期刊
MICROORGANISMS
卷 10, 期 10, 页码 -出版社
MDPI
DOI: 10.3390/microorganisms10102060
关键词
qPCR; Vibrio alginolyticus; Listonella anguillara; Vibrio harveyi; vibriosis; aquaculture
类别
资金
- FCT-Fundacao para a Ciencia e a Tecnologia, I.P. [UIDB/04292/2020, UIDP/04292/2020, POCI-01-0247-FEDER-035234]
- AlgaValor, Portugal 2020 program
This study presents the development and evaluation of a method for the early quantification of Vibrio alginolyticus, Listonella anguillara, and Vibrio harveyi using real-time polymerase chain reaction (qPCR), with a new primer pair targeting the groEL gene. The most efficient DNA extraction method was the DNeasy Blood and Tissue kit. Adjustment of the DNA control's standard curve to the relevant extraction matrices is important for accurate quantification using qPCR.
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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