Shirima Rudolph R, Maeda Daniel G, Kanju Edward, Ceasar Gloria, Tibazarwa Flora I, Legg James P
International Institute of Tropical Agriculture, P. O. Box 34441, Dar es Salaam, Tanzania; University of Dar es Salaam, P.O. Box 35179, Dar es Salaam, Tanzania.
University of Dar es Salaam, P.O. Box 35179, Dar es Salaam, Tanzania.
J Virol Methods. 2017 Jul;245:5-13. doi: 10.1016/j.jviromet.2017.03.003. Epub 2017 Mar 16.
Cassava brown streak disease (CBSD) is the most important virus disease of cassava and a major food security threat in Africa. Yearly economic losses of up to $100 million USD have been attributed to CBSD. The lack of information on plant-virus interactions has restricted progress in breeding for CBSD resistance. Virus quantification is becoming a major tool for the quick and reliable assessment of plant host resistance. Therefore, a protocol for specific absolute quantification of Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) was developed. CBSV and UCBSV coat protein (CP) specific standard templates: CBSV (pFer2, 826bp) and UCBSV (pUF1-R1-1, 732) respectively were generated and maintained in a TA cloning vector. These were used to construct standard curves using a TaqMan qPCR assay. Standard curves with acceptable amplification efficiencies (90-105%) and coefficients of determination (R) greater than 0.99 were obtained. Infected cassava plants were sampled from a screenhouse and the field and used to validate this assay. Results obtained by testing several screenhouse and field samples revealed consistent absolute quantification assays for different CBSV and UCBSV isolates. This study presents the first protocol for absolute quantification of CBSVs and is expected to accelerate screening for CBSD resistance and hence breeding for CBSD resistance. The use of the method presented here should improve the clarity of virus quantification data as the results obtained are not influenced by varietal, host, seasonal or environmental conditions. Screening efficiency will also be greatly improved as there is no need for the use of reference genes consequently allowing for a larger number of samples to be analyzed. This will increase experimental precision in a timely and cost effective manner.
木薯褐色条纹病（CBSD）是木薯最重要的病毒病，也是非洲主要的粮食安全威胁。据估计，每年因CBSD造成的经济损失高达1亿美元。由于缺乏关于植物与病毒相互作用的信息，木薯褐色条纹病抗性育种进展受限。病毒定量分析正成为快速可靠评估植物宿主抗性的主要工具。因此，开发了一种用于木薯褐色条纹病毒（CBSV）和乌干达木薯褐色条纹病毒（UCBSV）特异性绝对定量的方法。分别构建了CBSV和UCBSV外壳蛋白（CP）特异性标准模板：CBSV（pFer2，826bp）和UCBSV（pUF1-R1-1，732），并保存在TA克隆载体中。使用TaqMan qPCR分析法，以这些标准模板构建标准曲线。获得了扩增效率（90-105%）和决定系数（R）大于0.99的标准曲线。从温室和田间采集受感染的木薯植株样本，用于验证该分析方法。对多个温室和田间样本的检测结果表明，该方法对不同的CBSV和UCBSV分离株具有一致的绝对定量分析能力。本研究提出了首个CBSV绝对定量分析方法，有望加速木薯褐色条纹病抗性筛选及抗性育种进程。使用本文提出的方法应能提高病毒定量数据的清晰度，因为所获结果不受品种、宿主、季节或环境条件的影响。由于无需使用内参基因，从而可分析更多样本，筛选效率也将大大提高。这将及时且经济高效地提高实验精度。
