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
The closely related members of the Bacillus cereus-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 threshol...
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Frontiers Media S.A.
2022-06-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.930648/full |
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| author | Annemarie Busch Ulrich Schotte Nadja Jeßberger Hendrik Frentzel Madeleine Plötz Amir Abdulmawjood |
| author_facet | Annemarie Busch Ulrich Schotte Nadja Jeßberger Hendrik Frentzel Madeleine Plötz Amir Abdulmawjood |
| author_sort | Annemarie Busch |
| collection | DOAJ |
| description | The closely related members of the Bacillus cereus-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 B. cereus-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 groEL was used to determine the representatives of the B. cereus-group. Second, since bacteria in which nheB is present are basically capable of producing enterotoxins, this gene was chosen for detection. The specificity of the developed assay was 100% for B. cereus-group isolates and 93.7% for the detection of nheB. The analytical sensitivity was 0.1 pg DNA/μl. Using simplified DNA extraction by boiling, cell-based sensitivity was determined. Targeting groEL and nheB, 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 B. cereus-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 groEL-LAMP. After a 6-h incubation period, isolates bearing the toxin gene nheB could also be reliably detected. In addition, colony material was boiled and used as a LAMP template for simple detection. Specificity for the B. cereus-group was 100 and 93.22% detecting nheB. The study demonstrated that screening of food samples with the groEL/nheB-LAMP assay can be performed within 1 day, making it possible to detect critical levels of potentially NHE-toxin-producing cells of the B. cereus-group. |
| first_indexed | 2024-04-12T13:19:32Z |
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| id | doaj.art-59a7ca7ca6e3422e826ba42516f4a7cf |
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| publishDate | 2022-06-01 |
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| spelling | doaj.art-59a7ca7ca6e3422e826ba42516f4a7cf2022-12-22T03:31:34ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-06-011310.3389/fmicb.2022.930648930648Establishment and Validation of a Two-Step LAMP Assay for Detection of Bacillus cereus-Group Isolates in Food and Their Possibility of Non-haemolytic Enterotoxin ProductionAnnemarie Busch0Ulrich Schotte1Nadja Jeßberger2Hendrik Frentzel3Madeleine Plötz4Amir Abdulmawjood5Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Hanover, GermanyDepartment A (Veterinary Medicine), Central Institute of the Bundeswehr Medical Service Kiel, Kronshagen, GermanyInstitute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Hanover, GermanyUnit Bacterial Toxins, Food Service, Department of Biological Safety, German Federal Institute for Risk Assessment, Berlin, GermanyInstitute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Hanover, GermanyInstitute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Hanover, GermanyThe closely related members of the Bacillus cereus-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 B. cereus-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 groEL was used to determine the representatives of the B. cereus-group. Second, since bacteria in which nheB is present are basically capable of producing enterotoxins, this gene was chosen for detection. The specificity of the developed assay was 100% for B. cereus-group isolates and 93.7% for the detection of nheB. The analytical sensitivity was 0.1 pg DNA/μl. Using simplified DNA extraction by boiling, cell-based sensitivity was determined. Targeting groEL and nheB, 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 B. cereus-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 groEL-LAMP. After a 6-h incubation period, isolates bearing the toxin gene nheB could also be reliably detected. In addition, colony material was boiled and used as a LAMP template for simple detection. Specificity for the B. cereus-group was 100 and 93.22% detecting nheB. The study demonstrated that screening of food samples with the groEL/nheB-LAMP assay can be performed within 1 day, making it possible to detect critical levels of potentially NHE-toxin-producing cells of the B. cereus-group.https://www.frontiersin.org/articles/10.3389/fmicb.2022.930648/fullLAMPBacillus cereusnheBgroELtoxin productionfast detection |
| spellingShingle | Annemarie Busch Ulrich Schotte Nadja Jeßberger Hendrik Frentzel Madeleine Plötz Amir Abdulmawjood 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 Frontiers in Microbiology LAMP Bacillus cereus nheB groEL toxin production fast detection |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | LAMP Bacillus cereus nheB groEL toxin production fast detection |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.930648/full |
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