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

Abstract

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 <i>Vibrio alginolyticus</i>, <i>Listonella anguillara</i>, and <i>Vibrio harveyi</i> using a new primer pair targeting the <i>groEL</i> 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 <i>V. alginolyticus</i> and <i>L. anguillara</i>, and 48 CFU mL^-1 for <i>V. harveyi</i>. 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 <i>V. harveyi</i> 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.