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

Abstract

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.Winemaking involves a mixed culture of numerous microorganisms including fungal, yeast, and bacteria species (6). The principal bacteria present in wine are members of the lactic acid bacteria (LAB), acetic acid bacteria (AAB), and to a lesser extent species of bacilli (13). LAB and AAB are often present on the surface of the grape and can represent significant populations in musts (17). LAB play dual roles in wine fermentations: as agents of wine spoilage and as the main effector of secondary, or malolactic, fermentation. Most LAB found in wine, including members of Oenococcus, Lactobacillus, Pediococcus, and Leuconostoc, are microaerophilic and able to grow in the anaerobic environment of fermenting wine (24). In contrast, wine-related AAB, such as members of Gluconobacter or Gluconacetobacter, are obligately aerobic and loosely categorized as vinegar bacteria (7,24). Both AAB and LAB can produce acetic acid, a potential inhibitor of growth and alcohol production by Saccharomyces cerevisiae (18).Most bacterial species present in wine fermentations have been identified by traditional microbiological techniques involving cultivation. However, as observed with microbial ecological studies of other environments, cultivation-dependent methods often exhibit biases resulting in an incomplete representation of the true bacterial diversity present (1, 16). Applications of culture-independent molecular techniques to monitor the microbial successions of various food and beverage fermentations have revealed microbial constituents and microbial interactions not witnessed by previous plating analyses (14). One example of this is the recent use of epifluorescence microscopy to identify populations of viable but not culturable bacteria (both LAB and AAB) in aging wine (20).Denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis ...

Indigenous Microbial Community of Barley Greatly Influences Grain Germination and Malt Quality

“…Denaturing gradient gel electrophoresis (DGGE) is perhaps the most commonly used culture-independent fingerprinting technique for studying the response of community dynamics to environmental variations. This technique has also demonstrated its potential in food-related ecosystems 12,19 and has been applied in beverage fields such as whisky 54,55 and wine fermentations 34 . PCR-DGGE detects the predominant species of a community without discriminating living from dead cells or cells in a non-culturable state.…”

PCR and DGGE Detection Limits for Wine Spoilage Microbes

“…According to Lopez et al (2003), no specific primers have been reported for AAB and the WBAC1 GC -WBAC2 primers could successfully be used for the amplification of both LAB and AAB associated with wine. The PCR reactions were performed in a total volume of 50 μL, containing 1 x PCR buffer, 1.5 mM MgCl 2 , 0.8 mM dNTPs, 0.2 μM of each primer, 2.5 U Taq DNA polymerase (Super-Therm) and 3 μL of DNA template.…”

“…The V7 to V8 variable region of the 16S rRNA gene for the bacterial reference strains was amplified using the bacteriaspecific primers WBAC1 GC (5'-CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCC CCC GGT CGT CAG CTC GTG TCG TGA GA-3') and WBAC2 (5'-CCC GGG AAC GTA TTC ACC GCG-3') (GC clamp sequence is underlined) (320 bp fragment), which are based on the primers described by Lopez et al (2003). According to Lopez et al (2003), no specific primers have been reported for AAB and the WBAC1 GC -WBAC2 primers could successfully be used for the amplification of both LAB and AAB associated with wine.…”

“…The V3 variable region of the 16S ribosomal RNA (rRNA) gene for the bacterial reference strains, and the D1/D2 region of the 26S rRNA gene for the yeast reference strain were amplified using the universal primers HDA1 GC (5'-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC G ACT CCT ACG GGA GGC AGC AGT-3') and HDA2 (5'-GTA TTA CCG CGG CTG CTG GCA C-3') (250 bp fragment) (Lopez et al, 2003). To facilitate DGGE separation, a GC-rich sequence (GC clamp sequence is underlined) was attached to the forward primer.…”

“…Most microbial species in wine have been identified using conventional microbiological methods (Lopez et al, 2003). However, these methods have limitations in the identification and classification of microbes (Muyzer, 1999), and are often time consuming and labour intensive (Hernán-Gómez et al, 2000;Kopke et al, 2000).…”