Jinik Hwang1, Sung-Suk Suh2, Mirye Park1, Myung-Joo Oh3, Jong-Oh Kim3, Sukchan Lee4, Taek-Kyun Lee5. 1. South Sea Environment Research Department, Korea Institute of Ocean Science and Technology, Geoje 656-830, Republic of Korea; Korea University of Science and Technology, Daejeon 305-350, Republic of Korea. 2. South Sea Environment Research Department, Korea Institute of Ocean Science and Technology, Geoje 656-830, Republic of Korea. 3. Department of Aqualife Medicine, Chonnam National University, Yeosu 550-749, Republic of Korea. 4. Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea. 5. South Sea Environment Research Department, Korea Institute of Ocean Science and Technology, Geoje 656-830, Republic of Korea; Korea University of Science and Technology, Daejeon 305-350, Republic of Korea. Electronic address: tklee@kiost.ac.
Abstract
OBJECTIVE: To establish a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the identification of nervous necrosis virus (NNV) infection. METHODS: A set of synthesized primers was used to match the sequences of a specific region of the nnv gene from the National Center for Biotechnology Information database, not originating from NNV-infected fish, the efficiency and specificity of LAMP were measured dependent on the concentration of DNA polymerase and the reaction temperature and time. In addition, to determine species-specific LAMP primers, cross reactivity testing was applied to the reaction between NVV and other virus families including viral hemorrhagic septicemia virus and marine birnavirus. RESULTS: The optimized LAMP reaction carried out at 64 °C for 60 min, and above 4 U Bst DNA polymerase. The sensitivity of LAMP for the detection of nnv was thus about 10 times greater than the sensitivity of polymerase chain reaction. The LAMP assay primers were specific for the detection NNV infection in Epinephelus septemfasciatus. CONCLUSIONS: The development of LAMP primers based on genetic information from a public database, not virus-infected samples, may provide a very simple and convenient method to identify viral infection in aquatic organisms.
OBJECTIVE: To establish a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the identification of nervous necrosis virus (NNV) infection. METHODS: A set of synthesized primers was used to match the sequences of a specific region of the nnv gene from the National Center for Biotechnology Information database, not originating from NNV-infected fish, the efficiency and specificity of LAMP were measured dependent on the concentration of DNA polymerase and the reaction temperature and time. In addition, to determine species-specific LAMP primers, cross reactivity testing was applied to the reaction between NVV and other virus families including viral hemorrhagic septicemia virus and marine birnavirus. RESULTS: The optimized LAMP reaction carried out at 64 °C for 60 min, and above 4 U Bst DNA polymerase. The sensitivity of LAMP for the detection of nnv was thus about 10 times greater than the sensitivity of polymerase chain reaction. The LAMP assay primers were specific for the detection NNV infection in Epinephelus septemfasciatus. CONCLUSIONS: The development of LAMP primers based on genetic information from a public database, not virus-infected samples, may provide a very simple and convenient method to identify viral infection in aquatic organisms.
Authors: Maja A Zaczek-Moczydłowska; Letitia Mohamed-Smith; Anna Toldrà; Chantelle Hooper; Mònica Campàs; M Dolors Furones; Tim P Bean; Katrina Campbell Journal: Int J Mol Sci Date: 2020-09-09 Impact factor: 5.923