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Applied and Environmental Microbiology, June 2007, p. 3747-3751, Vol. 73, No. 11
0099-2240/07/$08.00+0     doi:10.1128/AEM.02642-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Quantitative Detection of Clostridium tyrobutyricum in Milk by Real-Time PCR{triangledown} ,{dagger}

Lorena López-Enríquez,1 David Rodríguez-Lázaro,2,3 and Marta Hernández1*

Laboratorio de Biología Molecular, Subdirección de Investigación y Tecnología, Instituto Tecnológico Agrario de Castilla y León (ITACyL), Valladolid, Spain,1 Division of Veterinary Pathology, Infection and Immunity, Faculty of Medical and Veterinary Sciences, University of Bristol, Langford, United Kingdom,2 Institute for Molecular Biology and Genetics (IBGM), Spanish National Research Council (CSIC), Valladolid, Spain3

Received 12 November 2006/ Accepted 8 April 2007

We developed a real-time PCR assay for the quantitative detection of Clostridium tyrobutyricum, which has been identified as the major causal agent of late blowing in cheese. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent in 40% of the reactions. The quantification was linear (R2 > 0.9995) over a 5-log dynamic range, down to 10 genome equivalents, with a PCR efficiency of >0.946. With optimized detergent treatment and enzymatic pretreatment of the sample before centrifugation and nucleic acid extraction, the assay counted down to 300 C. tyrobutyricum spores, with a relative accuracy of 82.98 to 107.68, and detected as few as 25 spores in 25 ml of artificially contaminated raw or ultrahigh-temperature-treated whole milk.

* Corresponding author. Mailing address: Instituto Tecnológico Agrario de Castilla y León (ITACyL), Carretera de Burgos, km. 119, 47071 Valladolid, Spain. Phone: 34 983 317542. Fax: 34 983 414780. E-mail: ita-herperma{at}itacyl.es

{triangledown} Published ahead of print on 20 April 2007.

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

Applied and Environmental Microbiology, June 2007, p. 3747-3751, Vol. 73, No. 11
0099-2240/07/$08.00+0     doi:10.1128/AEM.02642-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Oravcova, K., Lopez-Enriquez, L., Rodriguez-Lazaro, D., Hernandez, M. (2009). Mycoplasma agalactiae p40 Gene, a Novel Marker for Diagnosis of Contagious Agalactia in Sheep by Real-Time PCR: Assessment of Analytical Performance and In-House Validation Using Naturally Contaminated Milk Samples. J. Clin. Microbiol. 47: 445-450 [Abstract] [Full Text]  


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