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Journal of Clinical Microbiology, April 2008, p. 1307-1316, Vol. 46, No. 4
0095-1137/08/$08.00+0     doi:10.1128/JCM.01617-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Suppression-Subtractive Hybridization as a Strategy To Identify Taxon-Specific Sequences within the Mycoplasma mycoides Cluster: Design and Validation of an M. capricolum subsp. capricolum-Specific PCR Assay{triangledown}

Laure Maigre,1 Christine Citti,2 Marc Marenda,3 François Poumarat,1 and Florence Tardy1*

AFSSA-Lyon, 31 avenue Tony Garnier, 69364 Lyon, Cedex 07, France,1 UMR1225 INRA, ENVT Ecole Nationale Vétérinaire, 23 Chemin des Capelles BP 87614, 31076 Toulouse, Cedex 3, France,2 Faculty of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia3

Received 14 August 2007/ Returned for modification 20 September 2007/ Accepted 21 January 2008

The phylogenetically related Mycoplasma capricolum subsp. capricolum and M. mycoides subsp. mycoides biotype Large Colony are two small-ruminant pathogens involved in contagious agalactia. Their respective contributions to clinical outbreaks are not well documented, because they are difficult to differentiate with the current diagnostic techniques. In order to identify DNA sequences specific to one taxon or the other, a suppression-subtractive hybridization approach was developed. DNA fragments resulting from the reciprocal subtraction of the type strains were used as probes on a panel of M. capricolum subsp. capricolum and M. mycoides subsp. mycoides biotype Large Colony strains to assess their intrataxon specificity. Due to a high intrataxon polymorphism and important cross-reactions between taxa, a single DNA fragment was shown to be specific for M. capricolum subsp. capricolum and to be present in all M. capricolum subsp. capricolum field isolates tested in this study. A PCR assay targeting the corresponding gene (simpA51) was designed that resulted in a 560-bp amplification only in M. capricolum subsp. capricolum and in M. capricolum subsp. capripneumoniae (the etiological agent of contagious caprine pleuropneumonia). simpA51 was further improved to generate a multiplex PCR (multA51) that allows the differentiation of M. capricolum subsp. capripneumoniae from M. capricolum subsp. capricolum. Both the simpA51 and multA51 assays accurately identify M. capricolum subsp. capricolum among other mycoplasmas, including all members of the M. mycoides cluster. simpA51 and multA51 PCRs are proposed as sensitive and robust tools for the specific identification of M. capricolum subsp. capricolum and M. capricolum subsp. capripneumoniae.

* Corresponding author. Mailing address: AFSSA-Lyon, 31 avenue Tony Garnier, 69364 Lyon, Cedex 07, France. Phone: 33 4 78 69 68 43. Fax: 33 4 78 61 91 45. E-mail: f.tardy{at}afssa.fr

{triangledown} Published ahead of print on 30 January 2008.

Journal of Clinical Microbiology, April 2008, p. 1307-1316, Vol. 46, No. 4
0095-1137/08/$08.00+0     doi:10.1128/JCM.01617-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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