Improved Short-Sequence-Repeat Genotyping of Mycobacterium avium subsp paratuberculosis by Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry
Improved Short-Sequence-Repeat Genotyping of Mycobacterium avium subsp paratuberculosis by Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry
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DOI：
10.1128/aem.03212-13
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发表时间：
2014-01-01
影响因子：
4.4
通讯作者：
De Buck, Jeroen
中科院分区：
文献类型：
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作者：
Ahlstrom, Christina;Barkema, Herman W.;De Buck, Jeroen
单核苷酸重复区的精确序列分析是困难的，使使用短序列重复（SSR）作为细菌菌株区分的工具变得复杂。虽然鸟分枝杆菌鸟亚种（Mycobacterium avium subsp.）副结核分枝杆菌允许M.鸟亚种尽管副结核分枝杆菌分离株具有高区分能力，但由于测序单核苷酸重复序列的固有困难，大多数区分性基因座的进一步表征受到限制。在此，使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）作为桑格测序的替代方法来评价一种方法，以进一步区分优势分枝杆菌散布重复单位（MIRU）-可变数目串联重复（VNTR）M。鸟亚种在加拿大奶牛群中，通过针对高度歧视性的montubertide SSR位点，对副结核菌型（n = 37）进行了研究。首先，用两种限制性内切酶消化PCR扩增的DNA以产生含有SSR位点的足够小的片段。其次，进行MALDI-TOF MS以鉴定靶的质量，从而鉴定靶的重复长度。获得足够强的鉴别光谱，以确定重复长度高达15，使用传统测序技术超过11的限制的改进。与合成寡核苷酸和桑格测序结果的比较证实了有效且可重复的测定，其增加了对优势M的区分。鸟亚种副结核MIRU-VNTR型。因此，MALDI-TOF MS是一种可靠、快速和自动化的技术，可以准确地分离M。鸟亚种基于SSR的副结核病基因型。
Accurate sequence analysis of mononucleotide repeat regions is difficult, complicating the use of short sequence repeats (SSRs) as a tool for bacterial strain discrimination. Although multiple SSR loci in the genome of Mycobacterium avium subsp. paratuberculosis allow genotyping of M. avium subsp. paratuberculosis isolates with high discriminatory power, further characterization of the most discriminatory loci is limited due to inherent difficulties in sequencing mononucleotide repeats. Here, a method was evaluated using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as an alternative to Sanger sequencing to further differentiate the dominant mycobacterial interspersed repetitive-unit (MIRU)-variable-number tandem-repeat (VNTR) M. avium subsp. paratuberculosis type (n = 37) in Canadian dairy herds by targeting a highly discriminatory mononucleotide SSR locus. First, PCR-amplified DNA was digested with two restriction enzymes to yield a sufficiently small fragment containing the SSR locus. Second, MALDI-TOF MS was performed to identify the mass, and thus repeat length, of the target. Sufficiently intense, discriminating spectra were obtained to determine repeat lengths up to 15, an improvement over the limit of 11 using traditional sequencing techniques. Comparison to synthetic oligonucleotides and Sanger sequencing results confirmed a valid and reproducible assay that increased discrimination of the dominant M. avium subsp. paratuberculosis MIRU-VNTR type. Thus, MALDI-TOF MS was a reliable, fast, and automatable technique to accurately resolve M. avium subsp. paratuberculosis genotypes based on SSRs.