Institute for Biosecurity and Microbial Forensics, Oklahoma State University, Stillwater, OK, United States of America.
Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, United States of America.
PLoS One. 2023 May 8;18(5):e0272980. doi: 10.1371/journal.pone.0272980. eCollection 2023.
Twenty-four species of RNA viruses contain members infecting economically important crops that are classified within the genus Emaravirus, family Fimoviridae. There are at least two other non-classified species that may be added. Some of these viruses are spreading rapidly and cause economically important diseases on several crops, raising a need for a sensitive diagnostic technique for taxonomic and quarantine purposes. High-resolution melting (HRM) has shown to be reliable for the detection, discrimination, and diagnosis of several diseases of plants, animals, and humans. This research aimed to explore the ability to predict HRM outputs coupled to reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To approach this goal a pair of degenerate genus-specific primers were designed for endpoint RT-PCR and RT-qPCR-HRM and the species in the genus Emaravirus were selected to framework the development of the assays. Both nucleic acid amplification methods were able to detect in-vitro several members of seven Emaravirus species with sensitivity up to one fg of cDNA. Specific parameters for in-silico prediction of the melting temperatures of each expected emaravirus amplicon are compared to the data obtained in-vitro. A very distinct isolate of the High Plains wheat mosaic virus was also detected. The high-resolution DNA melting curves of the RT-PCR products predicted in-silico using uMeltSM allowed saving time while designing and developing the RT-qPCR-HRM assay since the approach avoided extensive searching for optimal HRM assay regions and rounds of HRM tests in-vitro for optimization. The resultant assay provides sensitive detection and reliable diagnosis for potentially any emaravirus, including new species or strains.
二十四种 RNA 病毒包含感染经济重要作物的成员，这些成员被归类为 Emaravirus 属，Fimoviridae 科。至少还有另外两个非分类物种可能会被添加。其中一些病毒传播迅速，在几种作物上引起重要的经济疾病，因此需要一种敏感的诊断技术用于分类和检疫目的。高分辨率熔解（HRM）已被证明可用于检测、区分和诊断几种植物、动物和人类疾病。本研究旨在探索与反转录定量聚合酶链反应（RT-qPCR）相结合预测 HRM 输出的能力。为了实现这一目标，设计了一对简并属特异性引物用于终点 RT-PCR 和 RT-qPCR-HRM，并选择 Emaravirus 属中的物种来构建检测方法。这两种核酸扩增方法都能够检测到体外七种 Emaravirus 种的多个成员，灵敏度高达 fg 的 cDNA。将每个预期的 emaravirus 扩增子的熔解温度的计算机预测的特定参数与体外获得的数据进行比较。还检测到了高平原小麦花叶病毒的一个非常独特的分离株。使用 uMeltSM 进行计算机预测的 RT-PCR 产物的高分辨率 DNA 熔解曲线允许在设计和开发 RT-qPCR-HRM 测定时节省时间，因为该方法避免了为优化而进行广泛的 HRM 测定区域和轮次的体外 HRM 测试。该测定法为潜在的任何 emaravirus 提供了敏感的检测和可靠的诊断，包括新的物种或菌株。
