Establishment and Validation of a Two-Step LAMP Assay for Detection of Bacillus cereus-Group Isolates in Food and Their Possibility of Non-haemolytic Enterotoxin Production

Abstract

The closely related members of the <i>Bacillus cereus</i>-group can mainly only be differentiated by whole genome sequencing. Among them, there are potentially toxin-producing bacteria. When consumed with food, these can cause vomiting or diarrhea and abdominal cramps. To date, although no EU-wide threshold exists, a bacterial count of 10⁵ CFU/g can be regarded as critical. Specific and rapid detection of the bacteria is difficult due to their close relationship, and no loop-mediated isothermal amplification (LAMP) assay has been developed so far to detect potentially toxin-producing members of the <i>B. cereus</i>-group. Aim of this study was to develop a LAMP method to detect critical cell counts specifically and rapidly of potentially non-haemolytic enterotoxin (NHE)-producing cells of this group. A two-step LAMP assay was developed. First, the target sequence <i>groEL</i> was used to determine the representatives of the <i>B. cereus</i>-group. Second, since bacteria in which <i>nheB</i> is present are basically capable of producing enterotoxins, this gene was chosen for detection. The specificity of the developed assay was 100% for <i>B. cereus</i>-group isolates and 93.7% for the detection of <i>nheB</i>. The analytical sensitivity was 0.1 pg DNA/μl. Using simplified DNA extraction by boiling, cell-based sensitivity was determined. Targeting <i>groEL</i> and <i>nheB</i>, 11.35-27.05 CFU/reaction and 11.35-270.5 CFU/reaction were detectable, respectively. Artificially contaminated samples were investigated to prove the application in foods. Direct detection of the critical value of <i>B. cereus</i>-group cells was possible in 83.3% of samples and detecting the toxin-gene 50% thereof. After a 6-h incubation period, the detection rate increased to 100 and 91.7%, respectively. Additionally, 100 natively contaminated food samples were tested, also quantitatively and culturally. Samples with relevant contamination levels were reliably detected using <i>groEL</i>-LAMP. After a 6-h incubation period, isolates bearing the toxin gene <i>nheB</i> could also be reliably detected. In addition, colony material was boiled and used as a LAMP template for simple detection. Specificity for the <i>B. cereus</i>-group was 100 and 93.22% detecting <i>nheB.</i> The study demonstrated that screening of food samples with the <i>groEL</i>/<i>nheB</i>-LAMP assay can be performed within 1 day, making it possible to detect critical levels of potentially NHE-toxin-producing cells of the <i>B. cereus</i>-group.