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Development of loop-mediated isothermal amplification for rapid detection of sporotrichosis caused by Sporothrix schenckii
BACKGROUND AND AIM: Sporothrix schenckii is the causative agent of sporotrichosis, which most commonly causes lymphocutaneous infections in immunocompromised hosts. This pathogen infects dogs, cats, cattle, and buffaloes and can potentially infect humans. Diagnosis by fungal culture is lengthy, and...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Veterinary World
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10421540/ https://www.ncbi.nlm.nih.gov/pubmed/37577200 http://dx.doi.org/10.14202/vetworld.2023.1356-1362 |
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author | Chupia, Vena Ninsuwon, Jirapat Intanon, Montira Pikulkaew, Surachai |
author_facet | Chupia, Vena Ninsuwon, Jirapat Intanon, Montira Pikulkaew, Surachai |
author_sort | Chupia, Vena |
collection | PubMed |
description | BACKGROUND AND AIM: Sporothrix schenckii is the causative agent of sporotrichosis, which most commonly causes lymphocutaneous infections in immunocompromised hosts. This pathogen infects dogs, cats, cattle, and buffaloes and can potentially infect humans. Diagnosis by fungal culture is lengthy, and although there are several clinical diagnoses and molecular methods, these are complicated and time-consuming for veterinarians. This study aimed to develop a visual diagnostic assay that is less time-consuming and can be used by veterinarians to screen for sporotrichosis. MATERIALS AND METHODS: To develop a loop-mediated isothermal amplification (LAMP) assay for sporotrichosis, primers specific for fragments of the 18S rRNA gene of S. schenckii were designed. Then, the time and temperature were optimized to successfully achieve LAMP. Ten-fold serial dilutions of DNA were used to determine the detection limit using both LAMP and nested polymerase chain reaction (nPCR) assays. RESULTS: The optimal LAMP conditions were incubation at 73°C for 30 min. Agarose gel electrophoresis revealed a ladder-like pattern of the LAMP product, and a sky-blue color indicated a positive result. A comparison of the LAMP assay with nPCR revealed that it was 10 times more sensitive than nPCR, with a detection limit of 10 pg. The use of a heat box compared with a thermocycler gave the same results. CONCLUSION: Loop-mediated isothermal amplification gives good results and may represent a future alternative diagnostic tool for screening fungal pathogens before the results of conventional fungal cultures are received. However, this method should be further studied to clarify its use with clinical samples. |
format | Online Article Text |
id | pubmed-10421540 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Veterinary World |
record_format | MEDLINE/PubMed |
spelling | pubmed-104215402023-08-12 Development of loop-mediated isothermal amplification for rapid detection of sporotrichosis caused by Sporothrix schenckii Chupia, Vena Ninsuwon, Jirapat Intanon, Montira Pikulkaew, Surachai Vet World Research Article BACKGROUND AND AIM: Sporothrix schenckii is the causative agent of sporotrichosis, which most commonly causes lymphocutaneous infections in immunocompromised hosts. This pathogen infects dogs, cats, cattle, and buffaloes and can potentially infect humans. Diagnosis by fungal culture is lengthy, and although there are several clinical diagnoses and molecular methods, these are complicated and time-consuming for veterinarians. This study aimed to develop a visual diagnostic assay that is less time-consuming and can be used by veterinarians to screen for sporotrichosis. MATERIALS AND METHODS: To develop a loop-mediated isothermal amplification (LAMP) assay for sporotrichosis, primers specific for fragments of the 18S rRNA gene of S. schenckii were designed. Then, the time and temperature were optimized to successfully achieve LAMP. Ten-fold serial dilutions of DNA were used to determine the detection limit using both LAMP and nested polymerase chain reaction (nPCR) assays. RESULTS: The optimal LAMP conditions were incubation at 73°C for 30 min. Agarose gel electrophoresis revealed a ladder-like pattern of the LAMP product, and a sky-blue color indicated a positive result. A comparison of the LAMP assay with nPCR revealed that it was 10 times more sensitive than nPCR, with a detection limit of 10 pg. The use of a heat box compared with a thermocycler gave the same results. CONCLUSION: Loop-mediated isothermal amplification gives good results and may represent a future alternative diagnostic tool for screening fungal pathogens before the results of conventional fungal cultures are received. However, this method should be further studied to clarify its use with clinical samples. Veterinary World 2023-06 2023-06-17 /pmc/articles/PMC10421540/ /pubmed/37577200 http://dx.doi.org/10.14202/vetworld.2023.1356-1362 Text en Copyright: © Chupia, 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 Chupia, Vena Ninsuwon, Jirapat Intanon, Montira Pikulkaew, Surachai Development of loop-mediated isothermal amplification for rapid detection of sporotrichosis caused by Sporothrix schenckii |
title | Development of loop-mediated isothermal amplification for rapid detection of sporotrichosis caused by Sporothrix schenckii |
title_full | Development of loop-mediated isothermal amplification for rapid detection of sporotrichosis caused by Sporothrix schenckii |
title_fullStr | Development of loop-mediated isothermal amplification for rapid detection of sporotrichosis caused by Sporothrix schenckii |
title_full_unstemmed | Development of loop-mediated isothermal amplification for rapid detection of sporotrichosis caused by Sporothrix schenckii |
title_short | Development of loop-mediated isothermal amplification for rapid detection of sporotrichosis caused by Sporothrix schenckii |
title_sort | development of loop-mediated isothermal amplification for rapid detection of sporotrichosis caused by sporothrix schenckii |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10421540/ https://www.ncbi.nlm.nih.gov/pubmed/37577200 http://dx.doi.org/10.14202/vetworld.2023.1356-1362 |
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