Evaluation of synthetic DNA probes for confirmation of Clostridium perfringens enterotoxin gene PCR products.
A new diagnostic reagent was developed that is capable of detecting the presence of Clostridium perfringens rapidly and accurately compared to the conventional methods. C. perfringens enterotoxin (cpe) gene is the gene of interest since it encodes the enterotoxin responsible for food poisoning. Two new cpe-specific labeled DNA probes were evaluated using Southern and dot blot hybridization. Bacterial DNA was amplified by a duplex PCR procedure. The results showed that 40 enterotoxin producing C. perfringens strains generated two bands of amplicons with sizes of 420 and 280 bp, whereas 40 non-enterotoxin producing strains produced a single band of 280 bp on agarose gel-electrophoresis. No bands were observed from 32 strains of Clostridium spp and other bacteria. Southern blot analysis using either cpe-specific DNA or oligonucleotide probe showed hybridization specifically to the 420 bp band in enterotoxin-positive C. perfringens. On the dot blot membrane, both cpe-specific DNA and oligonucleotide probes were able to hybridize specifically with the corresponding DNA templates but with different efficacy (100% vs 91.1%).
Tansuphasiri
Unchalee
U
Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok, Thailand. unchalee@loxley.co.th
Muadcheingka
Taniya
T
Choonharuangdej
Suwan
S
D015139:Blotting, Southern; D003016:Clostridium perfringens / Q000235:genetics* / Q000302:isolation & purification; D015342:DNA Probes; D004587:Electrophoresis, Agar Gel; D004768:Enterotoxins / Q000235:genetics* / Q000302:isolation & purification; D005517:Foodborne Diseases / Q000382:microbiology; D005798:Genes, Bacterial; D006801:Humans; D015151:Immunoblotting; D007202:Indicators and Reagents; D016133:Polymerase Chain Reaction
D015342:DNA Probes; D004768:Enterotoxins; D007202:Indicators and Reagents; C035598:enterotoxin, Clostridium