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将逆转录环介导的等温扩增技术(reverse transcription loop-mediated isothermal amplification,RT-LAMP)引入到鱼类的传染性造血器官坏死病病毒(infectious haematopoietic necrosis virus,IHNV)检测中,建立了简单、快速、灵敏的IHNV检测方法。针对IHNV的聚合酶蛋白基因(polymerase protein,L)设计特异性引物,以IHNV基因组RNA为模板,在反转录酶和Bst DNA聚合酶的作用下进行RT-LAMP,反应产物添加SYBR Green I荧光染料后,肉眼观察阳性样本表现为荧光绿色,阴性样本表现为红棕色。2%琼脂糖凝胶电泳检测结果与可视化检测结果一致。该方法实现了检测结果的可视化。特异性分析显示该方法不与传染性胰脏坏死病毒(infectious pancreatic necrosis virus,IPNV)及病毒性出血性败血症病毒(viral haemorrhagic septicaemia virus,VHSV)发生交叉反应,并且可检测不低于10 pfu的IHNV RNA。本研究所建立的IHNV RT-LAMP可视化检测方法具有灵敏度高、特异性好、成本低、不依赖任何专门的仪器设备的优点,其检测结果直观可视化,可实现现场高通量快速检测。
The reverse transcription loop-mediated isothermal amplification (RT-LAMP) was introduced into the detection of infectious haematopoietic necrosis virus (IHNV) in fish to establish a simple , Fast, sensitive IHNV detection method. Specific primers were designed for IHNV polymerase protein (L). RT-LAMP was performed using IHNV genomic RNA as a template under the action of reverse transcriptase and Bst DNA polymerase. The reaction product was supplemented with SYBR Green I fluorescence After staining, the positive samples showed macroscopic fluorescence green and the negative samples showed reddish brown. 2% agarose gel electrophoresis test results and visual test results. This method realizes the visualization of test results. Specific analysis showed that this method does not cross-react with infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV), and can detect not less than 10 pfu IHNV RNA. The visualization method of IHNV RT-LAMP established in this study has the advantages of high sensitivity, good specificity, low cost and independent of any specialized instrument. The visualization results of the test results can be used to quickly detect high-throughput on-site.