Rapid species identification and partial strain differentiation of Clostridium butyricum by PCR using 16S-23S rDNA intergenic spacer regions.
Some Clostridium butyricum strains have been used as probiotics for both humans and animals. Strain-specific identification is necessary for the manufacturing process of probiotics. The aim of this study was to determine whether there are sufficient genetic variations in 16S-23S intergenic spacer regions (ISRs) to discriminate C. butyricum at the biovar level. We amplified ISRs from five reference strains, a probiotic strain (MIYAIRI 588) and 22 isolates, and we classified them into four groups on the basis of amplification patterns (type A through D). However, amplification of ISRs is not sufficient for discriminating strains. Moreover, we compared genetic structures of these ISRs. Sequence analysis revealed that the size variations of ISRs were generated by the insertion of tRNA genes and unique sequences into the internal portion, while the external portions were highly conserved. On the basis of the highly conserved nucleotide sequences within the ISRs, we developed a PCR primer set specific to C. butyricum. In addition, the PCR primer designed from the unique inserted sequence in type B strain was useful to differentiate probiotic strains at the biovar level.
Nakanishi
Shusuke
S
Department of Molecular Bacteriology, Institute of Health Biosciences, The University of Tokushima Graduate School, Japan. s.nakanishi@miyarisan.com
Kuwahara
Tomomi
T
Nakayama
Haruyuki
H
Tanaka
Mamoru
M
Ohnishi
Yoshinari
Y
D015373:Bacterial Typing Techniques / Q000379:methods*; D001483:Base Sequence; D046972:Clostridium butyricum / Q000145:classification* / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000032:analysis* / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S