Design and evaluation of PCR primers for analysis of bacterial populations in wine by denaturing gradient gel electrophoresis.
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.
Lopez
Isabel
I
Department of Food and Agriculture, University of La Rioja, Logroño, Spain.
Ruiz-Larrea
Fernanda
F
Cocolin
Luca
L
Orr
Erica
E
Phister
Trevor
T
Marshall
Megan
M
VanderGheynst
Jean
J
Mills
David A
DA
D019342:Acetic Acid / Q000378:metabolism*; D001419:Bacteria / Q000145:classification* / Q000235:genetics; D017931:DNA Primers / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis; D004271:DNA, Fungal / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004275:DNA, Ribosomal / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005285:Fermentation; D019344:Lactic Acid / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013045:Species Specificity; D014920:Wine / Q000382:microbiology*
D017931:DNA Primers; D004269:DNA, Bacterial; D004271:DNA, Fungal; D018744:DNA, Plant; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D019344:Lactic Acid; D019342:Acetic Acid