National Institute for Microbial Forensics & Food and Agricultural Biosecurity (NIMFFAB), Oklahoma State University, 127 NCR Stillwater, OK 74074-3033, United State of America.
North Carolina State University, Raleigh, NC 27695, United States of America.
J Virol Methods. 2020 Apr;278:113823. doi: 10.1016/j.jviromet.2020.113823. Epub 2020 Jan 22.
The Great Plains of the United States is a region comprised of approximately 45 million hectares of grasslands where several economically important cereal crops are grown. Arthropod-transmitted, cereal-infecting viruses vary in incidence from year-to-year and are often difficult to detect in large acreages. To facilitate the detection of economically important viruses of cereals that often exist in co-infections, a multiplex reverse transcriptase PCR (RT-PCR) platform assay was developed. This method can be used in combination with high resolution melting (HRM) to detect and allow for discrimination between three arthropod-transmitted plant viruses; Wheat streak mosaic virus (WSMV), Maize mosaic virus (MMV) and Barley yellow dwarf virus (BYDV). Multiplex PCR in combination with HRM allowed for successful detection of WSMV, MMV, and BYDV, as well as discrimination between three BYDV species, BYDV-PAS, BYDV-PAV and BYDV-MAV. All primer pairs amplified products of the predicted size. The BYDV-RT-PCR primers amplified products of identical length for all three species of BYDV. HRM was then used to discriminate between these products by determining significant differences between the melting rates for each (p < 0.05). This study demonstrates the flexibility of combining multiplex PCR with HRM to increase the specificity of plant virus diagnostics based on the needs of the diagnostician performing the assay.
美国大平原是一个由大约 4500 万公顷草原组成的地区，这里种植着几种经济上重要的谷类作物。节肢动物传播的、感染谷类的病毒在不同年份的发病率不同，而且在大面积范围内往往很难检测到。为了便于检测经常存在共同感染的经济上重要的谷类病毒，开发了一种多重逆转录聚合酶链反应（RT-PCR）平台检测方法。这种方法可以与高分辨率熔解（HRM）结合使用，用于检测和区分三种节肢动物传播的植物病毒；小麦线条花叶病毒（WSMV）、玉米花叶病毒（MMV）和大麦黄矮病毒（BYDV）。多重 PCR 与 HRM 相结合，成功地检测到了 WSMV、MMV 和 BYDV，以及三种 BYDV 物种，BYDV-PAS、BYDV-PAV 和 BYDV-MAV 之间的区分。所有引物对都扩增出了预期大小的产物。BYDV-RT-PCR 引物对所有三种 BYDV 物种的扩增产物长度相同。然后，通过确定每个（p < 0.05）熔解率之间的显著差异，使用 HRM 来区分这些产物。本研究证明了将多重 PCR 与 HRM 相结合的灵活性，可根据进行检测的诊断人员的需求，提高基于植物病毒诊断的特异性。
