Aubakirova Karlygash P, Bakytzhanova Zhibek N, Rakhatkyzy Akbota, Yerbolova Laura S, Malakhova Natalya P, Galiakparov Nurbol N
Laboratory of Biotechnology and Molecular Genetics, M. Aitkhozhin Institute of Molecular Biology and Biochemistry, Almaty 050012, Kazakhstan.
Laboratory of Plant Bioengineering, M. Aitkhozhin Institute of Molecular Biology and Biochemistry, Almaty 050012, Kazakhstan.
Pathogens. 2025 Aug 6;14(8):782. doi: 10.3390/pathogens14080782.
Grapevine ( L.) cultivation is an important agricultural sector worldwide. Its expansion into new areas, like Kazakhstan, brings significant phytosanitary risks. Viroids, such as grapevine yellow speckle viroid 1 (GYSVd-1) and hop stunt viroid (HSVd), are RNA pathogens that threaten vineyard productivity. They can cause a progressive decline through latent infections. Traditional diagnostic methods are usually targeted and therefore not suitable for thorough surveillance. In contrast, modern high-throughput sequencing (HTS) methods often face challenges due to their high costs and complicated sample preparation, such as ribosomal RNA (rRNA) depletion. This study introduces a simplified diagnostic workflow that overcomes these barriers. We utilized the latest Oxford Nanopore V14 cDNA chemistry, which is designed to prevent internal priming, by substituting a targeted oligo(dT)VN priming strategy to facilitate the sequencing of non-polyadenylated viroids from total RNA extracts, completely bypassing the rRNA depletion step and use of random oligonucleotides for c DNA synthesis. This method effectively detects and identifies both GYSVd-1 and HSVd. This workflow significantly reduces the time, cost, and complexity of HTS-based diagnostics. It provides a powerful and scalable tool for establishing strong genomic surveillance and phytosanitary certification programs, which are essential for supporting the growing viticulture industry in Kazakhstan.
葡萄（Vitis vinifera L.）种植是全球重要的农业领域。其向哈萨克斯坦等新地区的扩张带来了重大的植物检疫风险。类病毒，如葡萄黄斑类病毒1（GYSVd - 1）和啤酒花矮化类病毒（HSVd），是威胁葡萄园生产力的RNA病原体。它们可通过潜伏感染导致产量逐渐下降。传统诊断方法通常具有针对性，因此不适用于全面监测。相比之下，现代高通量测序（HTS）方法由于成本高和样品制备复杂，如核糖体RNA（rRNA）去除，常常面临挑战。本研究引入了一种简化的诊断工作流程，克服了这些障碍。我们采用了最新的牛津纳米孔V14 cDNA化学技术，该技术旨在通过替代靶向寡聚（dT）VN引物策略来防止内部引物引发，以促进从总RNA提取物中对非多聚腺苷酸化类病毒进行测序，完全绕过rRNA去除步骤，并使用随机寡核苷酸进行cDNA合成。该方法能有效检测和鉴定GYSVd - 1和HSVd。此工作流程显著减少了基于HTS诊断的时间、成本和复杂性。它为建立强大的基因组监测和植物检疫认证计划提供了一个强大且可扩展的工具，这对于支持哈萨克斯坦不断发展的葡萄种植业至关重要。
