PCR and real-time PCR primers developed for detection and identification of <it>Bifidobacterium thermophilum </it>in faeces

• Main Authors: Mini Raffaella, Lacroix Christophe, Mathys Sophie, Meile Leo
• Format: Article
• Language: English
• Published: BMC 2008-10-01
• Series: BMC Microbiology
• Online Access: http://www.biomedcentral.com/1471-2180/8/179

<p>Abstract</p> <p>Background</p> <p>Culture-independent methods based on the 16S ribosomal RNA molecule are nowadays widely used for assessment of the composition of the intestinal microbiota, in relation to host health or probiotic efficacy. Because <it>Bifidobacterium thermophilum </it>was only recently isolated from human faeces until now, no specific real-time PCR (qPCR) assay has been developed for detection of this species as component of the bifidobacterial community of the human intestinal flora.</p> <p>Results</p> <p>Design of specific primers and probe was achieved based on comparison of 108 published bifidobacterial 16S rDNA sequences with the recently published sequence of the human faecal isolate <it>B. thermophilum </it>RBL67. Specificity of the primer was tested <it>in silico </it>by similarity search against the sequence database and confirmed experimentally by PCR amplification on 17 <it>Bifidobacterium </it>strains, representing 12 different species, and two <it>Lactobacillus </it>strains. The qPCR assay developed was linear for <it>B. thermophilum </it>RBL67 DNA quantities ranging from 0.02 ng/μl to 200 ng/μl and showed a detection limit of 10⁵cells per gram faeces. The application of this new qPCR assay allowed to detect the presence of <it>B. thermophilum </it>in one sample from a 6-month old breast-fed baby among 17 human faecal samples tested. Additionally, the specific qPCR primers in combination with selective plating experiments led to the isolation of F9K9, a faecal isolate from a 4-month old breast-fed baby. The 16S rDNA sequence of this isolate is 99.93% similar to that of <it>B. thermophilum </it>RBL67 and confirmed the applicability of the new qPCR assay in faecal samples.</p> <p>Conclusion</p> <p>A new <it>B. thermophilum</it>-specific qPCR assay was developed based on species-specific target nucleotides in the 16S rDNA. It can be used to further characterize the composition of the bifidobacterial community in the human gastrointestinal tract. Until recently, <it>B. thermophilum </it>was considered as a species of animal origin, but here we confirm with the application of this new PCR assay the presence of <it>B. thermophilum </it>strains in the human gut.</p>