CHEMILUMINESCENT ENZYME-IMMUNOASSAY FOR DETECTION OF PCR-AMPLIFIED ENTEROTOXIN-A FROM CLOSTRIDIUM-PERFRINGENS

A PCR protocol was developed for the rapid and specific detection of C lostridium perfringens strains harboring the enterotoxin A gene in art ificially contaminated ground beef, A biotinylated primer pair was des igned for amplification of a 750 bp fragment of the C. perfringens ent erotoxin A gene. A combination of 4 h enrichment incubation and nuclei c acid extraction, followed by 2 h of PCR amplification allowed detect ion at levels below 10 CFU of freshly grown cells in raw and cooked be ef samples. PCR amplified products were confirmed by a Southern hybrid ization assay using a digoxigenin-labeled internal probe, and two hybr idization ELISA protocols (PCR-ELISA) applying a streptavidin rapture step for the hybridized PCR products. Both enzyme immunoassays utilize d chemiluminescent detection with Lumiphos 530(TM) as substrate, after hybridization to an internal digoxigenin-labeled probe or a 5' conjug ated alkaline phosphatase-labeled probe. The PCR-ELISA resulted in fas ter confirmation of the PCR products while providing a level of sensit ivity comparable to Southern hybridization, and has potential for deve lopment into an automated method.