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Real-Time PCR Protocol for Detection and Quantification of Three Pathogenic Members of the Vibrionaceae Family

Vibriosis, an often-fatal disease induced by pathogenic members of the Vibrionaceae family, causes severe economic losses in aquacultures. To mitigate/avoid vibriosis outbursts, it is vital to detect and quantify these pathogens as early as possible. However, standard microbiological methods are tim...

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Autores principales: Costa, Cátia, Ferreira, Guilherme D., Simões, Marco, Silva, Joana L., Campos, Maria J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9609007/
https://www.ncbi.nlm.nih.gov/pubmed/36296336
http://dx.doi.org/10.3390/microorganisms10102060
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author Costa, Cátia
Ferreira, Guilherme D.
Simões, Marco
Silva, Joana L.
Campos, Maria J.
author_facet Costa, Cátia
Ferreira, Guilherme D.
Simões, Marco
Silva, Joana L.
Campos, Maria J.
author_sort Costa, Cátia
collection PubMed
description Vibriosis, an often-fatal disease induced by pathogenic members of the Vibrionaceae family, causes severe economic losses in aquacultures. To mitigate/avoid vibriosis outbursts, it is vital to detect and quantify these pathogens as early as possible. However, standard microbiological methods are time-consuming and often underestimate cell counts, which calls for the development of valid alternatives. In this study, real-time polymerase chain reaction (qPCR) was employed to detect the pathogenic species Vibrio alginolyticus, Listonella anguillara, and Vibrio harveyi using a new primer pair targeting the groEL gene. In addition, the DNA extraction efficiency of three methods, two commercial kits and the boiling method, was compared. The most efficient method was the DNeasy Blood and Tissue kit, with a detection limit ranging between 154 and 600 CFU mL(−1) in the case of V. alginolyticus and L. anguillara, and 48 CFU mL(−1) for V. harveyi. Thus, this study presents the development and evaluation of a method for the early quantification of all three species in saline suspensions. However, the results obtained by spiking a microalgae sample with V. harveyi emphasize the importance of adjusting the DNA control’s standard curve to the relevant extraction matrices, as it affects the DNA extraction efficiency and may hamper an accurate quantification with qPCR.
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spelling pubmed-96090072022-10-28 Real-Time PCR Protocol for Detection and Quantification of Three Pathogenic Members of the Vibrionaceae Family Costa, Cátia Ferreira, Guilherme D. Simões, Marco Silva, Joana L. Campos, Maria J. Microorganisms Article Vibriosis, an often-fatal disease induced by pathogenic members of the Vibrionaceae family, causes severe economic losses in aquacultures. To mitigate/avoid vibriosis outbursts, it is vital to detect and quantify these pathogens as early as possible. However, standard microbiological methods are time-consuming and often underestimate cell counts, which calls for the development of valid alternatives. In this study, real-time polymerase chain reaction (qPCR) was employed to detect the pathogenic species Vibrio alginolyticus, Listonella anguillara, and Vibrio harveyi using a new primer pair targeting the groEL gene. In addition, the DNA extraction efficiency of three methods, two commercial kits and the boiling method, was compared. The most efficient method was the DNeasy Blood and Tissue kit, with a detection limit ranging between 154 and 600 CFU mL(−1) in the case of V. alginolyticus and L. anguillara, and 48 CFU mL(−1) for V. harveyi. Thus, this study presents the development and evaluation of a method for the early quantification of all three species in saline suspensions. However, the results obtained by spiking a microalgae sample with V. harveyi emphasize the importance of adjusting the DNA control’s standard curve to the relevant extraction matrices, as it affects the DNA extraction efficiency and may hamper an accurate quantification with qPCR. MDPI 2022-10-18 /pmc/articles/PMC9609007/ /pubmed/36296336 http://dx.doi.org/10.3390/microorganisms10102060 Text en © 2022 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
Costa, Cátia
Ferreira, Guilherme D.
Simões, Marco
Silva, Joana L.
Campos, Maria J.
Real-Time PCR Protocol for Detection and Quantification of Three Pathogenic Members of the Vibrionaceae Family
title Real-Time PCR Protocol for Detection and Quantification of Three Pathogenic Members of the Vibrionaceae Family
title_full Real-Time PCR Protocol for Detection and Quantification of Three Pathogenic Members of the Vibrionaceae Family
title_fullStr Real-Time PCR Protocol for Detection and Quantification of Three Pathogenic Members of the Vibrionaceae Family
title_full_unstemmed Real-Time PCR Protocol for Detection and Quantification of Three Pathogenic Members of the Vibrionaceae Family
title_short Real-Time PCR Protocol for Detection and Quantification of Three Pathogenic Members of the Vibrionaceae Family
title_sort real-time pcr protocol for detection and quantification of three pathogenic members of the vibrionaceae family
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9609007/
https://www.ncbi.nlm.nih.gov/pubmed/36296336
http://dx.doi.org/10.3390/microorganisms10102060
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