High-resolution melting for analysis of short sequence repeats in Mycobacterium avium subsp. paratuberculosis

Authors

  • Matteo Ricchi,

    Corresponding author
    • Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, National Reference Centre for Paratuberculosis, Podenzano (PC), Italy
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  • Gianluca Barbieri,

    1. Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, National Reference Centre for Paratuberculosis, Podenzano (PC), Italy
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  • Giuliana Cammi,

    1. Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, National Reference Centre for Paratuberculosis, Podenzano (PC), Italy
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  • Chiara Anna Garbarino,

    1. Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, National Reference Centre for Paratuberculosis, Podenzano (PC), Italy
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  • Norma Arrigoni

    1. Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, National Reference Centre for Paratuberculosis, Podenzano (PC), Italy
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Correspondence: Matteo Ricchi, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Sezione Diagnostica di Piacenza, Strada Faggiola 1, 29027-loc. Gariga – Podenzano (PC), Italy. Tel.: +39 0523 524253; fax: +39 0523 523491; e-mail: matteo.ricchi@izsler.it

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.

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