甘薯羽状斑驳病毒(SPFMV)和甘薯褪绿矮化病毒(SPCSV)荧光定量RT-PCR检测方法的建立

Development of Detection Method for Sweet potato feathery mottle virus(SPFMV) and Sweet potato chlorotic stunt virus(SPCSV) Through Fluorescence Quantitative RT-PCR

【目的】甘薯病毒病(sweet potato virus disease,SPVD)是甘薯上的毁灭性病害之一,严重时可造成90%以上的产量损失。论文旨在对甘薯SPVD染病植株中甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)和甘薯褪绿矮化病毒(Sweet potato chlorotic stunt virus,SPCSV)2种病毒的复合侵染情况进行研究,建立SPVD荧光定量RT-PCR快速检测方法,为SPVD的早期预警和流行学研究提供技术手段。【方法】选取重庆地区具有不同SPVD典型症状的甘薯品种(系)叶片作为试验材料,对其进行症状和SPFMV、SPCSV的硝酸纤维素膜酶联免疫吸附(NCM-ELISA)检测;对供试材料叶片中的SPFMV外壳蛋白CP和SPCSV热激蛋白HSP70核苷酸序列进行克隆和序列分析,根据保守核苷酸序列设计荧光定量RT-PCR检测引物。以甘薯泛素(ubiquitin,UBI)和组蛋白(histone,H2B)编码基因为内参对供试样品进行荧光定量RT-PCR检测,与感染SPVD典型症状和NCM-ELISA检测结果进行比较,并对取自不同甘薯品种(系)、相同品种(系)不同单株以及相同植株不同部位叶片检测结果进行比较,筛选可快速、精确、定量检测SPVD的荧光定量RT-PCR检测方法。【结果】症状学诊断表明不同甘薯品种(系)的感病叶片可能表现出不同的典型症状特点,同一品种(系)的不同感病植株感病症状相似但也存在差异。序列分析结果表明供试材料中SPFMV至少存在2个株系类型(RC和EA),SPCSV至少存在1个株系类型(WA)。在15份供试材料中,10份材料由NCM-ELISA和荧光定量RT-PCR均检测到SPFMV和SPCSV的共同感染,且荧光定量RT-PCR检测结果与发病症状和NCM-ELISA检测结果相吻合,其检测结果可准确反映甘薯SPVD染病植株中2种病毒复合侵染情况。荧光定量RT-PCR检测结果还表明,不同品种(系)或相同品种(系)不同单株病叶中SPFMV CP与SPCSV HSP70转录水平存在差异;相同植株顶端幼嫩叶片和中部成熟叶片中SPFMV CP与SPCSV HSP70转录水平也存在差异。染病材料中SPFMV中CP转录水平均相对较高,是SPCSV的HSP70转录水平的3—556倍。4份材料经NCM-ELISA检测只有SPFMV而没有SPCSV感染,而荧光定量RT-PCR还可检测到其中2份材料中SPCSV HSP70转录水平,说明这2份材料中也存在SPFMV和SPCSV的共同感染,表明荧光定量RT-PCR比NCM-ELISA检测结果更灵敏、准确。【结论】筛选出可利用SPFMV CP与SPCSV HSP70转录水平进行检测的荧光定量RT-PCR检测引物,建立了SPVD荧光定量RT-PCR快速检测方法,为SPVD针对性地监测预警提供了极为有效的方法。在建立甘薯SPVD防御体系时,需综合考虑2种甘薯病毒的共侵染特性和甘薯品种差异,采取针对性的监测和预警机制。

【Objective】 Sweet potato virus disease（SPVD）, which could cause more than 90% of yield loss, is one of the most harmful diseases of the sweet potato. The objective of this study is to establish a rapid detection method of SPVD, provide a useful tool for the early warning of SPVD and epidemiological investigation, based on assay of co-infection of Sweet potato feathery mottle virus（SPFMV） and Sweet potato chlorotic stunt virus（SPCSV）.【Method】 The leaves of sweet potato cultivars/lines with typical SPVD symptoms were collected, and the co-infection of SPFMV and SPCSV were tested by typical symptoms analysis and Nitrocellulose Membrane Enzyme-Linked Immunosorbent Assay（NCM-ELISA）. The partial genes encoding coat protein（CP） of SPFMV and heat shock protein70（HSP70） of SPCSV were cloned from the diseased leaves. Fluorescence quantitative PCR（qR T-PCR） was used to detect SPFMV CP and SPCSV HSP70 by using primers designed according to the conservative regions, and by using sweet potato ubiquitin（UBI） and histone（H2B） as the reference genes. The rapid and accurate protocol for SPFMV and SPCSV detection were established by comparison with the symptoms and virus detection results by NCM-ELISA, and the detection results were also compared among leaves collected from different sweet potato cultivars/lines, among different plants collected from the same cultivar/line, and among top young leaves and mature leaves in the middle collected from the same plant. 【Result】 Symptoms analysis suggested that infected leaves from different cultivars/lines might exhibit different typical symptoms, and the symptoms of infected leaves from different plants of the same sweet potato cultivar/line were similar but exhibited differentiation. Cluster analysis showed that there were at least two types of SPFMV strains（EA and RC） and one type of SPCSV strain（WA） existing in the tested sweet potato leaves. Among the 15 tested cultivars/lines, 10 cultivars/lines were detected to be co-infected by SPFMV and SPCSV by using NCM-ELISA detection and qR T-PCR method. The results obtained from qR T-PCR detection corresponded to that from NCM-ELISA testing, indicating that the q RT-PCR detection method developed in this study could accurately reflect the co-infections degree of SPFMV and SPCSV in the sweet potato plants. Differential transcriptional levels of SPFMV CP and SPCSV HSP70 were detected in diseased leaves from different cultivars/lines or different plants of the same cultivar/line, and even in top young leaves and mature leaves in the middle from the same plant by using q RT-PCR. The detected transcriptional levels of SPFMV CP in the tested diseased leaves were higher than and were 3-556 times of that of SPCSV HSP70. In addition, q RT-PCR showed a transcriptional level of SPCSV HSP70 in two of four cultivars/lines which were detected to be only infected by SPFMV but not infected by SPCSV by NCM-ELISA detection, indicating the present q RT-PCR detection method was more sensitive and accurate when compared to the NCM-ELISA method.【Conclusion】 The appropriate PCR primers for q RT-PCR detection of SPVD were selected and a rapid method for SPVD detection was developed, which could provide a useful tool for the monitoring and early warning of SPVD. The characteristic of co-infection of two viruses and differentiation in typical symptoms among different cultivars/lines should be comprehensively analyzed and targeting approach should be concerned when a new monitoring or warning system of SPVD was established.