Frontiers in cellular and infection microbiology · 2026
Rapid detection of enterotoxigenic Bacillus cereus by loop-mediated isothermal amplification assay from food and feed samples.
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Haque Md Atiqul, Rahman Md Arifur, Islam Israt, Bristi Uditi Paul, Islam Farzana, Alam Nazmul, Sultana Tanjina, Mumin Jubayer, Ahmed Firoz, He Cheng, Islam Md Aminul
Аннотация
BACKGROUND: Foodborne illnesses caused by enterotoxigenic Bacillus cereus represent a significant public health concern globally. Rapid and simple detection methods are needed, particularly in resource-limited settings where conventional laboratory infrastructure is lacking. This study aimed to develop and validate a loop-mediated isothermal amplification (LAMP) assay targeting three major enterotoxin genes (nheA, cytK, and entFM) for the rapid detection of enterotoxigenic B. cereus in food and feed samples from Bangladesh.
METHODS: LAMP primers for the nheA gene were newly designed, while cytK and entFM primers were adapted from previous studies. Reaction conditions were optimized using B. cereus ATCC 14579. Analytical sensitivity was determined using serial dilutions of pure culture, spiked milk, and genomic DNA. Specificity was assessed against 20 bacterial strains, including Bacillus species and non-Bacillus foodborne pathogens. The assay was validated on 30 field samples (feed, milk, and eggs) and compared with conventional PCR.
RESULTS: Optimal LAMP amplification was achieved at 64°C for 40 min. The limit of detection was 100 fg/µL for genomic DNA, 9 × 102 CFU/mL for pure culture, and 9 × 103 CFU/mL for spiked milk-approximately 10,000-fold more sensitive than conventional PCR. The assay showed 100% specificity for enterotoxigenic Bacillus species, with no cross-amplification of non-target bacteria. In field samples, LAMP detected enterotoxin genes in 73.3% (22/30) for nheA, 53.3% (16/30) for cytK, and 80.0% (24/30) for entFM, compared to 63.3% (19/30), 40.0% (12/30), and 70.0% (21/30) by PCR, respectively. Overall diagnostic sensitivity and specificity against PCR were 96.1% and 66.7%, respectively, with substantial agreement (κ = 0.62).
CONCLUSION: Although LAMP assays for individual B. cereus toxin genes have been described previously, this study represents the first application of this specific nheA-cytK-entFM LAMP panel in the Bangladeshi food and feed context, providing a regionally relevant, field-deployable method for food safety monitoring.
METHODS: LAMP primers for the nheA gene were newly designed, while cytK and entFM primers were adapted from previous studies. Reaction conditions were optimized using B. cereus ATCC 14579. Analytical sensitivity was determined using serial dilutions of pure culture, spiked milk, and genomic DNA. Specificity was assessed against 20 bacterial strains, including Bacillus species and non-Bacillus foodborne pathogens. The assay was validated on 30 field samples (feed, milk, and eggs) and compared with conventional PCR.
RESULTS: Optimal LAMP amplification was achieved at 64°C for 40 min. The limit of detection was 100 fg/µL for genomic DNA, 9 × 102 CFU/mL for pure culture, and 9 × 103 CFU/mL for spiked milk-approximately 10,000-fold more sensitive than conventional PCR. The assay showed 100% specificity for enterotoxigenic Bacillus species, with no cross-amplification of non-target bacteria. In field samples, LAMP detected enterotoxin genes in 73.3% (22/30) for nheA, 53.3% (16/30) for cytK, and 80.0% (24/30) for entFM, compared to 63.3% (19/30), 40.0% (12/30), and 70.0% (21/30) by PCR, respectively. Overall diagnostic sensitivity and specificity against PCR were 96.1% and 66.7%, respectively, with substantial agreement (κ = 0.62).
CONCLUSION: Although LAMP assays for individual B. cereus toxin genes have been described previously, this study represents the first application of this specific nheA-cytK-entFM LAMP panel in the Bangladeshi food and feed context, providing a regionally relevant, field-deployable method for food safety monitoring.