title: Novel Single Nucleotide Polymorphism-Based Assay for Genotyping Mycobacterium avium subsp. paratuberculosis.
abstract: Typing of Mycobacterium avium subspecies paratuberculosis strains presents a challenge, since they are genetically monomorphic and traditional molecular techniques have limited discriminatory power. The recent advances and availability of whole-genome sequencing have extended possibilities for the characterization of Mycobacterium avium subspecies paratuberculosis, and whole-genome sequencing can provide a phylogenetic context to facilitate global epidemiology studies. In this study, we developed a single nucleotide polymorphism (SNP) assay based on PCR and restriction enzyme digestion or sequencing of the amplified product. The SNP analysis was performed using genome sequence data from 133 Mycobacterium avium subspecies paratuberculosis isolates with different genotypes from 8 different host species and 17 distinct geographic regions around the world. A total of 28,402 SNPs were identified among all of the isolates. The minimum number of SNPs required to distinguish between all of the 133 genomes was 93 and between only the type C isolates was 41. To reduce the number of SNPs and PCRs required, we adopted an approach based on sequential detection of SNPs and a decision tree. By the analysis of 14 SNPs Mycobacterium avium subspecies paratuberculosis isolates can be characterized within 14 phylogenetic groups with a higher discriminatory power than mycobacterial interspersed repetitive unit-variable number tandem repeat assay and other typing methods. Continuous updating of genome sequences is needed in order to better characterize new phylogenetic groups and SNP profiles. The novel SNP assay is a discriminative, simple, reproducible method and requires only basic laboratory equipment for the large-scale global typing of Mycobacterium avium subspecies paratuberculosis isolates.
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Penicuik, United Kingdom Unidade Estratégica de Investigação e Serviços em Produção e Saúde Animal, Instituto Nacional de Investigação Agrária e Veterinária, I.P. (INIAV, I.P.), Lisbon, Portugal celia.leao@iniav.pt j.inacio@brighton.ac.uk.
authors.forename: Célia
authors.initials: C
authors.lastname: Leão
authors.affiliation: Institute of Infection, Inflammation, and Immunity, University of Glasgow, Glasgow, United Kingdom.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Goldstone
authors.affiliation: Division of Infection and Immunity, University College London, London, United Kingdom.
authors.forename: Josephine
authors.identifier: http://orcid.org/0000-0002-0149-5768
authors.initials: J
authors.lastname: Bryant
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Penicuik, United Kingdom.
authors.forename: Joyce
authors.initials: J
authors.lastname: McLuckie
authors.affiliation: Unidade de Microbiologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, Portugal School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, United Kingdom celia.leao@iniav.pt j.inacio@brighton.ac.uk.
authors.forename: João
authors.identifier: http://orcid.org/0000-0001-7367-5007
authors.initials: J
authors.lastname: Inácio
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Penicuik, United Kingdom Institute of Infection, Inflammation, and Immunity, University of Glasgow, Glasgow, United Kingdom.
authors.forename: David G E
authors.initials: DG
authors.lastname: Smith
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Penicuik, United Kingdom.
authors.forename: Karen
authors.identifier: http://orcid.org/0000-0003-0270-9550
authors.initials: K
authors.lastname: Stevenson
mesh_terms: D016680:Genome, Bacterial; D060005:Genotyping Techniques / Q000379:methods*; D058889:Molecular Typing / Q000379:methods*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000145:classification* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA