Development of fluorescence and colorimetric LAMP-based assays for rapid and sensitive detection of toxigenic Bacillus cereus group strains in plant-based products

Abstract

Bacillus cereus represents a major concern for the microbiological safety of plant-based foods due to its ability to form heat-resistant spores and produce toxins associated with foodborne illness. Rapid, field-adapted tools for toxin gene detection are, therefore, needed to support surveillance and safety management. In this study, Loop-Mediated Isothermal Amplification (LAMP) assays targeting the emetic (cesA) and enterotoxigenic (hblD, nheB, cytK-1) genes of B. cereus were developed and their performance in plant-based products was evaluated. LAMP primer sets using qPCR equipment (qLAMP) were assessed for sensitivity and specificity with a diverse strain panel. Analytical sensitivity was also tested in spiked plant-based matrices. Seventy-two commercial plant-based foods and supplements, including dairy, meat, and fish analogues, were screened by colorimetric assays, with inconclusive samples subsequently analysed via qLAMP. No amplification occurred with non-Bacillus DNA. hblD, nheB, and cytK-1 assays showed 100% specificity to reference assignments, while cesA assay generated one apparent false positive. All assays successfully amplified target DNA in spiked matrices even at the lowest level of 101 spores × mL−1. The colorimetric analysis of commercial products showed mostly clearly positive (yellow) or negative (red) results. Some intermediate (orange) signals were confirmed by qLAMP as very low-level positives, while others showed no amplification. Among detected genes, nheB was the most frequent, followed by hblD, cytK-1, and cesA. Overall, LAMP assays provide rapid, sensitive, and specific detection of B. cereus toxin genes. The colorimetric method represents an effective first-line screening tool for plant-based foods, with qLAMP confirmation recommended for ambiguous results.