MULTIPLEX PCR ASSAY FOR DETECTION OF CLOSTRIDIUM-PERFRINGENS IN FECESAND INTESTINAL CONTENTS OF PIGS AND IN SWINE FEED

A multiplex polymerase chain reaction (PCR) assay, developed to detect the alpha-toxin and enterotoxin genes (cpa and cpe, respectively) of Clostridium perfringens, was used to identify enterotoxigenic isolates of this organism from feces and intestinal contents of pigs and from feed samples from pig farms in Iowa. The organism was grown on tryptos e-sulfite-cycloserine (TSC) agar, TSC agar without egg-yolk, sheep blo od a,oar, or in brain heart infusion broth or cooked meat medium. DNA was extracted by boiling and the PCR assay was carried out using reage nts from a commercial kit. The 319 bp amplification product of cpa and the 364 bp product of cpe were visualized under UV light after electr ophoresis in a 2% agarose gel containing ethidium bromide, The average sensitivity of the assay, determined on artificially contaminated fec es, was 9.2x10(4) colony forming units per gram. Assay of 97 isolates from feces and intestinal contents revealed cpa in 89, but all were ne gative for cpe. While 28% of the 442 total samples cultured yielded C. perfringens, only 5% of 298 fecal or intestinal contents samples were positive upon direct examination by the PCR assay. Ninety-one and eig ht-tenths % of isolates with the phenotype of C, perfringens were cpa positive by PCR. Forty-three percent of feed samples were culture posi tive, while 48.3% were PCR positive for cpa. None of these were cpe po sitive. We conclude that PCR is a useful assay for rapid detection of C. perfringens in feed, and for confirmation of the identity of isolat es presumed to be C. perfringens. (C) 1998 Elsevier Science B.V. All r ights reserved.