Loop-mediated isothermal Amplification (LAMP)법을 이용한 Vibrio alginolyticus의 신속 진단법 개발

Abstract

The development of rapid, accurate and sensitive diagnostic methods for the identification of pathogens is fundamental for treating and controlling, or even eradicating of infectious disease. Classical pathogenic identifications are based on their culture methods and their microscopic evaluation, which were know for its negative effects such as slow and sensitivetity in standaizing its growth and culture conditions. Therefore, the setting up of more rapid, sensitive and accurate diagnostic methods has long been desired in clinical molecular diagnosis of pathogens. The loop-mediated isothermal amplification method (LAMP), an PCR based diagnostic method based on autocycling strand displacement DNA synthesis in the presence of exonuclease-negative Bst DNA polymerase under isothermal condition. With the help of four specific primers that recognize six different sequences on a target DNA, LAMP has high specificity in pathogenic identification in short time. Since year 2000, the LAMP method has been tested in diagnosis of several viral, bacterial, fungal and protozoans species. Hence in the present study, LAMP is used as diagnostic tool in identification of the most dreadful aquatic pathogenic species Vibrio alginolyticus and to develop species specific LAMP primers, optimization of LAMP reaction conditions like annealing temperature, elongation time and other PCR chemical concentration such as MgSo4, dNTPs, Betaine and Bst polymerase. LAMP primers composed of two outer primers which initiate strand displacement and two inner primer which amplify "the loop" structure through the reaction. The LAMP products were shown a typical ladder-like patten on gel electrophoresis which indicated that stem-loop DNA with inverted repeats was amplified. The amplification was observed at 65℃ from 40 mins to 100 mins. Of which 100 mins elongation time was found to be good. The optimum MgSO4 concentration was 4mM, while the dNTPs concenteration lies in between 400~ 500 ?M. The optimized amount of Betaine was 1.0 M and Bst concentration was 8 U. The optimized LAMP primers were also checked for it specificity with other Vibrio species showed that the designed primers were very specific to Vibrio alginolyticus only. Results also reveals that the LAMP assay could be 10 fold sensitive than the conventional PCR in decting Vibrio alginolyticus. This could be the first report on using a rapid, and highly sensetive technique, LAMP assay, in the effective diagnosis of Vibrio alginolyticus a pathogenic bacteria and helps in the early detection of diseases particularly in aquaculture.