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Applied and Environmental Microbiology, November 2004, p. 6459-6465, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6459-6465.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Yuli Song,1* Chengxu Liu,1 and Sydney M. Finegold2,3,4

Research Service,1 Infectious Diseases Section, VA Medical Center West Los Angeles,2 Department of Medicine,3 Department of Microbiology, Immunology, and Molecular Genetics, UCLA School of Medicine, Los Angeles, California4

Received 11 February 2004/ Accepted 27 June 2004

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 108 CFU/g in autistic children and 4.8 x 108 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.

* Corresponding author. Mailing address: VA Medical Center West Los Angeles, 11301 Wilshire Blvd., Room E3-237, Bldg. 304, Los Angeles, CA 90073. Phone: (310) 478-3711, ext. 49151. Fax: (310) 268-4458. E-mail: yulis1{at}yahoo.com.

Applied and Environmental Microbiology, November 2004, p. 6459-6465, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6459-6465.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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