Detection of Enterotoxigenic Clostridium perfringens in Raw Beef by Polymerase Chain Reaction

Abstract

A polymerase chain reaction (PCR) procedure was developed for direct detection of Clostridium perfringens strains with potential for food poisoning in raw beef samples. An oligonucleotide primer pair was used to amplify a 364 base pair sequence internal to the C. perfringens enterotoxin gene. One milliliter portions of the meat homogenates were inoculated into cooked meat medium (CMM) or reduced Fluid Thioglycollate (FTG) medium and incubated at 37°C. Portions sampled at 2, 4, 6, 8 and 24 h of enrichment were assayed for detection of the enterotoxin sequence by PCR. Amplification of the 364 bp sequence could be detected in 6 h by agarose gel electrophoresis and as early as 2 h by hybridization to a 150 bp digoxigenin (DIG)-labeled probe. To increase the sensitivity of the detection assay a commercial chromosomal deoxyribonucleic acid (DNA) extraction assay was compared with a nested PCR approach. Both methods allowed detection of less than 1 log10 colony forming units (CFU)/g of C. perfringens strains harboring the enterotoxin gene, with no interference with the background microflora present in the raw ground beef.