| 管丽婷,乔光.火龙果主要病毒RT-PCR检测方法的建立及应用[J].中国南方果树,2019,48(5): |
| 火龙果主要病毒RT-PCR检测方法的建立及应用 |
| Development and Application of RT-PCR for Pathogen detection of Pitaya virus disease |
| 投稿时间：2018-11-23 修订日期：2018-12-28 |
| DOI： |
| 中文关键词: 火龙果 病毒 RT-PCR 检测 |
| 英文关键词:Pitaya Virus RT-PCR Detection |
| 基金项目:国家自然科学基金项目（面上项目，重点项目，重大项目） |
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| 摘要点击次数: 1863 |
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| 中文摘要: |
| 建立快速而稳定的火龙果病毒病分子检测体系，为火龙果病毒病的分子生物学鉴定提供技术支持。本文以感病的火龙果茎为研究材料，根据火龙果主要病毒病Pitaya virus X（PiVX）、Schlubergera virus X（SchVX）、Cactus virus X（CVX）及Zygocactus virus X（ZyVX）的保守序列设计特异性引物，运用RT-PCR技术进行扩增，将目的片段回收、克隆和测序。结果表明四种病毒序列与NCBI数据库里相应病毒核苷酸序列均具有较高的一致性。利用梯度稀释法依次将cDNA稀释为2、22、23、24、25、26、27倍，对各病毒灵敏度进行分析，结果显示，能检测出大部分病毒的火龙果cDNA最低浓度是45.75 ng·μL-1。应用该方法对贵州省罗甸县部分火龙果生产园内植株进行病原检测，结果显示该检测体系能特异、灵敏、稳定地检测出PiVX、SchVX、ZyVX及CVX这四种病毒。 |
| 英文摘要: |
| The objective of this study is to develop a rapid and stable molecular detection system for pitaya virus disease. The stems of pitaya were selected as the research materials. Based on the nucleotide sequence of the coat protein (CP) gene of the 4 main pitaya virus (PiVX, SchVX, ZyVX, CVX), the special primers were designed using Primer 5 respectively. The cDNA of pitaya infected by virus species were amplified by RT-PCR, and the target fragments were recovered, cloned and sequenced to verify the accuracy of RT-PCR detection. The result showed that four viral sequences were highly consistent with the corresponding viral nucleotide sequences in the NCBI database. To analyze the sensitivity of the RT-PCR, the cDNA which was prepared from the virus infected samples was diluted to 2-fold series. It was showed that the lowest concentration of most viruses detected was 45.75 ng·μL-1. The method was applied to the pathogen detection of pitaya in Luodian County, Guizhou Province. The results showed that the RT-PCR can stability detect PiVX, SchVX, ZyVX and CVX with high accuracy and sensitivity. |
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