Results

Conclusion