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

Mohar Lorbeg, Petra (author), Čanžek Majhenič, Andreja (author), Rogelj, Irena (author)

Enetrococci represent an imprtant 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 publushes so far, six primer pairs amplifying differnet variable regions of 16S rDNA were selected and applied in our DGGE analysis of 12 species belonging to genus Eneterococcus and eight other bacterial species often found in cheeses (seven lactobacilli and one Lactoccocus and eight other bacterial species often found in cheeses (seven lactobacilli and one Lactococcus lactis). When DGGE procedures were optimized, the same set of primers was used for DGGE analysis of five cheese samples. Our study demonstates 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 distincive 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.

• Language: English
• Keywords: mlečni izdelki, siri, mikrobiologija, enterokoki, molekularna genetika, rDNK, PCR-DGGE
• Work type: Not categorized
• Typology: 1.01 - Original Scientific Article
• Organization: BF - Biotechnical Faculty
• Year: 2009
• Number of pages: Str. 265-271
• Numbering: Vol. 76
• PID: 20.500.12556/RUL-36501
• UDC: 637.3:575
• ISSN on article: 0022-0299
• DOI: 10.1017/S0022029909003902
• COBISS.SI-ID: 2445704
• Publication date in RUL: 10.07.2015

Record is a part of a journal

• Title: Journal of Dairy Research
• Shortened title: J. Dairy Res.
• Publisher: Cambridge University Press
• ISSN: 0022-0299
• COBISS.SI-ID: 6400007