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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Bibliographic Details
Published in:Microorganisms (Basel) 2022-10, Vol.10 (10), p.2060
Main Authors: Costa, Cátia, Ferreira, Guilherme D, Simões, Marco, Silva, Joana L, Campos, Maria J
Format: Article
Language:English
Subjects:
Algae
Annealing
Aquaculture
Bacteria
Bacteria, Pathogenic
Deoxyribonucleic acid
Design
DNA
Economic impact
Genetic aspects
GroEL gene
Identification and classification
Listonella anguillara
Methods
Microorganisms
Outbursts
Pathogens
Polymerase chain reaction
Protocol
qPCR
Real time
Vibrio
Vibrio alginolyticus
Vibrio harveyi
Vibrionaceae
Vibriosis
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Summary: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.
ISSN:2076-2607
2076-2607
DOI:10.3390/microorganisms10102060