Sensitive quantification of Clostridium difficile cells by reverse transcription-quantitative PCR targeting rRNA molecules

Abstract

We established a sensitive and accurate quantification system for Clostridium difficile in human intestines, based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR). We newly developed a species-specific primer set for C. difficile targeting 23S rRNA gene sequences. Both the vegetative cells and the spores of C. difficile in human feces were quantified by RT-qPCR,with a lower detection limit of 102.4 cells/g of feces. In an analysis of the feces of residents (n=83; age, 85±8 years) and staff(n=19; age, 36±10 years) at a care facility for the elderly, C. difficile was detected by RT-qPCR in 43% of the residents (average count, log10 4.0±2.0 cells/gof feces) and 16% of the staff (average count, log10 2.2±0.1 cells/g of feces); these rates were far higher than those detected by qPCR (residents, 19%; staff, 0%) or selectivecultivation (residents, 18%; staff, 5%). Another analysis of healthy adults (n=63; age, 41±11 years) also revealedthe significant carriage rate of C. difficile in the intestines (detection rate, 13%; average count, log10 4.9±1.2 cells/g of feces). From these results, it was suggested that rRNA-targeted RT-qPCR should be an effective tool for analyzing population levels of C. difficile in the human intestine. © 2012, American Society for Microbiology.