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

Abstract

LAMP (Loop-mediated Isothermal Amplification)법은 PCR를 기반으로 등온에서 autocycling 가닥 변위 DNA 합성에 의존하며, Bst polymerase를 사용하여 진단하는 방법이다. 이것은 대상 DNA의 여섯 개의 배열을 인식하는 4개의 특정 primer의 도움을 받아 단시간 안에 병원체를 식별하는 높은 특이성을 지니고 있다. 본 연구에서는 LAMP로 수생에서 위험한 병원체인 Vibrio alginolyticus의 특별한 LAMP primer를 제작하였으며, 신속한 진단을 위해 MgSO₄, dNTP, Betaine, Bst polymerase의 최적 반응 조건의 특이성 및 기존의 PCR보다 10배 정도의 민감하다는 것을 확인하였다. 또한, 디자인 되어진 LAMP primer가 다른 Vibrio 종들 중 오직 V. alginolyticus에서만 반응한 것을 확인 할 수 있었다. 본 논문에서는 병원체 세균인 V. alginolyticus의 빠르고 민감한 효과적인 진단으로 양식 질병들을 조기에 발견할 수 있도록 개발하였다. Loop-mediated isothermal amplification (LAMP), a PCR-based diagnostic method, is based on autocycling strand displacement DNA synthesis in the presence of exonuclease-negative Bst DNA polymerase under isothermal conditions. With the help of four specific primers that recognize six different sequences of a target DNA, LAMP has high specificity in pathogenic identification in a short time. Hence, in the present study, LAMP is used as a diagnostic tool in the identification of the most dreadful aquatic pathogenic species, Vibrio alginolyticus, and to develop species-specific LAMP primers and optimization of LAMP reaction conditions such as annealing temperature, elongation time, and other PCR chemical concentrations, including MgSO₄, dNTPs, Betaine, and Bst polymerase. The optimized LAMP primers were also checked for specificity with other Vibrio species, which showed that the designed primers were very specific to V. alginolyticus After the first introduction of a species name like this one, the first part (“Vibrio” in this case) should be abbreviated to only the first letter.only. These are usually the most harmful pathogens of the Vibrio species that appear in shrimp and crabs. The results also revealed that the LAMP assay could be 10-fold more sensitive than conventional PCR in detecting V. alginolyticus. This could be the first report on using a rapid and highly sensitive technique, the LAMP assay, in the effective diagnosis of the pathogenic bacteria V. alginolyticus, which could help in the early detection of diseases, particularly in aquaculture.