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 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.

Loop-mediated isothermal amplification of the toxR gene coupled with lateral flow dipstick (LAMP-LFD) for the novel, rapid and specific visual detection of Vibrio harveyi

“…Detection of V. harveyi also has been reported to be performed using a more rapid and sensitive approach compared to conventional PCR known as real-time PCR by manipulating specific unique target DNA regions. The examples of the target region that have been used for the detection of this bacterium by real-time PCR are mreB (rod shape-determining) (Mougin et al, 2020), groEL (protein which belongs to the chaperonin family) (Costa et al, 2022) and 16S rRNA (small subunit ribosomal RNA) genes (Fukui and Sawabe, 2008). Although the PCRbased detection method is a very useful approach to detect V. harveyi, the method is still considered laborious, time-consuming and required skilled manpower making it not suitable to be used for point of care detection to get reliable information of the pathogen in the fields especially in the early stage for timely control measures.…”