Real-time PCR quantitation of clostridia in feces of autistic children

Appl Environ Microbiol. 2004 Nov;70(11):6459-65. doi: 10.1128/AEM.70.11.6459-6465.2004.

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.

Publication types

  • Evaluation Study
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Autistic Disorder / microbiology*
  • Child
  • Child, Preschool
  • Clostridium / genetics
  • Clostridium / isolation & purification*
  • Colony Count, Microbial
  • DNA Primers
  • DNA, Bacterial / analysis
  • Feces / microbiology*
  • Humans
  • Polymerase Chain Reaction / methods*
  • RNA, Ribosomal, 16S / genetics
  • Sensitivity and Specificity

Substances

  • DNA Primers
  • DNA, Bacterial
  • RNA, Ribosomal, 16S