title: Real-time PCR quantitation of clostridia in feces of autistic children.
abstract: Based on the hypothesis that intestinal clostridia play a role in late-onset autism, we have been characterizing clostridia from stools of autistic and control children. We applied the TaqMan real-time PCR procedure to detect and quantitate three Clostridium clusters and one Clostridium species, C. bolteae, in stool specimens. Group- and species-specific primers targeting the 16S rRNA genes were designed, and specificity of the primers was confirmed with DNA from related bacterial strains. In this procedure, a linear relationship exists between the threshold cycle (CT) fluorescence value and the number of bacterial cells (CFU). The assay showed high sensitivity: as few as 2 cells of members of cluster I, 6 cells of cluster XI, 4 cells of cluster XIVab, and 0.6 cell of C. bolteae could be detected per PCR. Analysis of the real-time PCR data indicated that the cell count differences between autistic and control children for C. bolteae and the following Clostridium groups were statistically significant: mean counts of C. bolteae and clusters I and XI in autistic children were 46-fold (P = 0.01), 9.0-fold (P = 0.014), and 3.5-fold (P = 0.004) greater than those in control children, respectively, but not for cluster XIVab (2.6 x 10(8) CFU/g in autistic children and 4.8 x 10(8) CFU/g in controls; respectively). More subjects need to be studied. The assay is a rapid and reliable method, and it should have great potential for quantitation of other bacteria in the intestinal tract.
authors.affiliation: Research Service, VA Medical Center West Los Angeles, Los Angeles, California 90073, USA. yulis1@yahoo.com
authors.forename: Yuli
authors.initials: Y
authors.lastname: Song
authors.forename: Chengxu
authors.initials: C
authors.lastname: Liu
authors.forename: Sydney M
authors.initials: SM
authors.lastname: Finegold
mesh_terms: D001321:Autistic Disorder / Q000382:microbiology*; D002648:Child; D002675:Child, Preschool; D003013:Clostridium / Q000235:genetics / Q000302:isolation & purification*; D015169:Colony Count, Microbial; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D005243:Feces / Q000382:microbiology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S