Research In Rapid Detection Methods Of Several Pathogens In Aquaculture

Vibrio alginolyticus, Vibrio anguillarum, Infectious spleen and kidney necrosis virus (ISKNV) and White spot syndrome virus (WSSV) have been considered as four of the most important pathogens in aquaculture because of their ecological and economic impacts in the world aquaculture industry. Accordingly, the establishment of practical diagnosic methods has long been desired. In this paper, the outer membrane protein K gene (OmpK) of V. alginolyticus was amplified by PCR and cloned into high efficient expression vector pET28a. The expression product was purified by an affinity chromatographic method. Polyclonal antibody (PAb) was obtained by immunizing mice with protein OmpK. Then, an indirect Enzyme-Linked Immunosorbent Assay (ELISA) for the rapid diagnosis of V. alginolyticus was developed using the PAb. The lowest V. alginolyticus suspension was 104 CFU. Cross reactions of antisera with other bacteria were detected, all results were negative. Loop-mediated isothermal amplification (LAMP) has been developed by Notomi et al. (2000), which is a novel nucleic acid-based detection technology. A set of primers were designed from the OmpK sequence of V. alginolyticus to develop LAMP assay for V. alginolyticus detection. The assay was optimised to amplify V. alginolyticus DNA by incubation at 65℃for only 1 h. LAMP amplification products had a ladder-like appearance when electrophoresed on an agarose gel. The detection limit of the LAMP assay was 38 CFU which was found to be higher than the commonly used PCR method. A 20-min LAMP method for amplification of the metalloprotease empA gene of V. anguillarum using biotin-labeled primer was combined with a chromatographic lateral flow dipstick (LFD) for rapid and simple visual detection of V. anguillarum-specific amplicons. The LFD process involved a 5-min post LAMP step for specific hybridization of DNA with an FITC-labeled DNA probe that confirmed the presence of specific, biotin-labeled V. anguillarum amplicons. The resulting DNA duplexes could be visualized trapped at the LFD strip test line within 5 min of sample exposure. Using the combined LAMP and LFD system, the total assay interval was approximately 30 min, excluding the time for DNA extraction. The detection limit of V. anguillarum by LAMP-LFD was 7.7 CFU per mL but PCR could detect up to 77 CFU per mL. The LAMP-LFD method could detect the presence of V. anguillarum from kidney, liver, spleen and muscle of experimentally infected ayu with V. anguillarum. The LAMP-LFD assay was established for ISKNV detection by using the primers designed from the DNA polymerase gene (DPOL) sequence of ISKNV. The detection limit of LAMP-LFD was as low as 10 copies viral DNA, which was 10 and 1000 times more sensitive than LAMP combined with agarose gel electrophoresis (LAMP-AGE) and PCR combined with agarose gel electrophoresis (PCR-AGE), respectively. A set of primers was designed from the VP26 gene sequence of WSSV to establish LAMP assay for WSSV detection. The assay was 10 times more sensitive than PCR and its result was observed by adding diluted fluorescent dye such as SYBR Green I. These results indicate that the sensitivity of the LAMP methods established in this paper were equal to that of the conventional PCR or even higher. In addition, because this assy is rapid, simple and dose not require sophisticated equipment, the LAMP assy can potentially be used for rapid clinical diagnosis.