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

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

Yuli Song  1 , Chengxu Liu, Sydney M Finegold
Affiliations

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

Yuli Song et al. Appl Environ Microbiol. 2004 Nov.

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.

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Figures

FIG. 1.
FIG. 1.
Standard curve generated by analysis of a dilution series of C. bolteae cells spiked in stools by real-time PCR. Quantitation was performed by determining the CT. The same experiment, which included a known C. bolteae-positive stool with an unknown count (•), was repeated six times. The CT values, the CFU/PCR determined, and R2 values of each experiment are listed in the inset. Means and the standard deviation were calculated from these six experiments. ⧫, C. bolteae standards.
FIG. 2.
FIG. 2.
Standard curves generated by analysis of a dilution series of DNA extracted from mixed cultures of representative strains for each Clostridium cluster by real-time PCR. The CT values are plotted against the corresponding cell numbers in the PCR. ▴, Clostridium cluster I; ⧫, Clostridium cluster XI; ▪, Clostridium cluster XIVab.
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