Abiopep S.L. Parque Científico de Murcia. Ctra. Madrid Km 388, Complejo Espinardo. Edificio R 2ª Planta, Espinardo, 30100 Murcia, Spain.
Centro de Edafología y Biología Aplicada del Segura, Consejo Superior de Investigaciones Científicas (CSIC), Campus Universitario de Espinardo, Edificio 25, Espinardo, 30100 Murcia, Spain.
Viruses. 2021 Aug 25;13(9):1680. doi: 10.3390/v13091680.
Plant viruses can evolve towards new pathogenic entities that may eventually cause outbreaks and become epidemics or even pandemics. Seven years ago, tomato brown rugose fruit virus (ToBRFV) emerged, overcoming the genetic resistance that had been employed for more than sixty years against tobamoviruses in tomato. Since then, ToBRFV has spread worldwide, producing significant losses in tomato crops. While new resistances are deployed, the only means of control is the implementation of effective prevention and eradication strategies. For this purpose, in this work, we have designed, assessed, and compared an array of tests for the specific and sensitive detection of the ToBRFV in leaf samples. First, two monoclonal antibodies were generated against a singular peptide of the ToBRFV coat protein; antibodies were utilized to devise a double-antibody-sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test that sensitively detects this virus and has no cross-reactivity with other related tobamoviruses. Second, a real-time quantitative PCR (RT-qPCR) test targeting the RNA-dependent replicase open reading frame (ORF) was designed, and its performance and specificity validated in comparison with the CaTa28 and CSP1325 tests recommended by plant protection authorities in Europe. Third, in line with the tendency to use field-deployable diagnostic techniques, we developed and tested two sets of loop-mediated isothermal amplification (LAMP) primers to double-check the detection of the movement protein ORF of ToBRFV, and one set that works as an internal control. Finally, we compared all of these methods by employing a collection of samples with different ToBRFV loads to evaluate the overall performance of each test.
植物病毒可以进化为新的病原体，最终可能导致疫情爆发，并成为流行病甚至大流行病。七年前，番茄褐色皱果病毒（ToBRFV）出现，克服了番茄中使用了六十多年的针对烟草花叶病毒的遗传抗性。自那时以来，ToBRFV 已在全球范围内传播，导致番茄作物遭受重大损失。虽然新的抗性正在部署，但唯一的控制手段是实施有效的预防和根除策略。为此，在这项工作中，我们设计、评估并比较了一系列用于检测叶片样本中 ToBRFV 的特定和敏感的测试方法。首先，我们针对 ToBRFV 外壳蛋白的单个肽段生成了两种单克隆抗体；利用这些抗体设计了一种双抗体夹心酶联免疫吸附测定（DAS-ELISA）测试，该测试可以灵敏地检测到该病毒，并且与其他相关烟草花叶病毒没有交叉反应。其次，设计了一种针对 RNA 依赖性复制酶开放阅读框（ORF）的实时定量 PCR（RT-qPCR）测试，并与欧洲植物保护机构推荐的 CaTa28 和 CSP1325 测试进行了比较，验证了其性能和特异性。第三，为了顺应使用现场可部署诊断技术的趋势，我们开发并测试了两套环介导等温扩增（LAMP）引物，以双重检查 ToBRFV 运动蛋白 ORF 的检测，并开发了一套作为内部对照的引物。最后，我们通过使用不同 ToBRFV 载量的样本集来比较所有这些方法，以评估每个测试的整体性能。
