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Applied and Environmental Microbiology, November 2006, p. 6972-6979, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.01303-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Quantitative Detection of Corynebacterium casei in Cheese by Real-Time PCR ^,

Christophe Monnet,^* Karine Correia, Anne-Sophie Sarthou, and Françoise Irlinger

Unité Mixte de Recherche Génie et Microbiologie des Procédés Alimentaires, Institut National de la Recherche Agronomique, 78850 Thiverval-Grignon, France

Received 7 June 2006/ Accepted 13 August 2006

The flora on the surface of smear-ripened cheeses is composed of numerous species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. Due to the absence of selective media, it is very difficult to quantify cheese surface bacteria, and, consequently, the ecology of the cheese surface microflora has not been extensively investigated. We developed a SYBR green I real-time PCR method to quantify Corynebacterium casei, a major species of smear-ripened cheeses, using primers designed to target the 16S rRNA gene. It was possible to recover C. casei genomic DNA from the cheese matrix with nearly the same yield that C. casei genomic DNA is recovered from cells recovered by centrifugation from liquid cultures. Quantification was linear over a range from 10⁵ to 10¹⁰ CFU per g of cheese. The specificity of the assay was demonstrated with DNA from species related to C. casei and from other bacteria and yeasts belonging to the cheese flora. Nine commercial cheeses were analyzed by real-time PCR, and six of them were found to contain more than 10⁵ CFU equivalents of C. casei per g. In two of them, the proportion of C. casei in the total bacterial flora was nearly 40%. The presence of C. casei in these samples was further confirmed by single-strand conformation polymorphism analysis and by a combined approach consisting of plate counting and 16S rRNA gene sequencing. We concluded that SYBR green I real-time PCR may be used as a reliable species-specific method for quantification of bacteria from the surface of cheeses.

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* Corresponding author. Mailing address: Unité Mixte de Recherche Génie et Microbiologie des Procédés Alimentaires, Institut National de la Recherche Agronomique, 78850 Thiverval-Grignon, France. Phone: 33 (0)1 30 81 54 91. Fax: 33 (0)1 30 81 55 97. E-mail: monnet{at}grignon.inra.fr.

Published ahead of print on 1 September 2006.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, November 2006, p. 6972-6979, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.01303-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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