传染性造血器官坏死病毒RT-LAMP检测方法的建立

Establishment of a RT-LAMP Method for Detection of IHNV

本试验建立了一套逆转录环介导等温扩增方法(reverse transcription loop-mediated isothermalamplification,RT-LAMP),用于实验室及现场鲑鳟鱼传染性造血器官坏死病毒(infectious haematopoieticnecrosis virus,IHNV)检测。针对IHNV 的糖蛋白(glycoprotein,G)基因设计特异性引物,以IHNV 基因组RNA 为模板,在反转录酶及Bst DNA 聚合酶的作用下进行RT-LAMP,结果阳性样本表现为荧光绿色,阴性样本仍为红棕色。反应条件优化结果表明,最适反应温度为67 ℃;特异性试验表明,仅IHNV 发生特异性扩增,鲤春病毒血症病毒(SVCV)、牙鲆弹状病毒(HRV)和病毒性出血性败血症病毒(VHSV)均不发生反应;敏感性试验表明,该LAMP 方法最低可检出5.0×10-7 μg 的IHNV 核酸。本研究所建立的IHNV检测方法成本低、操作简单、反应迅速,不依赖专门的仪器,同时具有较高的灵敏度和特异性,适合IHNV的现场检测和大批量样品检疫。

In this paper,a reverse transcription loop-mediated isothermal amplification(RT-LAMP)method was established to detect the infectious haematopoietic necrosis virus(IHNV)in trout. A pair of specific primers were designed based on the glycoprotein(G)gene sequence of IHNV. Taking the genomic RNA of IHNV as the template and under the action of reverse transcriptase and Bst DNA polymerase,the RT-LAMP assay was conducted. According to the results,the positive samples showed fluorescent green,and the negative samples showed reddish brown. Furthermore,it was indicated that,based on the optimized reaction conditions,the optimum reaction temperature was 67 ℃. The specificity test proved that,specific amplification only occurred in IHNV,and not in SVCV,HRV and VHSV. Based on the sensitivity test,5.0×10-7 μg of IHNV RNA could be detected by the established method. Therefore,it was concluded that the established method had a series of advantages,such as low cost,simple operation,rapid reaction,being independent from specialized instruments,strong specificity and high sensitivity,and it was appropriate for field detection of IHNV and inspection and quarantine of mass samples.