Nested polymerase chain reaction for detection of low levels of enterotoxigenic Clostridium perfringens in animal feces and meat.

A rapid and sensitive method for detecting enterotoxigenic Clostridium perfringens in animal feces and meat is described. The method consists of a combination of nested polymerase chain reaction (PCR) with enrichment culture of the sample. In the PCR, two pairs of oligonucleotide primers homologous to the C. perfringens enterotoxin (CPE) gene were used: the first primer pair amplified a 425-bp fragment and the second one amplified a 199-bp fragment within the fragment amplified by the first PCR. When the specificity and sensitivity of nested PCR in the detection of the CPE gene of isolated C. perfringens were compared with those of the single PCR, the former amplified specifically the 199-bp fragment and the sensitivity was about 10(3)-fold higher that that of the latter. The nested PCR combined with enrichment culture was applied to detecting the CPE gene in samples inoculated artificially with enterotoxigenic C. perfringens. This method detected within 22-26 hr the CPE gene in samples of animal feces and meat inoculated with fewer than 10(1) CFU/g of enterotoxigenic C. perfringens. When the method was applied to detection of indigenous enterotoxigenic C. perfringens in cattle feces, pig feces, beef and pork, C. perfringens was found in one case (10%) of 10 cattle fecal samples, and the PCR-amplified product corresponded to the fragment of the CPE gene in restriction endonucleases digestion pattern.