Real-Time PCR Quantitation of Clostridia in Feces of Autistic Children

• DOI: 10.1128/aem.70.11.6459-6465.2004
• Publication Date: 2004-11-04T21:58:18Z
• Authors: Yuli Song; Chengxu Liu; Sydney M. Finegold
• Keywords: Clostridium; DNA, Bacterial; 0303 health sciences; Colony Count, Microbial; Polymerase Chain Reaction; Sensitivity and Specificity; 3. Good health; Feces; 03 medical and health sciences; Child, Preschool; RNA, Ribosomal, 16S; Humans; Autistic Disorder; Child; DNA Primers

ABSTRACTBased 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 threeClostridiumclusters and oneClostridiumspecies,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 ofC. bolteaecould be detected per PCR. Analysis of the real-time PCR data indicated that the cell count differences between autistic and control children forC. bolteaeand the followingClostridiumgroups were statistically significant: mean counts ofC. bolteaeand 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 × 108CFU/g in autistic children and 4.8 × 108CFU/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.