High-resolution melt analysis for species identification of coagulase-negative staphylococci derived from bovine milk

Ajitkumar, P., Barkema, H.W., Zadoks, R.N. , Morck, D.W., van der Meer, F.J.U.M. and De Buck, J. (2013) High-resolution melt analysis for species identification of coagulase-negative staphylococci derived from bovine milk. Diagnostic Microbiology and Infectious Disease, 75(3), pp. 227-234. (doi: 10.1016/j.diagmicrobio.2012.11.008)

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Publisher's URL: http://dx.doi.org/10.1016/j.diagmicrobio.2012.11.008


Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens isolated from bovine milk. In this study, we report a rapid assay for species identification of CNS using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time polymerase chain reaction amplification of 16S rRNA gene fragment, spanning the variable region V1 and V2, was performed with a resulting amplicon of 215 bp. A library of distinct melt curves of reference strains of 13 common CNS species was created using HRMA. Sequencing of 16S rRNA and rpoB genes, and, when needed, tuf gene, of 100 CNS isolates obtained from Canadian Bovine Mastitis Research Network was done to determine their species identity, allowing for subsequent evaluation of the performance of HRMA for field isolates of bovine CNS. A combination of HRMA and sequencing revealed that Staphylococcus chromogenes, S. xylosus, S. simulans, and S. sciuri had multiple genotypes, complicating their resolution by HRMA. As the 3 genotypes of S. chromogenes had distinct melt curves, the 3 distinct genotypes were employed as reference strains in a blinded trial of 156 CNS isolates to identify S. chromogenes. HRMA correctly identified all S. chromogenes isolates which were later confirmed by sequencing. Staphylococcus chromogenes (68%) was most frequently found among the CNS isolates, followed by S. haemolyticus (10%) and S. xylosus (6%). The present study revealed that HRMA of 16S rRNA gene (V1–V2) could be used as a rapid, efficient, low-cost, and minimally cumbersome technique for S. chromogenes identification, the most common CNS derived from bovine milk.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Zadoks, Professor Ruth
Authors: Ajitkumar, P., Barkema, H.W., Zadoks, R.N., Morck, D.W., van der Meer, F.J.U.M., and De Buck, J.
College/School:College of Medical Veterinary and Life Sciences > School of Biodiversity, One Health & Veterinary Medicine
Journal Name:Diagnostic Microbiology and Infectious Disease
ISSN (Online):1879-0070
Published Online:27 December 2012

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