[Show abstract][Hide abstract] ABSTRACT: A total of 16 Bifidobacterium species were assayed by polymerase chain reaction (PCR) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) methods targeted on a 770-bp region of the tuf gene. Based on this sequence, a genus-specific primer set and 12 primer sets for 12 Bifidobacterium species including those previously reported for six probiotic species were developed. On the other hand, when these 16 Bifidobacterium species were subjected to PCR-DGGE analysis, 13 product migration patterns were obtained. PCR products for strains in pairs of B. adolescentis/B. thermophilum, B. longum/B. magnum and B. lactis/B. gallinarum migrated the same distance on the DGGE gel. Combined with species-specific PCR primers specific to B. adolescentis, B. longum and B. lactis, all of the 16 Bifidobacterium species could be identified. In addition, the subspecies of B. animalis, i.e., B. animalls and B. lactis, could be discriminated. This study indicated that the tuf gene is highly useful for the molecular detection of different Bifidobacterium species. Using the PCR and PCR-DGGE methods, 16 Bifidobacterium species, including those from probiotic products and those from other origins, could be rapidly identified. Copyright
Journal of Food and Drug Analysis 06/2013; 21(2):177-183. DOI:10.1016/j.jfda.2013.05.008 · 0.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Due to the increasing use of bifidobacteria in probiotic products, it is essential to establish a rapid method for the qualitative and quantitative assay of the bifidobacteria in commercial products. In this study, partial sequences of the tuf gene for 18 Bifidobacterium strains belonging to 14 species were determined. Alignment of these sequences showed that the similarities among these Bifidobacterium species were 82.24% to 99.72%. Based on these tuf gene sequences, 6 primer sets were designed for the polymerase chain reaction (PCR) assay of B. animalis subsp. animalis, B. animalis subsp. lactis, B. bifidum, B. breve, B. longum subsp. infantis, B. longum subsp. longum, and the genus of Bifidobacterium, respectively. These Bifidobacterium species are common probiotic species present in dairy and probiotic products. When each target Bifidobacterium spp. was assayed with the designed primers, PCR product with expected size was generated. In addition, for each target species, more than 70 bacterial strains other than the target species, including strains of other Bifidobacterium species, strains of Lactobacillus spp., Enterococcus spp., and other bacterial species, all generated negative results. PCR assay with primers specific to B. animalis subsp. lactis and B. longum subsp. longum confirmed the presence of these Bifidobacterium species in commercial yogurt products. In addition, for each product, enumeration of the bifidobacteria cells by culture method with BIM-25 agar and the quantitative real-time PCR showed similar cell counts. Such results indicated that within 15-d storage (4 °C) after manufacture, all the bifidobacteria cells originally present in yogurt products were viable and culturable during the storage.
[Show abstract][Hide abstract] ABSTRACT: PCR primers specific for the detection of Lactobacillus acidophilus, Lactobacillus casei group, Lactobacillus delbrueckii, and Bifidobacterium longum were designed based on the elongation factor Tu gene (tuf). The specificity of these four primer sets were confirmed by PCR with 88 bacterial strains of Lactobacillus, Enterococcus, Bifidobacterium, and other bacterial species. Results indicated that these primer sets generated predicted PCR products of 397, 230, 202, and 161 bp for L. acidophilus, L. delbrueckii, L. casei group, and B. longum, respectively. Bacterial species other than the target organisms tested did not generate false-positive results. When these four primer sets were combined for the simultaneous detection of the lactic acid bacteria (LAB) in fermented milk products including yogurt, the LAB species listed on the labels of these products could be identified without the preenrichment step. The identification limit for each LAB strain with this multiplex PCR method was N X 10(3) CFU/ml in milk samples. The results of our multiplex PCR method were confirmed by PCR assay using primers based on the 16S rDNA or the 16S-23S intergenic spacer region and by biochemical tests using the API 50 CHL kit. When this multiplex PCR method was used with the determination of counts of total viable LAB and bifidobacteria, the quality of commercial fermented milk products could be assured.
Journal of food protection 02/2009; 72(1):93-100. · 1.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: aBSTRaCT Staphylococcal intoxications are often associated with staphylococcal enterotoxins produced by Staphylococcus aureus. However, several studies revealed that other Staphylococcus species, such as S. intermedius and S. warneri, could produce enterotoxins as well. To facilitate the identification of a mixed culture while tracing the causative staphylococci of food poison, a polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method was developed to differentiate Staphylococcus species using Staphylococcus-specific primers targeting the elongation factor Tu gene (tuf gene). Eleven tested species were differentiated to 10 separated patterns. Variance of the patterns between strains within a species was analyzed using 9 strains of S. aureus and several strains of other species. It was shown that strains within a species migrated the same distance in the DGGE assay. When mixed cultures of different Staphylococcus species in milk were subjected to DNA extraction and PCR-DGGE, the resulted patterns faithfully corresponded to the species of the mixed cultures, including those of potential to secret enterotoxins.
Journal of Food and Drug Analysis 08/2008; 16(4):44-51. · 0.62 Impact Factor