Chemiluminescent enzyme immunoassay for detection of PCR-amplified enterotoxin A from Clostridium perfringens

Abstract

A PCR protocol was developed for the rapid and specific detection of Clostridium perfringens strains harboring the enterotoxin A gene in artificially contaminated ground beef. A biotinylated primer pair was designed for amplification of a 750 bp fragment of the C. perfringens enterotoxin A gene. A combination of 4 h enrichment incubation and nucleic acid extraction, followed by 2 h of PCR amplification allowed detection at levels below 10 CFU of freshly grown cells in raw and cooked beef samples. PCR amplified products were confirmed by a Southern hybridization assay using a digoxigenin-labeled internal probe, and two hybridization ELISA protocols (PCR-ELISA) applying a streptavidin capture step for the hybridized PCR products. Both enzyme immunoassays utilized chemiluminescent detection with Lumiphos 530 ™ as substrate, after hybridization to an internal digoxigenin-labeled probe or a 5′ conjugated alkaline phosphatase-labeled probe. The PCR-ELISA resulted in faster confirmation of the PCR products while providing a level of sensitivity comparable to Southern hybridization, and has potential for development into an automated method.