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张盼1, 2，兰新芝3，乔奇1, 2，张德胜1, 2，秦艳红1, 2，田雨婷1, 2，王爽1, 2，张振臣1, 2*.甘薯病毒病害(SPVD)的多重RT-PCR检测方法及其应用[J].植物保护,2013,39(2):86-90.

甘薯病毒病害(SPVD)的多重RT-PCR检测方法及其应用

Development and application of a multiplex RT-PCR detection method for sweet potato virus disease (SPVD)

DOI：

中文关键词: 甘薯病毒病害甘薯羽状斑驳病毒甘薯褪绿矮化病毒多重RT-PCR

英文关键词:sweet potato virus diseases (SPVD) Sweet potato feathery mottle virus (SPFMV) Sweet potato chlorotic stunt virus (SPCSV) multiplex RT PCR

基金项目:

作者	单位
张盼1, 2，兰新芝3，乔奇1, 2，张德胜1, 2，秦艳红1, 2，田雨婷1, 2，王爽1, 2，张振臣1, 2*	1.河南省农业科学院植物保护研究所，郑州450002； 2.河南省农作物病虫害防治重点实验室,郑州450002； 3.河南省新县农业局植保站，新县465500

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中文摘要:

根据甘薯褪绿矮化病毒（Sweet potato chlorotic stunt virus， SPCSV）热激蛋白（Hsp70）基因和甘薯羽状斑驳病毒（Sweet potato feathery mottle virus， SPFMV）外壳蛋白（CP）基因核苷酸序列的保守区域设计了4对引物，以单一RT PCR反应体系为基础，分别对影响多重RT-PCR扩增的退火温度、Taq- DNA聚合酶浓度、dNTPs浓度、Mg2+浓度、引物浓度等条件进行了优化，建立了能同时检测SPVD两种病原的多重RT-PCR方法。该方法能有效区分SPFMV的主要株系类型。灵敏性试验表明，建立的多重RT-PCR方法对SPFMV-CH2、SPFMV-CH和SPCSV的最低检测浓度分别为1.42×104、1.32×104拷贝/μL和2.47×104拷贝/μL。该方法可用于甘薯叶片和薯块中病毒的检测，为SPVD的监测预警提供了一个有用的工具。

英文摘要:

A multiplex reverse transcription polymerase chain reaction (multiplex RT PCR) method was developed for the simultaneous detection and discrimination of Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV), the two pathogens of SPVD. Four compatible sets of primers specific for each virus were designed in conserved regions of the Hsp70 gene of SPCSV and the coat protein (CP) gene of SPFMV for multiplex RT PCR assay. The main impact factors of multiplex RT PCR including annealing temperature, Taq DNA polymerase, dNTPs, Mg2+ and primers concentration were optimized for the highest sensitivity and specificity. The multiplex RT-PCR method could distinguish the type of the main strains of SPFMV effectively. Sensitivity tests showed that the minimum detectable concentration of SPFMV CH2, SPFMV CH and SPCSV were 1.42×104copies/μL, 1.32×104 copies/μL and 2.47×104 copies /μL, respectively. This method could be used for the detection of virus in sweet potato plants and tubers, which provided a useful tool for the monitoring and early warning of SPVD.

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