Microbial Sciences, Pests & Diseases, Agriculture Victoria, 110 Natimuk Road, Horsham, Victoria 3400, Australia.
Australian Grains Genebank, Agriculture Victoria, 110 Natimuk Road, Horsham, Victoria 3400, Australia.
Viruses. 2021 Mar 30;13(4):583. doi: 10.3390/v13040583.
Globally, high-throughput sequencing (HTS) has been used for virus detection in germplasm certification programs. However, sequencing costs have impeded its implementation as a routine diagnostic certification tool. In this study, the targeted genome sequencing (TG-Seq) approach was developed to simultaneously detect multiple (four) viral species of; (PEBV), (CMV), (BYMV) and (PSbMV). TG-Seq detected all the expected viral amplicons within multiplex PCR (mPCR) reactions. In contrast, the expected PCR amplicons were not detected by gel electrophoresis (GE). For example, for CMV, GE only detected RNA1 and RNA2 while TG-Seq detected all the three RNA components of CMV. In an mPCR to amplify all four viruses, TG-Seq readily detected each virus with more than 732,277 sequence reads mapping to each amplicon. In addition, TG-Seq also detected all four amplicons within a 10 serial dilution that were not detectable by GE. Our current findings reveal that the TG-Seq approach offers significant potential and is a highly sensitive targeted approach for detecting multiple plant viruses within a given biological sample. This is the first study describing direct HTS of plant virus mPCR products. These findings have major implications for grain germplasm healthy certification programs and biosecurity management in relation to pathogen entry into Australia and elsewhere.
全球范围内，高通量测序（HTS）已被用于种质认证计划中的病毒检测。然而，测序成本阻碍了其作为常规诊断认证工具的实施。在本研究中，开发了靶向基因组测序（TG-Seq）方法，以同时检测四种病毒：（PEBV）、（CMV）、（BYMV）和（PSbMV）。TG-Seq 可在多重 PCR（mPCR）反应中检测到所有预期的病毒扩增子。相比之下，凝胶电泳（GE）未检测到预期的 PCR 扩增子。例如，对于 CMV，GE 仅检测到 RNA1 和 RNA2，而 TG-Seq 则检测到 CMV 的所有三个 RNA 成分。在扩增所有四种病毒的 mPCR 中，TG-Seq 可轻松检测到每个病毒，超过 732,277 个序列读长映射到每个扩增子。此外，TG-Seq 还可检测到在 GE 无法检测到的 10 个连续稀释度内的所有四个扩增子。我们目前的研究结果表明，TG-Seq 方法具有重要的潜力，是一种高度敏感的靶向方法，可用于检测给定生物样本中的多种植物病毒。这是第一个描述植物病毒 mPCR 产物直接 HTS 的研究。这些发现对谷物种质健康认证计划以及与病原体进入澳大利亚和其他地区相关的生物安全管理具有重大意义。
