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Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples
Published on: October 18, 2019
Detection of enterotoxigenic Clostridium perfringens in meat samples by using molecular methods
Ikuko Kaneko1, Kazuaki Miyamoto, Kanako Mimura
1Department of Microbiology, Wakayama Medical University School of Medicine, Kimiidera, Wakayama 641-0012, Japan.
Abstract:
To prevent food-borne bacterial diseases and to trace bacterial contamination events to foods, microbial source tracking (MST) methods provide important epidemiological information. To apply molecular methods to MST, it is necessary not only to amplify bacterial cells to detection limit levels but also to prepare DNA with reduced inhibitory compounds and contamination. Isolates carrying the Clostridium perfringens enterotoxin gene (cpe) on the chromosome or a plasmid rank among the most important food-borne pathogens. Previous surveys indicated that cpe-positive C. perfringens isolates are present in only ∼5% of nonoutbreak food samples and then only at low numbers, usually less than 3 cells/g. In this study, four molecular assays for the detection of cpe-positive C. perfringens isolates, i.e., ordinary PCR, nested PCR, real-time PCR, and loop-mediated isothermal amplification (LAMP), were developed and evaluated for their reliability using purified DNA. For use in the artificial contamination of meat samples, DNA templates were prepared by three different commercial DNA preparation kits. The four molecular assays always detected cpe when >10³ cells/g of cpe-positive C. perfringens were present, using any kit. Of three tested commercial DNA preparation kits, the InstaGene matrix kit appeared to be most suitable for the testing of a large number of samples. By using the InstaGene matrix kit, the four molecular assays efficiently detected cpe using DNA prepared from enrichment culture specimens of meat samples contaminated with low numbers of cpe-positive C. perfringens vegetative cells or spores. Overall, the current study developed molecular assay protocols for MST to detect the contamination of foods with low numbers of cells, and at a low frequency, of cpe-positive C. perfringens isolates.
Insights
This study developed molecular assays to detect low levels of Clostridium perfringens foodborne pathogens. The InstaGene matrix kit proved most effective for preparing DNA from contaminated meat samples for reliable detection.
Area of Science:
- Food safety and microbiology
- Molecular diagnostics
- Epidemiology
Background:
- Microbial source tracking (MST) is crucial for identifying foodborne bacterial pathogens and tracing contamination events.
- Clostridium perfringens isolates carrying the enterotoxin gene (cpe) are significant foodborne pathogens, often found at low levels (<3 cells/g) in non-outbreak food samples.
- Effective DNA extraction and sensitive molecular detection methods are necessary for accurate MST.
Purpose of the Study:
- To develop and evaluate four molecular assays (PCR, nested PCR, real-time PCR, LAMP) for detecting cpe-positive Clostridium perfringens.
- To assess the suitability of different DNA preparation kits for analyzing artificially contaminated meat samples.
- To establish reliable protocols for microbial source tracking of low-level C. perfringens contamination in food.
Main Methods:
- Development and validation of ordinary PCR, nested PCR, real-time PCR, and loop-mediated isothermal amplification (LAMP) assays.
- Artificial contamination of meat samples with known concentrations of cpe-positive C. perfringens.
- DNA extraction using three commercial kits, followed by molecular assay performance evaluation.
- Testing DNA prepared from enrichment cultures of contaminated meat samples.
Main Results:
- All four molecular assays consistently detected cpe when cpe-positive C. perfringens levels exceeded 10³ cells/g, irrespective of the DNA preparation kit used.
- The InstaGene matrix kit demonstrated superior suitability for large-scale sample processing compared to two other kits.
- The optimized protocols successfully detected cpe in DNA from enrichment cultures of meat samples with low C. perfringens contamination (vegetative cells or spores).
Conclusions:
- The study successfully developed and validated molecular assay protocols for microbial source tracking of cpe-positive Clostridium perfringens.
- The developed methods are effective in detecting low numbers and low frequencies of these foodborne pathogens in food matrices.
- The InstaGene matrix kit is recommended for DNA preparation in large-scale food safety testing involving C. perfringens detection.
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