Related Experiment Video
Updated: Jul 19, 2026
Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples
Published on: October 18, 2019
Enterotoxigenic Bacillus spp. DNA fingerprint revealed in naturally contaminated nonfat dry milk powder using rep-PCR
Robin M Cooper1, John L McKillip
1Roche Diagnostics Corporation, Indianapolis, IN 46256, USA.
Abstract:
Dry milk powders and functional ingredients frequently contain high levels of viable bacterial spores, some of which may result in growth of toxigenic Bacillus spp. in reconstituted and temperature-abused foods. Samples from nonfat dry milk (NFDM), infant milk formula (IMF), coffee creamer, lecithin, and cocoa powder were subjected to a short heat treatment followed by enrichment in tryptone phosphate glucose yeast extract (TPGY) broth at 32 degrees C for 12-25 hours to obtain cell densities of 10(6) CFU ml(-1). DNA was extracted using a modification of established protocol, leading to the development of an optimized method for each food system. Purified DNA was amplified by rep-PCR using extragenic sequence-targeting primers and optimized for each food. PCR fingerprints from each food were analyzed electrophoretically for banding patterns earlier correlated to that of enterotoxigenic Bacillus spp. and Bacillus cereus positive control DNA fingerprints. Reverse passive latex agglutination (RPLA) and Bacillus Diarrhoeal Enterotoxin Enzyme Linked Immunosorbent Assay (Tecra Diagnostics) confirmed the presence of HBL and NHE enterotoxin production in NFDM, Coffee creamer, infant milk formula, and two lecithin samples but not in cocoa powder. These results demonstrate the utility of rep-PCR not only as a tool for bacterial genotyping, but a unique means of quality control and hygiene monitoring in food microbiology.
Insights
This study developed a rep-PCR method to detect toxigenic Bacillus in dry food ingredients. The method identified enterotoxin-producing bacteria in nonfat dry milk, infant milk formula, and coffee creamer, aiding food safety.
Area of Science:
- Food Microbiology
- Molecular Biology
- Food Safety
Background:
- Dry milk powders and functional ingredients can harbor toxigenic bacterial spores.
- These spores may grow in reconstituted and temperature-abused foods, posing health risks.
- Accurate detection methods are crucial for food quality control.
Purpose of the Study:
- To optimize and validate a rep-PCR method for detecting toxigenic Bacillus species in various food matrices.
- To assess the presence of enterotoxin production in common dry food ingredients.
- To establish rep-PCR as a tool for bacterial genotyping and food hygiene monitoring.
Main Methods:
- Heat treatment and enrichment of food samples (NFDM, IMF, coffee creamer, lecithin, cocoa powder) in TPGY broth.
- DNA extraction and purification optimized for each food matrix.
- Repetitive sequence-based PCR (rep-PCR) amplification using extragenic sequence-targeting primers.
- Electrophoretic analysis of PCR fingerprints and comparison with known enterotoxigenic Bacillus DNA.
- Confirmation of enterotoxin production using Reverse Passive Latex Agglutination (RPLA) and ELISA.
Main Results:
- Optimized rep-PCR methods were developed for analyzing DNA from different food systems.
- PCR fingerprints correlated with enterotoxigenic Bacillus species and Bacillus cereus.
- Enterotoxin production (HBL and NHE) was confirmed in nonfat dry milk, coffee creamer, infant milk formula, and lecithin samples.
- Cocoa powder samples did not show evidence of enterotoxin production.
Conclusions:
- Rep-PCR is a valuable tool for bacterial genotyping in food microbiology.
- The developed rep-PCR method offers a unique means for quality control and hygiene monitoring in food production.
- This technique aids in identifying potential risks associated with toxigenic Bacillus contamination in dry food ingredients.
