Improved Short-Sequence-Repeat Genotyping of Mycobacterium avium subsp. paratuberculosis by Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Abstract

ABSTRACTAccurate 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 ofMycobacterium aviumsubsp.paratuberculosisallow genotyping ofM. aviumsubsp.paratuberculosisisolates 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. aviumsubsp.paratuberculosistype (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 dominantM. aviumsubsp.paratuberculosisMIRU-VNTR type. Thus, MALDI-TOF MS was a reliable, fast, and automatable technique to accurately resolveM. aviumsubsp.paratuberculosisgenotypes based on SSRs.