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KMID : 0545120120220071021
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Journal of Microbiology and Biotechnology
2012 Volume.22 No. 7 p.1021 ~ p.1028
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Development of a Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Detecting Nervous Necrosis Virus in Olive Flounder Paralichthys olivaceus
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Rungkarn Suebsing
Oh Myung-Joo
Kim Jeong-Ho
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Abstract
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In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid, sensitive, and inexpensive detection of nervous necrosis virus (NNV) in olive flounder, Paralichthys olivaceus, in Korea. A set of six specific primers was designed to target the RNA 2 gene encoding the coat protein of Korean NNV strains. The RT-LAMP reaction successfully detected NNV after 30 min at 65oC. When the sensitivities among RT-LAMP, RT-PCR, and nested RTPCR were compared, the RT-LAMP was shown to be able to detect the RNA template at 2.58 × 10-2 TCID50/ml, whereas the RT-PCR and nested RT-PCR were only able to detect the RNA template at 2.58 × 102 TCID50/ml and 2.58 TCID50/ml, respectively. Thus, the sensitivity of the RT-LAMP assay was higher than those of the RT-PCR assays. In the specificity test of the RT-LAMP, 2 genotypes of NNVs (SJNNV and RGNNV) were positive; however, no other fish viruses were positive with the primers, indicating that the RT-LAMP assay is only specific to NNV. A total of 102 olive flounder were collected from hatcheries between 2009 and 2011. The occurrence of NNV in olive flounder was determined to be 53.9% (55/ 102) by the RT-LAMP. On the other hand, the prevalence based on the nested RT-PCR and RT-PCR results was 33.8% (34/102) and 20.6% (21/102), respectively. This result indicates that the RT-LAMP assay developed in this study is suitable for early field diagnosis of NNV with high sensitivity.
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KEYWORD
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nervous necrosis virus, olive flounder, Paralichthys olivaceus, RT-LAMP, RT-PCR
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