Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based single nucleotide polymorphism genotyping assay using iPLEX gold technology for identification of Mycobacterium tuberculosis complex species and lineages

Abstract

The major goal of the present study was to investigate the potential use of a novel single nucleotide polymorphism (SNP) genotyping technology, called iPLEX Gold (Sequenom), for the simultaneous analysis of 16 SNPs that have been previously validated as useful for identification of Mycobacterium tuberculosis complex (MTBC) species and classification of MTBC isolates into distinct genetic lineages, known as principal genetic groups (PGGs) and SNP cluster groups (SCGs). In this context, we developed a 16-plex iPLEX assay based on an allele-specific-primer single-base-extension reaction using the iPLEX Gold kit (Sequenom), followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis on the commercially available Sequenom MassARRAY platform. This assay was tested on a panel of 55 well-characterized MTBC strains that were also genotyped for the same loci using the previously reported SNaPshot assay, as well as 10 non-MTBC mycobacteria and 4 bacteria not belonging to the genus Mycobacterium. All MTBC samples were successfully analyzed with the iPLEX assay, which yielded clear allelic data for 99.9% of the SNPs (879 out of 880). No false-positive results were obtained with the negative controls. Compared to the SNaPshot assay, the newly developed 16-plex iPLEX assay produced fully concordant results that allowed reliable differentiation of MTBC species and recognition of lineages, thus demonstrating its potential value in diagnostic, epidemiological, and evolutionary applications. Compared to the SNaPshot approach, the implementation of the iPLEX technology could offer a higher throughput and could be a more flexible and cost-effective option for microbiology laboratories.

Fig. 1.

Principle of SNP analysis by using two independent SNP genotyping technologies: the SNaPshot technology (Applied Biosystems) and the iPLEX technology (Sequenom). These genotyping methods comprise three main steps, namely, PCR amplification of the DNA region containing the polymorphic site (step 1), an allelic discrimination reaction consisting of an SBE reaction (step 2), and detection of allele-specific products (step 3).

Fig. 2.

M. tuberculosis H37Rv and M. bovis CIP102426 electropherograms and mass spectra obtained using the 16-plex SNaPshot assay and 16-plex iPLEX assay, respectively. Electropherograms (A.a. and B.a.) were generated with GeneMapper (version 4) software (AB) and show the relative fluorescence units (RFUs) versus the measured size (in nucleotides) of the SBE products relative to the GeneScan-120 LIZ internal size standard (AB). Mass spectra (A.b. and B.b.) were generated using MassARRAY Typer (version 4.0.5) software (Sequenom) and show the relative intensity versus the mass of the analytes. Mutated alleles are indicated by arrows.