We first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of a Clostridium perfringens-specific real-time PCR (rtPCR) assay based on the cpa gene (cpa rtPCR) by using a bacterial strain panel composed of C. perfringens and non-C. perfringens Clostridium strains. All non-C. perfringens Clostridium strains tested negative, whereas all C. perfringens strains tested positive with the cpa rtPCR, for an analytical specificity and ubiquity of 100%. The cpa rtPCR assay was then used to confirm the identity of 116 putative C. perfringens isolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar and cpa rtPCR were identified by sequencing the 16S rRNA and cpa genes. Four mCP ؊ /rtPCR ؉ colonies were identified as C. perfringens, whereas 3 mCP ؉ /rtPCR ؊ colonies were identified as non-C. perfringens. The cpa rtPCR was negative with all 51 non-C. perfringens strains and positive with 64 of 65 C. perfringens strains. Finally, we compared mCP agar and a CRENAME (concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment) procedure plus cpa rtPCR (CRENAME ؉ cpa rtPCR) for their abilities to detect C. perfringens spores in drinking water. CRENAME ؉ cpa rtPCR detected as few as one C. perfringens CFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME ؉ cpa rtPCR also allows the simultaneous and sensitive detection of Escherichia coli and C. perfringens from the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection.T he presence of Escherichia coli in drinking water indicates a recent fecal contamination, as well as a risk of waterborne gastrointestinal diseases (1). Under some circumstances, however, it has been shown that this microorganism inadequately indicates the presence of viruses and protozoan parasites of human health significance (2). For example, while chlorine in water rapidly inactivates E. coli, it leaves most disinfection-resistant pathogens almost unaffected for several hours (3). According to Payment and Franco (3), Clostridium perfringens is a suitable indicator of human enteric viruses, Giardia cysts, and Cryptosporidium oocysts in finished water and can also be used in the assessment of water treatment processes due to the resistance of Clostridium spores to chlorine. Furthermore, the presence of C. perfringens in water is also associated with fecal contamination, and it has been evaluated and utilized as an alternative indicator of fecal pollution (4-14). Thus, a simple and reliable culture-based method to isolate and enumerate C. perfringens, the membrane filtration method on mCP agar, has been elaborated and evaluated to monitor the presence of C. perfringens in water (15)(16)(17)(18)(19)(20). In the European Union, the mCP a...