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
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
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DOI：
10.1128/jcm.00744-11
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发表时间：
2011-09-01
影响因子：
9.4
通讯作者：
Ludes, B.
中科院分区：
文献类型：
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作者：
Bouakaze, C.;Keyser, C.;Ludes, B.
本研究的主要目的是探索一种名为IPLEX Gold(Sequenom)的新的单核苷酸多态(SNP)基因分型技术的潜在用途，该技术用于同时分析16个SNP，这些SNP先前已被证实有助于鉴定结核分枝杆菌复合体(MTBC)物种，并将MTBC分离株划分为不同的遗传谱系，称为主要遗传组(PGGs)和SNP聚类组(SCGs)。在此背景下，我们利用IPLEX Gold Kit(Sequenom)建立了一种基于等位基因特异性引物单碱基延伸反应的16-plex IPLEX分析方法，然后在商用的Sequenom Massarray平台上进行基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOF MS)分析。这项检测是在55株特征良好的MTBC菌株以及10株非MTBC分枝杆菌和4株不属于分枝杆菌属的细菌上进行的，这些菌株也使用了先前报道的快照分析方法对相同的基因座进行了基因分型。所有的MTBC样本都被成功地用IPLEX分析，得到了99.9%的SNPs的明确等位基因数据(879个)。阴性对照无假阳性结果。与快照分析相比，新开发的16-plex IPLEX分析产生了完全一致的结果，允许可靠地区分MTBC物种和识别谱系，从而显示其在诊断、流行病学和进化应用中的潜在价值。与快照方法相比，IPLEX技术的实施可以提供更高的吞吐量，并可能成为微生物实验室更灵活和更具成本效益的选择。
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.