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 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 530TM 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.
Baez
L A
LA
US Department of Agriculture, Eastern Regional Research Center, Wyndmoor, PA 19038, USA.
Juneja
V K
VK
Sackitey
S K
SK
D001427:Bacterial Toxins / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence; D002135:Calcium-Binding Proteins; D003016:Clostridium perfringens / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D008163:Luminescent Measurements; D008460:Meat / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D010738:Type C Phospholipases
D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens