Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis
Open Access
- 1 January 2005
- journal article
- research article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 71 (1), 29-38
- https://doi.org/10.1128/aem.71.1.29-38.2005
Abstract
A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes , could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g.Keywords
This publication has 27 references indexed in Scilit:
- Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial CommunitiesApplied and Environmental Microbiology, 2003
- Identification of the Bacterial Microflora in Dairy Products by Temporal Temperature Gradient Gel ElectrophoresisApplied and Environmental Microbiology, 2002
- Molecular Method To Assess the Diversity of Burkholderia Species in Environmental SamplesApplied and Environmental Microbiology, 2002
- Molecular Diversity of Lactobacillus spp. and Other Lactic Acid Bacteria in the Human Intestine as Determined by Specific Amplification of 16S Ribosomal DNAApplied and Environmental Microbiology, 2002
- Differentiation of lactate-fermenting, gas-producing Clostridium spp. isolated from milkInternational Journal of Food Microbiology, 1998
- Isolation and detection of Clostridium tyrobutyricum cells in semi-soft and hard cheeses using the polymerase chain reactionJournal of Dairy Research, 1997
- CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choiceNucleic Acids Research, 1994
- The Phylogeny of the Genus Clostridium: Proposal of Five New Genera and Eleven New Species CombinationsInternational Journal of Systematic and Evolutionary Microbiology, 1994
- Temperature-gradient gel electrophoresisBiophysical Chemistry, 1987
- Méthode d'extraction rapide des acides gras volatils libres des fromagesLe Lait, 1986