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Development of loop-mediated isothermal amplification-lateral flow dipstick as a rapid screening test for detecting Listeria monocytogenes in frozen food products using a specific region on the ferrous iron transport protein B gene

BACKGROUND AND AIM: Listeria monocytogenes is a critical foodborne pathogen that infects pregnant females and their newborns and older adults and individuals with comorbidities. It contaminates fresh vegetables, fruits, ready-to-eat foods, and frozen food products consumed by individuals. The cultur...

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Autores principales: Srisawat, Wimvipa, Saengthongpinit, Chalermkiat, Nuchchanart, Wirawan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Veterinary World 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9047130/
https://www.ncbi.nlm.nih.gov/pubmed/35497940
http://dx.doi.org/10.14202/vetworld.2022.590-601
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author Srisawat, Wimvipa
Saengthongpinit, Chalermkiat
Nuchchanart, Wirawan
author_facet Srisawat, Wimvipa
Saengthongpinit, Chalermkiat
Nuchchanart, Wirawan
author_sort Srisawat, Wimvipa
collection PubMed
description BACKGROUND AND AIM: Listeria monocytogenes is a critical foodborne pathogen that infects pregnant females and their newborns and older adults and individuals with comorbidities. It contaminates fresh vegetables, fruits, ready-to-eat foods, and frozen food products consumed by individuals. The culture conventional detection methods for L. monocytogenes are time-consuming, taking 4 days. This study aimed to describe the development and comparison of loop-mediated isothermal amplification (LAMP)- lateral flow dipstick (LFD), LAMP assay to PCR, and conventional culture for detecting L. monocytogenes in frozen food products. MATERIALS AND METHODS: Five LAMP primer sets, including F3, B3, forward inner primer, and backward inner primer, were designed from a specific region on ferrous iron transport protein B gene (feoB gene) to amplify LAMP products. The DNA probe was created, and the detection limit was determined in pure culture and purified DNA, as well as the detection in 20 frozen food product samples. RESULTS: The LMfeoB4 LAMP primer sets and DNA probe were LAMP products amplified at 60°C for 50 min. The specificity of the assay revealed no cross-reactivity with other pathogenic bacteria. The limit of detection (LOD) of the LAMP-LFD and LAMP assays using purified genomic DNA was 219 fg/μL both in LAMP and LAMP-LFD assays. The LOD of LAMP and LAMP-LFD assays in pure culture was 4.3×10(2) colony-forming unit (CFU)/mL and 43 CFU/mL, respectively. The LOD of the LAMP-LFD assay using artificially inoculated chicken in frozen food samples with pre-enrichment was 3.2×10(2) CFU/mL. The LAMP-LFD was also more sensitive than the LAMP assay and polymerase chain reaction. Finally, LAMP-LFD revealed no false positives in any of the 20 frozen food product samples. CONCLUSION: LAMP-LFD assay using a specific region on the feoB gene to detect L. monocytogenes was highly specific, sensitive, faster, and convenient, making it a valuable tool for the monitoring and rapid screening of L. monocytogenes in frozen food products. This technique is applicable to the development of detection technologies for other pathogens in food products.
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spelling pubmed-90471302022-04-29 Development of loop-mediated isothermal amplification-lateral flow dipstick as a rapid screening test for detecting Listeria monocytogenes in frozen food products using a specific region on the ferrous iron transport protein B gene Srisawat, Wimvipa Saengthongpinit, Chalermkiat Nuchchanart, Wirawan Vet World Research Article BACKGROUND AND AIM: Listeria monocytogenes is a critical foodborne pathogen that infects pregnant females and their newborns and older adults and individuals with comorbidities. It contaminates fresh vegetables, fruits, ready-to-eat foods, and frozen food products consumed by individuals. The culture conventional detection methods for L. monocytogenes are time-consuming, taking 4 days. This study aimed to describe the development and comparison of loop-mediated isothermal amplification (LAMP)- lateral flow dipstick (LFD), LAMP assay to PCR, and conventional culture for detecting L. monocytogenes in frozen food products. MATERIALS AND METHODS: Five LAMP primer sets, including F3, B3, forward inner primer, and backward inner primer, were designed from a specific region on ferrous iron transport protein B gene (feoB gene) to amplify LAMP products. The DNA probe was created, and the detection limit was determined in pure culture and purified DNA, as well as the detection in 20 frozen food product samples. RESULTS: The LMfeoB4 LAMP primer sets and DNA probe were LAMP products amplified at 60°C for 50 min. The specificity of the assay revealed no cross-reactivity with other pathogenic bacteria. The limit of detection (LOD) of the LAMP-LFD and LAMP assays using purified genomic DNA was 219 fg/μL both in LAMP and LAMP-LFD assays. The LOD of LAMP and LAMP-LFD assays in pure culture was 4.3×10(2) colony-forming unit (CFU)/mL and 43 CFU/mL, respectively. The LOD of the LAMP-LFD assay using artificially inoculated chicken in frozen food samples with pre-enrichment was 3.2×10(2) CFU/mL. The LAMP-LFD was also more sensitive than the LAMP assay and polymerase chain reaction. Finally, LAMP-LFD revealed no false positives in any of the 20 frozen food product samples. CONCLUSION: LAMP-LFD assay using a specific region on the feoB gene to detect L. monocytogenes was highly specific, sensitive, faster, and convenient, making it a valuable tool for the monitoring and rapid screening of L. monocytogenes in frozen food products. This technique is applicable to the development of detection technologies for other pathogens in food products. Veterinary World 2022-03 2022-03-12 /pmc/articles/PMC9047130/ /pubmed/35497940 http://dx.doi.org/10.14202/vetworld.2022.590-601 Text en Copyright: © Srisawat, et al. https://creativecommons.org/licenses/by/4.0/Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Srisawat, Wimvipa
Saengthongpinit, Chalermkiat
Nuchchanart, Wirawan
Development of loop-mediated isothermal amplification-lateral flow dipstick as a rapid screening test for detecting Listeria monocytogenes in frozen food products using a specific region on the ferrous iron transport protein B gene
title Development of loop-mediated isothermal amplification-lateral flow dipstick as a rapid screening test for detecting Listeria monocytogenes in frozen food products using a specific region on the ferrous iron transport protein B gene
title_full Development of loop-mediated isothermal amplification-lateral flow dipstick as a rapid screening test for detecting Listeria monocytogenes in frozen food products using a specific region on the ferrous iron transport protein B gene
title_fullStr Development of loop-mediated isothermal amplification-lateral flow dipstick as a rapid screening test for detecting Listeria monocytogenes in frozen food products using a specific region on the ferrous iron transport protein B gene
title_full_unstemmed Development of loop-mediated isothermal amplification-lateral flow dipstick as a rapid screening test for detecting Listeria monocytogenes in frozen food products using a specific region on the ferrous iron transport protein B gene
title_short Development of loop-mediated isothermal amplification-lateral flow dipstick as a rapid screening test for detecting Listeria monocytogenes in frozen food products using a specific region on the ferrous iron transport protein B gene
title_sort development of loop-mediated isothermal amplification-lateral flow dipstick as a rapid screening test for detecting listeria monocytogenes in frozen food products using a specific region on the ferrous iron transport protein b gene
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9047130/
https://www.ncbi.nlm.nih.gov/pubmed/35497940
http://dx.doi.org/10.14202/vetworld.2022.590-601
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