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Applied and Environmental Microbiology, November 2003, p. 6801-6807, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6801-6807.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

Isabel Lopez,1 Fernanda Ruiz-Larrea,1 Luca Cocolin,2 Erica Orr,3 Trevor Phister,3 Megan Marshall,4 Jean VanderGheynst,4 and David A. Mills3*

Department of Food and Agriculture, University of La Rioja, Logroño, Spain,1 Dipartimento di Scienze degli Alimenti, Universita' degli Studi di Udine, Udine, Italy,2 Department of Viticulture and Enology,3 Department of Biological and Agricultural Engineering, University of California, Davis, California 956164

Received 27 March 2003/ Accepted 24 August 2003

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.

* Corresponding author. Mailing address: Department of Viticulture and Enology, University of California, Davis, CA 95616. Phone: (530) 754-7821. Fax: (530) 752-0382. E-mail: damills{at}ucdavis.edu.

Applied and Environmental Microbiology, November 2003, p. 6801-6807, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6801-6807.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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