Detection of Clostridium piliforme by enzymatic assay of amplified cDNA segment in microtitration plates

We developed a simple enzymatic assay system using reverse transcription-polymerase chain reaction products in a microtitration plate to detect Clostridium piliforme in tissue specimens. The reverse transcription-polymerase chain reaction was performed with a biotin-labeled primer directed in a highly conserved area of the C. piliforme 16S rRNA gene. The amplified cDNA was hybridized to an alkaline phosphatase-labeled DNA probe in a microtube, and the mixture was applied to a microtitration well precoated with streptavidin. Then hybridization signals in the microtitration plate were visualized by reaction with substrate for the alkaline phosphatase. When an optical density > 0.200 at 405 nm was considered a positive result, we found that the system could detect the RNA from as few as 10 organisms. The system was also capable of detecting organisms from the liver, mesenteric lymph nodes, and ileum of experimentally infected mice. In the ileum the C. piliforme gene was detected from 1 to 21 days after infection. This result suggests that the intestinal epithelium is one of the sites of persistent infection. We conclude that this enzymatic assay system of reverse transcription-polymerase chain reaction products is simple, rapid, and convenient for the detection of C. piliforme.