Development of duplex PCR assay for rapid detection of enterotoxigenic isolates of Clostridium perfringens

A duplex PCR assay was developed for the rapid and specific amplification of the alpha-toxin (phospholipase C, plc) gene and the enterotoxin (cpe) gene from Clostridium perfringens. Two pairs of primers were newly designed for the species identification and also for the differentiation between enterotoxin-positive and enterotoxin-negative C. perfringens strains in a single reaction. The detection by agarose gel electrophoresis yielded 2 bands of 280-bp of plc and 420-bp of cpe for all four enterotoxin-positive reference strains tested without the need for further hybridization, and one band of 280-bp of plc for all seven enterotoxin-negative reference strains. While 50 strains of other Clostridium species and other bacteria tested by PCR were negative for both genes. The detection limit, as measured with purified DNA was 10 fg or as few as 4 organisms could be detected. This assay was used to identify primary fecal spore isolates from 244 fecal specimens of patients with diarrhea. Of total 432 colonies from 144 positive growth cultures determined, 21 revealed both plc and cpe genes and 411 were positive for plc gene only. This suggested a prevalent of 5% of all C. perfringens strains that carry the enterotoxin gene. The results indicate the duplex PCR as a simple, sensitive, specific, cost-effective and time saving assay for detection of potentially enterotoxigenic isolates of C. perfringens, and has potential application for epidemiological investigations of food poisoning outbreaks and quality control of food products for humans and animal feeds.