Characterization of human intestinal bifidobacteria using competitive PCR and PCR-TTGE.
In this study, a competitive PCR was developed to estimate the quantity of bifidobacteria in human faecal samples using two 16S rRNA gene Bifidobacterium genus-specific primers, Bif164f and Bif662r. A PCR-temporal temperature gradient gel electrophoresis (TTGE) with the same primers also allowed us to describe the Bifidobacterium species present in these faecal samples. The PCR product obtained from the competitor had 467 bp, and was 47 bp shorter than the PCR products obtained from Bifidobacterium strains. The number of bifidobacterial cells was linear from 10 to 10(8) cells per PCR assay. Taking into account the dilutions of the extracted DNA, the linear range was over 8 x 10(5) bifidobacteria g(-1) of faeces. Reproducibility was assessed from 10 independent DNA extractions from the same stool and the coefficient of variation was 0.5%. When the competitive PCR was compared with the culture method, a similar count of seven out of nine Bifidobacterium pure cultures were obtained, or had a difference inferior or equal to 1 log(10). In faecal samples, the enumeration of Bifidobacterium genus in most cases gave higher results with competitive PCR than with culture on selective Columbia-Beerens agar pH 5 (P < 0.05). In conclusion, this competitive PCR allows a rapid, highly specific and reproducible quantification of Bifidobacterium genus in faecal samples. TTGE fragments co-migrating with B. longum CIP64.63 fragment were found in 10 out of 11 faecal samples. Bifidobacterium adolescentis and B. bifidum were detected in five out of 11 subjects. Thus, cPCR and PCR-TTGE can be associated in order to characterize human faecal bifidobacteria.
Mangin
Irène
I
Laboratoire de Biologie, Conservatoire national des arts et métiers, Paris, France. irene.mangin@cnam.fr
Suau
Antonia
A
Magne
Fabien
F
Garrido
Daniel
D
Gotteland
Martin
M
Neut
Christel
C
Pochart
Philippe
P
D001644:Bifidobacterium / Q000235:genetics / Q000302:isolation & purification*; D015169:Colony Count, Microbial / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005243:Feces / Q000382:microbiology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S