College of Life Science and Engineering, Foshan University, 18 Jiangwan Street, Foshan, 528231, Guangdong Province, China.
Faculty of Science, Kafrelsheikh University, Kafr El-Sheikh, Egypt.
Sci Rep. 2018 Sep 19;8(1):14039. doi: 10.1038/s41598-018-32473-4.
Here we present a visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting the gene encoding the σB major outer-capsid protein of novel duck reovirus (NDRV). A set of primers, composed of two outer primers, two inner primers and two loop primers, was designed based on the gene of interest. The LAMP reaction was conducted in a traditional laboratory water bath at 65 °C for 50 min. We compared the performance of calcein/Mn and SYBR Green I dyes, as well as electrophoresis on agarose gel stained with GoldView nucleic acid dye to detect the RT-LAMP-amplified products and all assays could be employed to discriminate between positive and negative specimens in visible or UV light. Our data showed that there is no cross-reaction with other viruses and the RT-LAMP technique displayed high sensitivity for detecting NDRV with a minimal detection limit of 200 fg RNA input. This assay was more sensitive than conventional PCR in detecting NDRV both in natural and experimental infection. In conclusion, the RT-LAMP technique was remarkably sensitive, specific, rapid, simple and profitable for the identification of NDRV.
在这里，我们介绍了一种可视化逆转录环介导等温扩增（RT-LAMP）检测方法，用于检测新型鸭呼肠孤病毒（NDRV）编码 σB 主要外壳蛋白的基因。根据目的基因，设计了一组由两个外引物、两个内引物和两个环引物组成的引物。LAMP 反应在传统的实验室水浴中于 65°C 进行 50 分钟。我们比较了钙黄绿素/Mn 和 SYBR Green I 染料以及琼脂糖凝胶电泳染色的金纳米核酸染料在检测 RT-LAMP 扩增产物方面的性能，所有检测方法都可以用于在可见光或紫外光下区分阳性和阴性样本。我们的数据表明，该方法与其他病毒没有交叉反应，RT-LAMP 技术对 NDRV 的检测具有很高的灵敏度，最小检测限为 200 fg RNA 输入。与传统 PCR 相比，该方法在自然感染和实验感染中对 NDRV 的检测更为敏感。总之，RT-LAMP 技术在 NDRV 的鉴定中具有显著的敏感性、特异性、快速性、简单性和经济性。
