Moving towards on-site detection of Shiga toxin-producing Escherichia coli in ready-to-eat leafy greens
Rapid identification of Shiga toxin-producing Escherichia coli, or STEC, is of utmost importance to assure the innocuousness of the foodstuffs. STEC have been implicated in outbreaks associated with different types of foods however, among them, ready-to-eat (RTE) vegetables are particularly problema...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2024-01-01
|
| Series: | Current Research in Food Science |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S266592712400042X |
| _version_ | 1797262827871797248 |
|---|---|
| author | Ana Costa-Ribeiro Alexandre Lamas Azucena Mora Marta Prado Alejandro Garrido-Maestu |
| author_facet | Ana Costa-Ribeiro Alexandre Lamas Azucena Mora Marta Prado Alejandro Garrido-Maestu |
| author_sort | Ana Costa-Ribeiro |
| collection | DOAJ |
| description | Rapid identification of Shiga toxin-producing Escherichia coli, or STEC, is of utmost importance to assure the innocuousness of the foodstuffs. STEC have been implicated in outbreaks associated with different types of foods however, among them, ready-to-eat (RTE) vegetables are particularly problematic as they are consumed raw, and are rich in compounds that inhibit DNA-based detection methods such as qPCR. In the present study a novel method based on Loop-mediated isothermal amplification (LAMP) to overcome the limitations associated with current molecular methods for the detection of STEC in RTE vegetables targeting stx1 and stx2 genes. In this sense, LAMP demonstrated to be more robust against inhibitory substances in food. In this study, a comprehensive enrichment protocol was combined with four inexpensive DNA extraction protocols. The one based on silica purification enhanced the performance of the method, therefore it was selected for its implementation in the final method. Additionally, three different detection chemistries were compared, namely real-time fluorescence detection, and two end-point colorimetric strategies, one based on the addition of SYBR Green, and the other based on a commercial colorimetric master mix. After optimization, all three chemistries demonstrated suitable for the detection of STEC in spiked RTE salad samples, as it was possible to reach a LOD50 of 0.9, 1.4, and 7.0 CFU/25 g for the real-time, SYBR and CC LAMP assays respectively. All the performance parameters reached values higher than 90 %, when compared to a reference method based on multiplex qPCR. More specifically, the analytical sensitivity was 100, 90.0 and 100 % for real-time, SYBR and CC LAMP respectively, the specificity 100 % for all three assays, and accuracy 100, 96 and 100 %. Finally, a high degree of concordance was also obtained (1, 0.92 and 1 respectively). Considering the current technological advances, the method reported, using any of the three detection strategies, demonstrated suitable for their implementation in decentralized settings, with low equipment resources. |
| first_indexed | 2024-04-25T00:03:18Z |
| format | Article |
| id | doaj.art-0a6ed0f2eef34aeabced7adfaf2964a0 |
| institution | Directory Open Access Journal |
| issn | 2665-9271 |
| language | English |
| last_indexed | 2024-04-25T00:03:18Z |
| publishDate | 2024-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Current Research in Food Science |
| spelling | doaj.art-0a6ed0f2eef34aeabced7adfaf2964a02024-03-14T06:16:04ZengElsevierCurrent Research in Food Science2665-92712024-01-018100716Moving towards on-site detection of Shiga toxin-producing Escherichia coli in ready-to-eat leafy greensAna Costa-Ribeiro0Alexandre Lamas1Azucena Mora2Marta Prado3Alejandro Garrido-Maestu4International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330, Braga, Portugal; Department of Biochemistry, Genetics and Immunology, University of Vigo, 36310, Vigo, SpainFood Hygiene, Inspection and Control Laboratory (Lhica), Department of Analytical Chemistry, Nutrition, and Bromatology, Veterinary School, Campus Terra, Universidade de Santiago de Compostela (USC), 27002, Lugo, SpainLaboratorio de Referencia de E. coli (LREC), Dpto. de Microbioloxía e Parasitoloxía, Facultade de Veterinaria, Universidade de Santiago de Compostela (USC), Lugo, Spain; Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Santiago, SpainInternational Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330, Braga, Portugal; Food Hygiene, Inspection and Control Laboratory (Lhica), Department of Analytical Chemistry, Nutrition, and