Colorimetric and real-time loop-mediated isothermal amplification (cr-LAMP) assays were developed targeting the coat protein region of genomic DNA of Wheat dwarf virus (WDV). The method was optimized relating to primer selection, determination of isothermal temperature, and determination of incubation duration. The specificity of the assay was tested using potential hosts of WDV and Maize streak virus (MSV) from Mastrevirus genus. No cross-reaction took place with MSV or with the potential hosts tested. For the naked eye observation of positive LAMP reaction, WarmStart 2X Master Mix from NEB (New England Biolabs) was used, where the change in colour from pink to yellowish points out a positive result. The amplification was monitored in real-time by smart-DART platform (Diagenetix Inc, USA) that allows connecting to a tablet or smartphone via bluetooth. The detection sensitivity of the LAMP assay was 100-fold more sensitive than conventional PCR after optimization of the cr-LAMP reaction conditions. The novel LAMP assay showed a high specificity in distinction of WDV detection from potential hosts and MSV. The cr-LAMP is considered to be fast and effective assay since it only requires very basic equipment and the results can be evaluated easily by using colorimetric and real time approaches. The simple, low-power, handheld smart-DART platform and colorimetric detection both have a major powerful advantage of the LAMP reaction in that it can be enabled in agricultural industries, where laboratory capacity is often rudimentary.

开发了比色法和实时环介导等温扩增 (cr-LAMP) 检测，以针对小麦矮病毒 (WDV) 基因组 DNA 的外壳蛋白区域。该方法在引物选择、等温温度的确定和孵育时间的确定方面进行了优化。使用来自Mastrevirus的 WDV 和玉米条纹病毒 (MSV) 的潜在宿主测试了该测定的特异性属。没有与 MSV 或测试的潜在宿主发生交叉反应。对于阳性 LAMP 反应的肉眼观察，使用来自 NEB（新英格兰生物实验室）的 WarmStart 2X Master Mix，其中颜色从粉红色变为黄色表明阳性结果。放大由允许通过蓝牙连接到平板电脑或智能手机的 smart-DART 平台（美国 Diagenetix Inc）实时监控。在优化 cr-LAMP 反应条件后，LAMP 检测的检测灵敏度比常规 PCR 高 100 倍。新型 LAMP 检测在区分 WDV 检测与潜在宿主和 MSV 方面具有高度特异性。cr-LAMP 被认为是快速有效的检测方法，因为它只需要非常基本的设备，并且可以使用比色法和实时方法轻松评估结果。简单、低功耗、手持式智能 DART 平台和比色检测都具有 LAMP 反应的主要强大优势，因为它可以在实验室能力通常不成熟的农业行业中启用。