Okra yellow vein mosaic disease (OYVMD) causes serious loss in okra production in Sri Lanka. Therefore, screening of resistant okra verities is an essential need to control the disease. As the available qualitative and semi-quantitative methods failed to detect latent infection the present study aimed to develop a quantitative PCR (qPCR) assay to detect and quantify one of the OYVMD causing agent, symptom modulating satellite molecules. A pair of primers targeting a portion of βC1 gene of BYVMBs was designed and used to quantify of BYVMBs by absolute quantification method using SYBR Green I chemistry. Standard curves were prepared using series of dilutions of known copy number plasmids carrying target sequence. The mean amplification efficiency was 95% and the coefficient of determination was 0.994. The method was tested to find out the relation between symptoms and betasatellite titre in range of severity of OYVMD symptoms; the betasatellite titre increased with increasing severity. Interestingly, the method was able to detect BYVMBs present in apparently healthy plants growing in an infected field at a concentration which was not able to detect in end point PCR. Betasatellite titre was also measured in different ages of leaves and different positions. On average, the betasatellite titre in younger leaves was higher than in mature leaves and there were no significant variations in betasatellite titre in different position in each leaf. The assay was also tested as a tool to screen for resistant okra varieties; among the eight varieties tested no BYVMBs were detected in variety Maha F1. Varieties TV8 and MI5 had significantly higher copy number than rest of the varieties. The qPCR protocol described in this study is a useful method to detect and quantify BYVMBs in okra, especially for plant samples with betasatellite titre lower than the detection limit of conventional methods.

中文翻译：

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通过qPCR检测和定量测定与秋葵黄脉花叶病有关的β卫星。

黄秋葵黄脉花叶病（OYVMD）导致斯里兰卡秋葵产严重损失。因此，筛选抗性秋葵属植物是控制该疾病的必要条件。由于可用的定性和半定量方法未能检测到潜伏感染，因此本研究旨在开发一种定量PCR（qPCR）检测方法，以检测和定量一种OYVMD致病因子，即症状调节卫星分子。设计了一对靶向BYVMB的βC1基因的一部分的引物，并使用SYBR Green I化学通过绝对定量方法将其用于BYVMB的定量。使用一系列带有靶序列的已知拷贝数质粒的稀释液制备标准曲线。平均扩增效率为95％，测定系数为0.994。测试了该方法，以发现症状与β-卫星滴度在OYVMD症状严重程度之间的关系。β卫星滴度随着严重程度的增加而增加。有趣的是，该方法能够检测在感染场中生长的看起来健康的植物中存在的BYVMB，其浓度无法在终点PCR中检测到。还测量了不同年龄的叶片和不同位置上的β卫星滴度。平均而言，年轻叶片中的β-卫星滴度高于成熟叶片，并且每片叶片中不同位置的β-卫星滴度没有显着变化。该试验还作为筛选抗性秋葵品种的工具进行了测试；在测试的八个品种中，没有在Maha F1品种中检测到BYVMB。TV8和MI5品种的拷贝数明显高于其余品种。本研究中描述的qPCR方案是检测和定量秋葵中BYVMB的有用方法，特别是对于β卫星滴度低于常规方法检出限的植物样品。