99A-22 |
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G.-B. KIM1, J.-H. Choi2, B.-C. Park2, and B. Lee1. (1) Dept. of Food Science & Agricultural Chemistry, McGill Univ., Macdonald Campus, 21111 Lakeshore Rd., Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada, (2) Research Center of Bioscience and Biotechnology, CTCBIO Inc., 93 Okeum-dong, Songpa-ku, Seoul, 138-858, South Korea Although the genus Bifidobacterium can be easily distinguished from other bacterial groups by fructose-6-phosphate phosphoketolase (F6PPK) activity, the F6PPK assay itself is a time-consuming procedure and sometimes it leads to doubtful results. Therefore, it would be highly desirable to develop a molecular technique for the rapid, reliable and accurate identification of Bifidobacterium strains based on the F6PPK gene locus. The F6PPK gene sequences of two Bifidobacterium species for which the nucleotide sequences are available from the GenBank were aligned and compared. Based on the four conserved regions, two pairs of primers for amplifying regions of 600bp and 950bp were designed. To evaluate the feasibility, PCR-based methods with these primers were tested for the identification of 30 Bifidobacterium strains derived from 9 species of human and 8 species of animal origin. The first primer set allowed the amplification of partial F6PPK gene sequences (600bp) from all the strains tested, while the second primer set produced PCR amplicons from 26 out of 30 strains. For the comparison, B. bifidum ATCC11863 was selected and PCR cloning was carried out to obtain the complete open reading frame (ORF) of F6PPK gene. The predicted ORF of 2478 nt was present with 82% and 93% DNA sequence identity and 92% and 98% deduced amino acid sequence similarity to F6PPKs of B. lactis DSM10140 and B. longum NCC2705, respectively. This new primer set based on the highly conserved area of F6PPK gene was very specific in identifying all the Bifidobacterium strains tested. In addition, the lower level of homology in F6PPK gene sequences (82~93%) than those of 16S rRNA among Bifidobacterium species suggested that the F6PPK locus could be a potential alternative to the16S rRNA sequence for comparative phylogenetic analysis of the genus Bifidobacterium.
Session 99A, Biotechnology: General
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