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

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)-variablenumber 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.

Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp. paratuberculosis on a national and herd level

“…A single colony was subsequently substreaked onto 7H11 agar and incubated for 4–8 weeks prior to DNA extraction. DNA was extracted using a modified protocol of the Qiagen DNeasy blood and tissue kit (Qiagen, Mississauga, ON, Canada), as described previously [ 24 ].…”

Knowledge gaps that hamper prevention and control ofMycobacterium aviumsubspeciesparatuberculosisinfection

“…Diversity among isolates has been reported based on VNTR typing (Fritsch, Luyven, Köhler, Lutz, & Möbius, ; Sohal et al., ; Stevenson et al., ; Thibault et al., ), with most isolates being one of two dominant VNTR types. MIRU‐VNTR genotyping does not, however, accurately reflect phylogenetic relationships, and repeat sequences are subject to homoplasy (Ahlstrom, Barkema, & De Buck, ; Bryant et al., ). Current VNTR typing includes loci that are too unstable and unreliable to be used for a molecular epidemiological analysis of MAP (Ahlstrom et al., ).…”

Typing of Mycobacterium avium Subspecies paratuberculosis Isolates from Newfoundland Using Fragment Analysis

“…In previous studies, single or combined molecular methods have been used to obtain epidemiological data regarding Map strain types [ 14 – 17 ]. Most of the previously used strain typing methods are expensive, time consuming, lack discriminatory capabilities and sometimes do not provide consistent results [ 14 , 15 , 18 ]. Despite these limitations, the information obtained from such studies is essential for identifying sources and transmission routes more accurately.…”

“…When combined with information on host genetics, strain typing studies can also be used to determine strain pathogenicity and host resistance. Molecular techniques, especially DNA short-sequence-repeat (SSR) analysis has been shown to be a powerful tool for discriminating between Map isolates at the genetic level [ 5 , 14 , 15 , 16 , 18 , 19 ]. Due to differences in the numbers of nucleotide repeats associated with SSRs from different Map isolates, the relatedness and prevalence of Map strains can be monitored within/between farms and the environment [ 14 , 19 ].…”

“…Due to differences in the numbers of nucleotide repeats associated with SSRs from different Map isolates, the relatedness and prevalence of Map strains can be monitored within/between farms and the environment [ 14 , 19 ]. One major problem with conventional methods for SSR analyses such as the use of Sanger sequencing, is that they are prone to artifacts and failure due to challenges associated with determining the DNA sequences of the repeats, with the most recent technology being capable of analyzing repeats up to 15 bp using a mass spectrometry based approach [ 18 ]. Therefore, there is need for developing cheap, reliable and reproducible methods for Map SSR analysis, which can accurately measure repeats over 15 bp in length.…”