A Membrane-Array Method to Detect Specific Human Intestinal Bacteria in Fecal Samples Using Reverse Transcriptase-PCR and Chemiluminescence

A membrane-based oligonucleotide array was used to detect predominant bacterial species in human fecal samples. Digoxygenin-labeled 16S rDNA probes were generated by PCR from DNA that had been extracted from fecal samples or slurries. These probes were hybridized to an array of 120 oligonucleotides with sequences specific for 40 different bacterial species commonly found in human feces, followed by color development using an alkaline phosphatase-conjugated antibody and NBT /BCIP. Twenty of the species were detected by this method, but E. coli, which was present at $\~$1 $\times 10$^5$ CFU per gram feces, was not detected. To improve the sensitivity of this assay, reverse transcriptase-PCR was used to generate probes from RNA extracted from fecal cultures. Coupled with a chemiluminescence detection method, this approach lowered the detection limit for E. coli from $\~1$ $\times 10$^6$ to ${\leq}$ 1 $\times 10$^5$ These results indicate that the membrane-array method with reverse transcriptase-PCR and chemiluminescence detection can simultaneously identify bacterial species present in fecal samples at cell concentrations as low as${\leq}$ 1 $\times 10$^5$ CFU per gram.