用于大麻哈鱼传染性造血器官坏死病毒的逆转录酶环介导等温扩增检测法

Reverse transcriptase loop-mediated isothermal amplification assay for infectious hematopoietic necrosis virus in Oncorhynchus keta.

作者信息

Suebsing Rungkarn, Jeon Chan-Hyeok, Oh Myung-Joo, Kim Jeong-Ho

机构信息

Faculty of Marine Bioscience & Technology, Gangneung-Wonju National University, Gangneung 210-702, South Korea.

出版信息

Dis Aquat Organ. 2011 Mar 16;94(1):1-8. doi: 10.3354/dao02310.

DOI:10.3354/dao02310

PMID:21553563

Abstract

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.

摘要

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。