title: High-resolution melting for analysis of short sequence repeats in Mycobacterium avium subsp. paratuberculosis.
abstract: Analysis of micro- and minisatellite loci is widely used in sub-typing of Mycobacterium avium subsp. paratuberculosis. Microsatellite (short sequence repeat, SSR) loci have shown highest discriminatory power, but direct sequencing of amplicons is required for correct assignment of the repeat number. We developed an alternative method to sequencing, focusing on the SSR8 locus (constituted by GGT triplets from three to six repeats). The approach is based on asymmetric quantitative PCR, followed by high-resolution melting analysis with unlabelled probes (UP-HRM). Data showed perfect concordance between direct sequencing and UP-HRM, which is faster, simpler and more cost effective.
authors.affiliation: Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, National Reference Centre for Paratuberculosis, Podenzano (PC), Italy. matteo.ricchi@izsler.it
authors.forename: Matteo
authors.initials: M
authors.lastname: Ricchi
authors.forename: Gianluca
authors.initials: G
authors.lastname: Barbieri
authors.forename: Giuliana
authors.initials: G
authors.lastname: Cammi
authors.forename: Chiara Anna
authors.initials: CA
authors.lastname: Garbarino
authors.forename: Norma
authors.initials: N
authors.lastname: Arrigoni
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics*; D058889:Molecular Typing / Q000379:methods*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012091:Repetitive Sequences, Nucleic Acid; D044366:Transition Temperature
chemical_list: D004269:DNA, Bacterial