Design and evaluation of a 16S rRNA-targeted oligonucleotide probe for specific detection and quantitation of human faecal Bacteroides populations.
Colonic Bacteroides include several species which, by their population level and activities, are significant contributers to the metabolic activity and health of man and animals. Yet, the understanding of their ecology has been hampered by the lack of highly specific and reliable enumeration techniques. Based on 16S rRNA sequence comparisons within the available database, we have designed an 18-mer oligonucleotide that targets a region common to-and specific for the Bacteroides-Porphyromonas-Prevotella group. We have tested the specificity of the probe and its usefulness for studies of human faecal samples. Under experimentally optimized hybridization conditions, the probe was shown to similarly recognize the rDNA obtained from 40 strains representing 8 species of the Bacteroides-Porphyromonas-Prevotella group. Importantly, it did not recognize 31 strains of microorganisms representing 8 genera of the dominant human faecal microbiota. Among selected colonies of dominant microorganisms of the faecal flora of two human individuals, strains identified as B. vulgatus by immunoblots using a species-specific monoclonal antibody were all detected by the probe. Colony hybridization was used to enumerate total Bacteroides-group microorganisms in faecal specimen from children and adults. The probe described therein was further used in quantitative RNA blots to monitor fluctuations of the Bacteroides-group versus Bifidobacterium genus in frozen faecal samples from a child between 85 and 125 days of age. It will be applicable to similar investigations of other anaerobic environments.
Doré
J
J
Laboratoire d'Ecologie et Physiologie du Système digestif, Institut National de la Recherche Agronomique, Jouy-en-Josas, France. dore@jouy.inra.fr
Sghir
A
A
Hannequart-Gramet
G
G
Corthier
G
G
Pochart
P
P
D000328:Adult; D001439:Bacteroides / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001644:Bifidobacterium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis* / Q000302:isolation & purification; D005069:Evaluation Studies as Topic; D005243:Feces / Q000382:microbiology*; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007223:Infant; D008297:Male; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity; D013997:Time Factors
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S