Bromatology, Veterinary School, Campus Terra, Universidade de Santiago de Compostela (USC), 27002, Lugo, SpainInternational Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330, Braga, Portugal; Laboratory of Microbiology and Technology of Marine Products (MicroTEC), Instituto de Investigaciones Marinas (IIM), CSIC, Eduardo Cabello, 6, 36208, Vigo, Spain; Corresponding author. International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330 Braga, Portugal.Rapid identification of Shiga toxin-producing Escherichia coli, or STEC, is of utmost importance to assure the innocuousness of the foodstuffs. STEC have been implicated in outbreaks associated with different types of foods however, among them, ready-to-eat (RTE) vegetables are particularly problematic as they are consumed raw, and are rich in compounds that inhibit DNA-based detection methods such as qPCR. In the present study a novel method based on Loop-mediated isothermal amplification (LAMP) to overcome the limitations associated with current molecular methods for the detection of STEC in RTE vegetables targeting stx1 and stx2 genes. In this sense, LAMP demonstrated to be more robust against inhibitory substances in food. In this study, a comprehensive enrichment protocol was combined with four inexpensive DNA extraction protocols. The one based on silica purification enhanced the performance of the method, therefore it was selected for its implementation in the final method. Additionally, three different detection chemistries were compared, namely real-time fluorescence detection, and two end-point colorimetric strategies, one based on the addition of SYBR Green, and the other based on a commercial colorimetric master mix. After optimization, all three chemistries demonstrated suitable for the detection of STEC in spiked RTE salad samples, as it was possible to reach a LOD50 of 0.9, 1.4, and 7.0 CFU/25 g for the real-time, SYBR and CC LAMP assays respectively. All the performance parameters reached values higher than 90 %, when compared to a reference method based on multiplex qPCR. More specifically, the analytical sensitivity was 100, 90.0 and 100 % for real-time, SYBR and CC LAMP respectively, the specificity 100 % for all three assays, and accuracy 100, 96 and 100 %. Finally, a high degree of concordance was also obtained (1, 0.92 and 1 respectively). Considering the current technological advances, the method reported, using any of the three detection strategies, demonstrated suitable for their implementation in decentralized settings, with low equipment resources.http://www.sciencedirect.com/science/article/pii/S266592712400042XSTECShiga toxin-producing E. colistx1stx2Point-of-careLoop-mediated isothermal amplification |
| spellingShingle | Ana Costa-Ribeiro Alexandre Lamas Azucena Mora Marta Prado Alejandro Garrido-Maestu Moving towards on-site detection of Shiga toxin-producing Escherichia coli in ready-to-eat leafy greens Current Research in Food Science STEC Shiga toxin-producing E. coli stx1 stx2 Point-of-care Loop-mediated isothermal amplification |
| title | Moving towards on-site detection of Shiga toxin-producing Escherichia coli in ready-to-eat leafy greens |
| title_full | Moving towards on-site detection of Shiga toxin-producing Escherichia coli in ready-to-eat leafy greens |
| title_fullStr | Moving towards on-site detection of Shiga toxin-producing Escherichia coli in ready-to-eat leafy greens |
| title_full_unstemmed | Moving towards on-site detection of Shiga toxin-producing Escherichia coli in ready-to-eat leafy greens |
| title_short | Moving towards on-site detection of Shiga toxin-producing Escherichia coli in ready-to-eat leafy greens |
| title_sort | moving towards on site detection of shiga toxin producing escherichia coli in ready to eat leafy greens |
| topic | STEC Shiga toxin-producing E. coli stx1 stx2 Point-of-care Loop-mediated isothermal amplification |
| url | http://www.sciencedirect.com/science/article/pii/S266592712400042X |
| work_keys_str_mv | AT anacostaribeiro movingtowardsonsitedetectionofshigatoxinproducingescherichiacoliinreadytoeatleafygreens AT alexandrelamas movingtowardsonsitedetectionofshigatoxinproducingescherichiacoliinreadytoeatleafygreens AT azucenamora movingtowardsonsitedetectionofshigatoxinproducingescherichiacoliinreadytoeatleafygreens AT martaprado movingtowardsonsitedetectionofshigatoxinproducingescherichiacoliinreadytoeatleafygreens AT alejandrogarridomaestu movingtowardsonsitedetectionofshigatoxinproducingescherichiacoliinreadytoeatleafygreens |