Abstract: Objective To establish the specific and sensitive real time fluorescence quantitative PCR(RT-PCR)assay for the rapid detection of Clostridium difficile,evaluate two standard curves of quantitative using pure bacterial culture or same concentration of bacteria in feces and quantitatively measure C. difficile counts in feces of mice infected with C. difficile. Methods The specific primers and probe for Taqman based PCR were designed based on 16S rRNA sequence of C. difficile. A TaqMan probe based RT-PCR assay was established,and its specificity,sensitivity and standard curve were assessed. The standard curve based on pure bacterial gradient dilution or the same concentration gradient bacteria in feces were compared to determine the optimal quantitative RT-PCR. Antibiotic-treated C57BL/6 mice were infected with virulent C. difficile strain NAP1/027 to establish C. difficile infected(CDI)mouse model. The amounts of C. difficile in fecal samples of CDI mice were detected by using quantitative RT-PCR and culture method. Results The specificity and sensitivity of this established TaqMan probe based RT-PCR were high. The correlation coefficient and slope value of standard curve were 0.999 8 and-3.400 4 respectively. There was no significant difference in counts of C. difficile in artificial prepared mimic fecal samples using two standard curves of quantitative RT-PCR based on pure bacterial gradient dilution or the same concentration gradient bacteria in feces. Antibiotic cocktail pretreated CDI mouse model was established. The results showed that the quantitative RT-PCR can detect the amount of C. difficile in mice feces with high efficiency and accuracy and can replace the culture method. Conclusion The quantitative RT-PCR is reliable and efficient for the detection of colonization of C. difficile in CDI mouse model by using standard curve based on pure bacteria compared with the culture method. This RT-PCR assay can be used to detect the C. difficile in CDI mouse model.