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Establishment of a Rapid LAMP Assay for Aeromonas hydrophila and Comparison with the Application of qPCR

The development of an exceptionally sensitive diagnostic technique for early identification of aquaculture diseases, specifically Aeromonas hydrophila, is essential for efficient management of disease outbreaks at aquaculture locations. In this research, a swift and sensitive diagnostic assay employ...

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Autores principales: Gao, Zihui, Yang, Chunhua, Zhang, Xiaobo, Hu, Bing, Zhang, Huang, Zhang, Zhihong, Kuang, Wendong, Zheng, Qiuyue, Cao, Jijuan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10383757/
https://www.ncbi.nlm.nih.gov/pubmed/37512548
http://dx.doi.org/10.3390/metabo13070841
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author Gao, Zihui
Yang, Chunhua
Zhang, Xiaobo
Hu, Bing
Zhang, Huang
Zhang, Zhihong
Kuang, Wendong
Zheng, Qiuyue
Cao, Jijuan
author_facet Gao, Zihui
Yang, Chunhua
Zhang, Xiaobo
Hu, Bing
Zhang, Huang
Zhang, Zhihong
Kuang, Wendong
Zheng, Qiuyue
Cao, Jijuan
author_sort Gao, Zihui
collection PubMed
description The development of an exceptionally sensitive diagnostic technique for early identification of aquaculture diseases, specifically Aeromonas hydrophila, is essential for efficient management of disease outbreaks at aquaculture locations. In this research, a swift and sensitive diagnostic assay employing Loop-mediated isothermal amplification (LAMP) of Aeromonas hydrophila was devised and compared to the conventional qPCR method documented by Rong Wang. Validation of the diagnostic assay was carried out using actual samples obtained from aquaculture fish. The findings revealed that based on the rapid detection of crude bacterial genomic DNA, the fluorescent LAMP assay possessed a lower limit of detection (LOD) of 0.559 ng/μL (0.315–1.693, 95% CI), while the LOD for qPCR stood at 4.301 ng/μL (2.084–8.876, 95% CI). Both techniques demonstrated outstanding specificity, exhibiting no cross-reactivity with bacteria from the same or closely related genera. A total of 74 fish samples suspected to be infected with the fish disease were gathered, with 26 and 23 samples testing positive for Aeromonas hydrophila via LAMP and qPCR, respectively. The concordance analysis for LAMP and qPCR methods generated a Kappa value of 0.909 (0.778–1.000, 95% CI), signifying a high degree of diagnostic consensus. This study highlights that the LAMP assay eliminates the thermal cycle temperature change process of qPCR, uses lysate to crudely extract bacterial genomic DNA, and can complete the detection within 40 min, rendering it a practical and efficient alternative for monitoring disease outbreaks at aquaculture sites.
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spelling pubmed-103837572023-07-30 Establishment of a Rapid LAMP Assay for Aeromonas hydrophila and Comparison with the Application of qPCR Gao, Zihui Yang, Chunhua Zhang, Xiaobo Hu, Bing Zhang, Huang Zhang, Zhihong Kuang, Wendong Zheng, Qiuyue Cao, Jijuan Metabolites Article The development of an exceptionally sensitive diagnostic technique for early identification of aquaculture diseases, specifically Aeromonas hydrophila, is essential for efficient management of disease outbreaks at aquaculture locations. In this research, a swift and sensitive diagnostic assay employing Loop-mediated isothermal amplification (LAMP) of Aeromonas hydrophila was devised and compared to the conventional qPCR method documented by Rong Wang. Validation of the diagnostic assay was carried out using actual samples obtained from aquaculture fish. The findings revealed that based on the rapid detection of crude bacterial genomic DNA, the fluorescent LAMP assay possessed a lower limit of detection (LOD) of 0.559 ng/μL (0.315–1.693, 95% CI), while the LOD for qPCR stood at 4.301 ng/μL (2.084–8.876, 95% CI). Both techniques demonstrated outstanding specificity, exhibiting no cross-reactivity with bacteria from the same or closely related genera. A total of 74 fish samples suspected to be infected with the fish disease were gathered, with 26 and 23 samples testing positive for Aeromonas hydrophila via LAMP and qPCR, respectively. The concordance analysis for LAMP and qPCR methods generated a Kappa value of 0.909 (0.778–1.000, 95% CI), signifying a high degree of diagnostic consensus. This study highlights that the LAMP assay eliminates the thermal cycle temperature change process of qPCR, uses lysate to crudely extract bacterial genomic DNA, and can complete the detection within 40 min, rendering it a practical and efficient alternative for monitoring disease outbreaks at aquaculture sites. MDPI 2023-07-12 /pmc/articles/PMC10383757/ /pubmed/37512548 http://dx.doi.org/10.3390/metabo13070841 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Gao, Zihui
Yang, Chunhua
Zhang, Xiaobo
Hu, Bing
Zhang, Huang
Zhang, Zhihong
Kuang, Wendong
Zheng, Qiuyue
Cao, Jijuan
Establishment of a Rapid LAMP Assay for Aeromonas hydrophila and Comparison with the Application of qPCR
title Establishment of a Rapid LAMP Assay for Aeromonas hydrophila and Comparison with the Application of qPCR
title_full Establishment of a Rapid LAMP Assay for Aeromonas hydrophila and Comparison with the Application of qPCR
title_fullStr Establishment of a Rapid LAMP Assay for Aeromonas hydrophila and Comparison with the Application of qPCR
title_full_unstemmed Establishment of a Rapid LAMP Assay for Aeromonas hydrophila and Comparison with the Application of qPCR
title_short Establishment of a Rapid LAMP Assay for Aeromonas hydrophila and Comparison with the Application of qPCR
title_sort establishment of a rapid lamp assay for aeromonas hydrophila and comparison with the application of qpcr
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10383757/
https://www.ncbi.nlm.nih.gov/pubmed/37512548
http://dx.doi.org/10.3390/metabo13070841
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