Abstract
본 연구에서는 어체내 IHNV 모니터링에 LAMP법의 사용이 가능 하는지를 검토하기 위해 IHNV를 무지개송어에 인위적으로 감염시킨 후 시간 경과에 따라 LAMP법과 어류세포를 사용한 분리배양법을 이용하여 IHNV 검사를 실시하였다. IHNV를 <TEX>$10^{6.5}\;TCID_{50}$</TEX>/fish, <TEX>$10^{5.5}\;TCID_{50}$</TEX>/fish, <TEX>$10^{4.5}\;TCID_{50}$</TEX>/fish로 복강 주사한 결과, 40%, 0%, 0%의 누적폐사율이 관찰되었다. 폐사어 및 IHNV 접종 후 16일과 28일째에 각 실험구에서 채집한 생존어 5마리를 대상으로 한 IHNV 검사 결과, 폐사어에서 IHNV가 100% (8/8 마리) 분리되었고 (감염가: <TEX>$10^{4.3}-10^{6.8}\;TCID_{50}/ml$</TEX>), RT-LAMP법에서도 100% 검출되었다. 16일째 생존한 개체를 대상으로 한 IHNV 검사 결과에서는 <TEX>$10^{6.5}\;TCID_{50}$</TEX>/fish, <TEX>$10^{5.5}\;TCID_{50}$</TEX>/fish, <TEX>$10^{4.5}\;TCID_{50}$</TEX>/fish의 IHNV로 접종한 실험구에서 각각 60% (3/5 마리, 감염가: <TEX>$10^{2.8}-10^{5.05}\;TCID_{50}/ml$</TEX>), 20% (1/5 마리, <TEX>$10^{1.05}\;TCID_{50}/ml$</TEX>), 60% (3/5 마리, <TEX>$10^{1.05}-10^{4.8}\;TCID_{50}/ml$</TEX>) 의 검출율을 보였으나 LAMP법에서는 20% (1/5 마리), 0% (0/5 마리), 20% (1/5 마리) 의 검출율을 나타내었다. 28일째 생존한 개체 및 대조구의 어류에서는 IHNV가 분리 검출되지 않았다. 이상의 연구결과로 LAMP법은 IHNV-생존어에서 바이러스를 모니터링 하는데 한계가 있으나 병어로부터 IHNV를 검출하는데 유용하게 사용될 수 있을 것으로 사료되었다. A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was evaluated to monitor infectious hematopoietic necrosis virus (IHNV) from artificially infected rainbow trout Oncorhynchus mykiss. The cumulative mortalities of fish challenged with IHNV at <TEX>$10^{6.5}\;TCID_{50}$</TEX>/fish, <TEX>$10^{5.5}\;TCID_{50}$</TEX>/fish and <TEX>$10^{4.5}\;TCID_{50}$</TEX>/fish were 40%, 0% and 0%, respectively. Dead fish and survivors at 16 and 28 d post-challenge in each group were employed for IHNV detection by RT-LAMP assay and virus isolation using BF-2 cells. IHNV from <TEX>$10^{4.3}$</TEX> to <TEX>$10^{6.8}\;TCID_{50}/ml$</TEX> was isolated from all the dead fish and also detected in all of the examined dead fish by RT-LAMP assay. In survivors at 16 d, 60% (3/5 fish, <TEX>$10^{2.8}-10^{5.05}\;TCID_{50}/ml$</TEX>), 20% (1/5 fish, <TEX>$10^{1.05}\;TCID_{50}/ml$</TEX>) and 60% (3/5 fish, <TEX>$10^{1.05}-10^{4.8}\;TCID_{50}/ml$</TEX>) were found to be IHNV-positive by virus isolation in fish challenged with IHNV at <TEX>$10^{6.5}\;TCID_{50}$</TEX>/fish, <TEX>$10^{5.5}\;TCID_{50}$</TEX>/fish and <TEX>$10^{4.5}\;TCID_{50}$</TEX>/fish, respectively, while 20% (1/5 fish), 0% (0/5 fish) and 20% (1/5 fish) were IHNV-positive by RT-LAMP assay. No IHNV was detected in the survivors at 28 d and control fish. These results indicate that the RT-LAMP assay is useful for detection of IHNV in diseased fish although it is not enough to monitor virus in IHNV-survivors.
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