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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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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. |
format | Online Article Text |
id | pubmed-9609007 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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