Application of QCM DNA biosensor to detect a marine derived pathogenic virus VHSV

摘要

Each year infectious diseases cause substantial economic losses for public and commercial aquaculture ventures. VHS is one of most serious viral diseases caused by VHSV and damaging both fresh and marine fish species (Skall et al., 2005). Detecting VHSV has been dependent on the conventional methods which are time consuming and need highly skilled techniques (Skall et al., 2005). Thus this study aims to apply a sensitive QCM biosensor for detecting VHSV infection in fish to reduce detection time. Quartz micro balance (QCM) biosensors are working by measuring mass changes on sensor chip surfaces (Clark et al., 1987). Biological pathogens can be attached to the receptors immobilized surface of the biosensor chip and form an absorbed rigid thin film. This absorbed rigid films leads to the mass change of the electrodes and this mass changes leads to the change in the resonant frequency of the biosensor chip material. The receptors to detect pathogens can be any substances specifically binding ligands. Normally antibodies can be used as a receptor to detect pathogens but it has limitations as a proteinrnwhich is unstable in ambient environment and in regeneration process. Recently researchers are seeking more stable receptors for example peptides, DNA, PNA, aptamers, and so on (Minunni et al., 2003, Tombelli et al., 2006). In this study we tried to develop a QCM DNA sensor to detect VHSV genome consisted by single strand RNA. We targeted to detect a main gene in VHSV genome i.e. G protein and constructed a probe specific for VHSV G protein RNA. We also attempted three different methods to immobilize probe DNA on quartz surface coated with gold: immobilization of thiol labelled probe DNA on naked gold surface (Tombelli et al., 2006), immobilization of amino labelled probe DNA on gold surface prepared as carboxyl chip using MPA followed by EDC/NHS activation, and immobilization of biotin labelled probe DNA on gold surface after immobilising avidin on carboxyl chip prior to biotin (Aung et al., 2008). As a result, immobilization method using avidin-biotin interaction was most efficient to detect target DNA. The QCM biosensor chip was outstanding for regeneration and sensitivity.