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Controlled multiplex PCR of enterotoxigenic Clostridium perfringens strains in food samples
H Schoepe1, H Potschka, T Schlapp
1Institut für Hygiene und Infektionskrankheiten der Tiere der Justus-Liebig Universität Giessen, Frankfurter Str. 87-89, Giessen, 35392, Germany.
Abstract:
A controlled multiplex polymerase chain reaction (PCR) for the detection of Clostridium(C.) perfringens enterotoxin gene (cpe) was established and compared with an in vitro antigenic detection method. Thecpe PCR and the classical method of electric immunodiffusion gave identical results. The predicted specific amplicon of the cpe gene was generated from all of the tested enterotoxigenic C. perfringens strains. In contrast, cultures of any other Clostridium species tested by PCR were negative (100% sensitivity, 100% specificity). Addition of an alphatoxin (plc) gene specific PCR as an in process control reaction was performed in order to prevent false negative PCR results. The PCR detection limit was 0.5 ng of genomic C. perfringens DNA per ml of bouillon culture. By contaminating minced meat with C. perfringens reference strains, the multiplex PCR was established as a tool for routine diagnostic laboratories. The detection limit was approximately 3.0x10(5)C. perfringens cells per gram meat. The results demonstrate the multiplex PCR as an easy, specific, sensitive and time saving diagnostic procedure. Application of this improved method should enhance the knowledge concerning epidemiological aspects of food borne diseases caused by enterotoxigenic C. perfringens.
Insights
A new multiplex PCR method accurately detects the Clostridium perfringens enterotoxin gene (cpe). This sensitive and specific diagnostic tool aids in identifying foodborne illnesses caused by enterotoxigenic C. perfringens.
Area of Science:
- Microbiology
- Molecular Biology
- Food Safety
Background:
- Clostridium perfringens is a significant cause of foodborne illness.
- Accurate detection of the enterotoxin gene (cpe) is crucial for diagnosis.
- Existing detection methods may have limitations.
Purpose of the Study:
- To establish and validate a multiplex polymerase chain reaction (PCR) for detecting the C. perfringens cpe gene.
- To compare the multiplex PCR with an existing in vitro antigenic detection method.
- To assess the sensitivity, specificity, and practical application of the developed PCR assay.
Main Methods:
- Development of a controlled multiplex PCR assay targeting the C. perfringens cpe gene.
- Inclusion of an alphatoxin (plc) gene PCR as an in-process control.
- Comparison with electric immunodiffusion for antigenic detection.
- Testing on various Clostridium species and spiked minced meat samples.
Main Results:
- The multiplex PCR demonstrated 100% sensitivity and 100% specificity, matching electric immunodiffusion results.
- The assay successfully amplified the cpe gene from enterotoxigenic C. perfringens strains only.
- Detection limits were established at 0.5 ng genomic DNA/ml and approximately 3.0x10^5 cells/g in minced meat.
- The assay proved effective as a routine diagnostic tool.
Conclusions:
- The developed multiplex PCR is a reliable, sensitive, specific, and time-saving method for C. perfringens enterotoxin gene detection.
- This assay can be readily implemented in routine diagnostic laboratories.
- The method will aid in understanding the epidemiology of foodborne diseases linked to enterotoxigenic C. perfringens.