Clostridium perfringens is considered an important index of water pollution and a useful marker to alert water companies to the possible presence of other stress-resistant pathogens. In Europe, C. perfringens has been used in conjunction with other sulphite reducing clostridia to monitor fecal contamination in water since 1960s.

The standard methodology used for C. perfringens detection in water is primarily based on bacterial growth on specific culture media and subsequent confirmation by a series of biochemical tests (ISO 6461 and ISO 7937). Compared to traditional media, m-CP agar provides faster results with increased selectivity and specificity, although its low recovery rate makes TSC media more useful than m-CP. However, TSC requires the use of biochemical tests for presumptive colonies confirmation, presenting a number of disadvantages such as long time-to-results, and frequent ambiguous results.

For those water-testing laboratories using conventional TSC culture isolation methods, we propose a rapid and more specific methodology for the confirmation of presumptive C. perfringens colonies using conventional PCR that, in addition, reduces the presence of false positives. This methodology was tested in 341 environmental water samples from which, 23 out of the total confirmed colonies were positive using biochemical tests and negative by PCR, and identified afterwards as C. sporogenes. The final objective of this work is to extend the simultaneous PCR confirmation method to a set of 10 additional bacteria frequently studied in water, using the same thermal cycler program and the corresponding specific primer set.