Jimenez Jenyfer, Leiva Ana Maria, Olaya Cristian, Acosta-Trujillo Daniela, Cuellar Wilmer Jose
Virology Laboratory, Crops for Nutrition and Health, International Center for Tropical Agriculture (CIAT), AA 6713, Cali, Colombia.
MethodsX. 2021 Aug 21;8:101496. doi: 10.1016/j.mex.2021.101496. eCollection 2021.
Our group works on the detection and characterization of cassava viruses, supporting projects that involve large scale pathogen surveillance activities and resistance screening assays in multiple and remote locations. In order to comply with these applications, nucleic acid isolation protocols need to be cost effective, adjusted for samples that will stand long distance transport and harsh storage conditions, while maximizing the yield and quality of the nucleic acid extracts obtained. The method we describe here has been widely used and validated using different downstream tests (including, but not limited to, Rolling Circle Amplification and Illumina and Nanopore sequencing), but is currently unpublished. The protocol begins with milligram amounts of dry leaf samples stored in silica gel, does not require liquid Nitrogen nor phenol extraction and produces an average of 2.11 µg of nucleic acids per mg of dry tissue.•DNA purity estimations reveal OD260/280 ratios above 2.0 and OD260/230 ratios above 1.7, even for samples stored in silica gel for several months.•The high quality of the extracts is suitable for detection of DNA and RNA viruses, with high efficiency.•We suggest this method could be used as part of a gold standard kit for virus detection in cassava.
我们的团队致力于木薯病毒的检测与特性分析，为涉及多个偏远地区大规模病原体监测活动和抗性筛选试验的项目提供支持。为了满足这些应用需求，核酸提取方案需要具有成本效益，针对能够经受长途运输和恶劣储存条件的样本进行调整，同时要最大限度地提高所获得核酸提取物的产量和质量。我们在此描述的方法已被广泛应用，并通过不同的下游测试（包括但不限于滚环扩增、Illumina测序和纳米孔测序）进行了验证，但目前尚未发表。该方案从存储在硅胶中的毫克级干叶样本开始，不需要液氮或酚提取，每毫克干组织平均可产生2.11微克核酸。
DNA纯度评估显示，即使是在硅胶中储存数月的样本，其OD260/280比值也高于2.0，OD260/230比值高于1.7。
提取物的高质量适合高效检测DNA和RNA病毒。
我们建议该方法可作为木薯病毒检测金标准试剂盒的一部分使用。
