Suebsing Rungkarn, Jeon Chan-Hyeok, Oh Myung-Joo, Kim Jeong-Ho
Faculty of Marine Bioscience & Technology, Gangneung-Wonju National University, Gangneung 210-702, South Korea.
Dis Aquat Organ. 2011 Mar 16;94(1):1-8. doi: 10.3354/dao02310.
A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting infectious hematopoietic necrosis virus (IHNV) from chum salmon Oncorhynchus keta in South Korea with high specificity, sensitivity and rapidity. A set of 6 IHNV-specific primers was designed, based on the G-protein sequence of IHNV (PRT strain), recognizing 8 distinct sequences of the target RNA. The assay was optimized to detect IHNV at 63 degrees C for 30 min. The limit of detection was 0.01 fg of RNA extracted from IHNV-infected CHSE-214 cells, compared with 1.0 fg for nested RT-PCR. The applicability of this RT-LAMP assay was further tested by comparison with nested RT-PCR using field samples. Of 473 samples tested, 191 samples (40.38%) were IHNV-positive by RT-LAMP, whereas 162 samples (34.25%) were IHNV-positive by nested RT-PCR. These results indicate that, because of its high sensitivity and rapidity, the RT-LAMP assay is useful for early diagnosis of IHN.
开发了一种逆转录酶环介导等温扩增（RT-LAMP）检测方法，用于从韩国的大麻哈鱼（Oncorhynchus keta）中检测传染性造血坏死病毒（IHNV），该方法具有高特异性、高灵敏度和快速性。基于IHNV（PRT株）的G蛋白序列设计了一组6条IHNV特异性引物，可识别目标RNA的8个不同序列。该检测方法优化为在63℃下检测IHNV 30分钟。检测限为从感染IHNV的CHSE-214细胞中提取的0.01 fg RNA，而巢式RT-PCR的检测限为1.0 fg。通过使用现场样本与巢式RT-PCR进行比较，进一步测试了该RT-LAMP检测方法的适用性。在检测的473个样本中，RT-LAMP检测出191个样本（40.38%）为IHNV阳性，而巢式RT-PCR检测出162个样本（34.25%）为IHNV阳性。这些结果表明，由于其高灵敏度和快速性，RT-LAMP检测方法可用于IHN的早期诊断。
