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 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 , , and using a new primer pair targeting the 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 in the case of and , and 48 CFU mL for . 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 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.