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Evaluation of different primers for PCR-DGGE analysis of cheese-associated enterococci

Published online by Cambridge University Press:  02 March 2009

Petra Mohar Lorbeg ,
Andreja Čanžek Majhenič  and
Irena Rogelj
Petra Mohar Lorbeg*
Affiliation:
University of Ljubljana, Biotechnical Faculty, Zootechnical Department, Chair of Dairy Science, Groblje 3, 1230Domžale, Slovenia
Andreja Čanžek Majhenič
Affiliation:
University of Ljubljana, Biotechnical Faculty, Zootechnical Department, Chair of Dairy Science, Groblje 3, 1230Domžale, Slovenia
Irena Rogelj
Affiliation:
University of Ljubljana, Biotechnical Faculty, Zootechnical Department, Chair of Dairy Science, Groblje 3, 1230Domžale, Slovenia
*
*For correspondence; e-mail: petra.mohar@bfro.uni-lj.si
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Abstract

Enterococci represent an important part of bacterial microbiota in different types of artisanal cheeses, made from either raw or pasteurized milk. Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) of ribosomal DNA is currently one of the most frequently used fingerprinting method to study diversity and dynamics of microbial communities and also a tool for microbial identification. Among several primer pairs for DGGE analysis published so far, six primer pairs amplifying different variable regions of 16S rDNA were selected and applied in our DGGE analysis of 12 species belonging to genus Enterococcus and eight other bacterial species often found in cheeses (seven lactobacilli and one Lactoccocus lactis). When DGGE procedures were optimized, the same set of primers was used for DGGE analysis of five cheese samples. Our study demonstrates that the use of different primer pairs generate significant differences in DGGE analysis of enterococcal population, consequently, appropriate primers regarding the purpose of analysis can be selected. For differentiation and identification of pure enterococcal isolates, primer pair P1V1/P2V1 showed the most promising results since all 12 enterococcal isolates gave distinctive DGGE fingerprints, but with multiple bands patterns; therefore, these primers do not seem to be appropriate for identification of enterococcal species in mixed cultures. Use of primer pairs HDA1/HDA2 and V3f/V3r amplifying V3 region showed better potential for detection and identification of enterococci in mixed communities, but since some bacterial species showed the same fingerprint, for clear identification combination of DGGE and some other method (e.g. species specific PCR) or combined DGGE analysis using two primer pairs generating distinctive results should be used.

Keywords

16S rDNAidentificationPCR-DGGE
Type
Research Article
Information
Journal of Dairy Research , Volume 76 , Issue 3 , August 2009 , pp. 265 - 271
Copyright
Copyright © Proprietors of Journal of Dairy Research 2009

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