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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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Autores principales: Chupia, Vena, Ninsuwon, Jirapat, Intanon, Montira, Pikulkaew, Surachai
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Veterinary World 2023
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.
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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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