Mark Deogratius, Tairo Fred, Ndunguru Joseph, Kweka Elisiana, Saggaf Maliha, Bachwenkizi Hilda, Chiunga Evangelista, Lusana James Leonard, Sikazwe Geofrey, Maghembe Reuben
Tanzania Agricultural Research Institute, 6226, Dar-es-Salaam, Tanzania.
Tanzania Plant Health and Pesticides Authority, Arusha, P.O.Box 3024, Tanzania.
Plant Methods. 2024 May 8;20(1):64. doi: 10.1186/s13007-024-01175-6.
Cassava leaf samples degrade quickly during storage and transportation from distant areas. Proper sampling and efficient, low-cost storage methods are critical for obtaining sufficient quality DNA and RNA for plant virus epidemiology and improving disease control understanding. This is useful when samples are collected from remote areas far from a laboratory or in developing countries where money and materials for virus diagnostics are scarce.
The effect of sample storage duration on nucleic acid (N.A.) quality on virus detection was investigated in this study. A simple, rapid, and cost-effective CTAB-based approach (M3) for single N.A. extraction was optimized and tested alongside two existing CTAB-based methods (M1 and M2) for N.A. extraction from fresh and herbarium cassava leaves stored for; 1, 8, 26, and 56 months. The amount and quality of DNA and RNA were determined using Nanodrop 2000 c U.V.-vis Spectrophotometer and agarose gel electrophoreses. The sample degradation rate was estimated using a simple mathematical model in Matlab computational software. The results show no significant difference in mean DNA concentration between M1 and M2 but a significant difference between M3 and the other two methods at p < 0.005. The mean DNA concentration extracted using M3 was higher for 1 and 8 months of leave storage. M3 and M2 produced high concentrations at 26 and 56 months of leave storage. Using a developed scale for quality score, M3 and M2 produced high-quality DNA from fresh samples. All methods produced poor-quality DNA and RNA at 8 and 26 months of leave storage and no visual bands at the age of 56 months. Statistically, there was a significant difference in the mean DNA quality between M1 and M2, but there was no significant difference between M3 and the other two methods at p < 0.005. However, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) were readily detected by RT-PCR from RNA isolated using M3. The quality of DNA declined per storage time at 0.0493 and 0.0521/month, while RNA was 0.0678 and 0.0744/month. Compared to the existing two methods, modified CTAB extracted enough high-quality N.A. in one-third the time of the existing two methods.
Our method provides cost-effective, quick, and simple processing of fresh and dry samples, which will quicken and guide the decision on when and what type of sample to process for plant disease management and surveillance actions.
木薯叶片样本在从偏远地区储存和运输过程中会迅速降解。合适的采样以及高效、低成本的储存方法对于获取足够质量的DNA和RNA以用于植物病毒流行病学研究及增进对疾病控制的理解至关重要。当从远离实验室的偏远地区或病毒诊断资金和材料稀缺的发展中国家采集样本时，这一点很有用。
本研究调查了样本储存时间对核酸（NA）质量及病毒检测的影响。优化了一种基于CTAB的简单、快速且经济高效的单一NA提取方法（M3），并将其与另外两种现有的基于CTAB的方法（M1和M2）一起进行测试，用于从储存了1、8、26和56个月的新鲜木薯叶片及腊叶标本中提取NA。使用Nanodrop 2000 c紫外可见分光光度计和琼脂糖凝胶电泳测定DNA和RNA的量及质量。使用Matlab计算软件中的一个简单数学模型估计样本降解率。结果显示，M1和M2之间的平均DNA浓度无显著差异，但M3与其他两种方法之间在p < 0.005时有显著差异。使用M3提取的平均DNA浓度在叶片储存1个月和8个月时较高。M3和M2在叶片储存26个月和56个月时产生高浓度DNA。使用制定的质量评分标准，M3和M2从新鲜样本中产生高质量DNA。所有方法在叶片储存8个月和26个月时产生的DNA和RNA质量较差，在56个月时无可见条带。从统计学上看，M1和M2之间的平均DNA质量有显著差异，但M3与其他两种方法之间在p < 0.005时无显著差异。然而，通过RT-PCR很容易从使用M3分离的RNA中检测到木薯褐色条纹病毒（CBSV）和乌干达木薯褐色条纹病毒（UCBSV）。DNA质量随储存时间每月下降0.0493和0.0521，而RNA为0.0678和0.0744。与现有的两种方法相比，改良的CTAB在现有两种方法三分之一的时间内提取了足够的高质量NA。
我们的方法为新鲜和干燥样本提供了经济高效、快速且简单的处理方式，这将加快并指导关于何时以及处理何种类型样本以进行植物病害管理和监测行动的决策。
