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• W2034395940 endingPage "539" @default.
• W2034395940 startingPage "534" @default.
• W2034395940 abstract "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." @default.
• W2034395940 created "2016-06-24" @default.
• W2034395940 creator A5021842123 @default.
• W2034395940 creator A5058471690 @default.
• W2034395940 creator A5062115118 @default.
• W2034395940 date "2014-01-15" @default.
• W2034395940 modified "2023-09-25" @default.
• W2034395940 title "Improved Short-Sequence-Repeat Genotyping of Mycobacterium avium subsp. paratuberculosis by Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry" @default.
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• W2034395940 doi "https://doi.org/10.1128/aem.03212-13" @default.
• W2034395940 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3911078" @default.
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