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 wellcharacterized 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. (1,8,12,37). Although M. tuberculosis is the predominant causative agent of human tuberculosis, each member of this complex has been implicated in human infection, except M. mungi so far (8,26). Moreover, two members, M. bovis, the causative agent of zoonotic bovine tuberculosis, and M. canettii, an unusual member responsible for rare tuberculosis cases almost always exposed to Africa, are naturally resistant to pyrazinamide, a first-line antituberculous drug (18,34). Therefore, the rapid and reliable identification of MTBC isolates to the species level is of prime importance for timely selection of appropriate patient antibiotic treatment and also for epidemiological and public health considerations (36). Furthermore, various studies have recently identified distinct phylogenetic groupings within the human-adapted members of the MTBC (i.e., M. tuberculosis and M. africanum species), all of which are congruent (2, 5, 10, 14-16, 19, 20, 32). As shown in Table 1, these MTBC members are currently classified into six major phylogenetic lineages, two of which are composed of M. africanum strains. These six major lineages were first identified by analysis of genomic deletions or large sequence polymorphisms (LSPs), but they are highly congruent to the ones defined by single nucleotide polymorphisms (SNPs)...

Molecular Epidemiology of Mycoplasma pneumoniae: Genotyping Using Single Nucleotide Polymorphisms and SNaPshot Technology

“…A major advantage of this technology is that the eight SNP sites can be simultaneously analyzed, significantly reducing the cost and analysis time, as the automated fluorescent capillary electrophoresis analysis of minisequencing products requires only 30 min. A recent study described the simultaneous investigation of 16 SNPs in a single 16-plex SNaPshot assay (40), supporting the fact that SNaPshot assays can be readily multiplexed to a level higher than suggested, as the manufacturer recommends a limit of 10 SNPs per assay. Thus, this flexibility facilitates updating the data set with additional new SNPs.…”

Mass spectrometry: a revolution in clinical microbiology?

“…[83] , Mycobacterium sp. [84,85] ) have been reported. All these developments are promising notably since the publication by Gaillot et al on cost-effectiveness of MS.…”

“…MassARRAY iPLEX is more efficient than a sequencing method; however, the analysis by MALDI-TOF MS is much faster than the analysis by capillary electrophoresis, requiring a few seconds for the former one and up to several minutes for the latter one [85] . The iPLEX assay is suitable for high-throughput analysis, as either 96 or 384 samples can be analyzed on the same chip.…”

Clinical Laboratory Diagnostics for Mycobacterium tuberculosis

“…The use of mass spectrometry for the identification of Mtb complex from culture isolates may replace current standards such as biochemical analysis, nucleic acid hybridization probes, and DNA sequencing due to the ability of mass spectrometry to rapidly and accurately identify Mtb in a cost-effective manner while minimizing technologist hands-on time and effort (Saleeb et al, 2011). Although still in it's infancy in the diagnostic microbiology laboratory, this technology may also have utility in predicting drug resistance patterns and evaluating epidemiologic groups (Bouakaze et al, 2011;Massire et al, 2011;Schurch et al, 2011).…”