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马铃薯纺锤块茎类病毒(potato spindle tuber viroid,PSTVd)是中国重要检疫性病原物之一，能够侵染茄科等多个科的植物，导致严重病害，具有高度侵袭性和潜伏期长的特点，极易传播。本研究针对PSTVd基因组，建立了基于反转录(reverse transcription,RT)-重组酶聚合酶扩增(recombinase polymerase amplification,RPA)和CRISPR/Cas系统(CRISPR/Cas12a)的RT-RPA-CRISPR/Cas12a可视化核酸检测系统。结果表明，整个过程可在45 min内完成，只有PSTVd质粒检测结果呈阳性，作为对照的5种茄科植物病毒与空白对照检测结果均呈阴性反应，检测灵敏度为4.134×10-7ng/μL。本研究建立的RT-RPA-CRISPR/Cas12a可视化核酸检测方法特异性强、灵敏度高，操作简便，适用于田间或口岸现场，利于PSTVd的监测与暴发流行的早期预警。
Abstract：Potato spindle tuber viroid(PSTVd) is one of the most important quarantine pathogens in China,and can cause diseases in many plant species of multiple families including Solanaceae. PSTVd is transmitted easily due to its high infectivity and the long latent period of the disease caused. We have developed a visual nucleic acid detection system that targets the genome of PSTVd based on reverse transcription(RT)-RPA(recombinase polymerase amplification) and CRISPR/Cas detection system. The results demonstrated that the entire detection process could be completed within 45 minutes,and only PSTVd plasmid tested positive,while both the five other viruses infecting solanaceous plants and the blank control were tested negative in the reactions. The detection sensitivity was 4.134×10-7ng/μL. The RT-RPACRISPR/Cas12a detection method established in this research is specific,sensitive,and easy to perform,thus it has the potential to be applied to on-site or in-field detection of PSTVd,facilitating the monitoring and early forcasting of the viroid disease outbreaks.
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基本信息:
DOI：10.19662/j.cnki.issn1005-2755.2025.03.004
中图分类号:S41-30
引用信息:
[1]徐司琦,韩春雪,王欣宇等.利用RT-RPA-CRISPR/Cas12a方法检测马铃薯纺锤块茎类病毒[J].植物检疫,2025,39(03):22-28.DOI:10.19662/j.cnki.issn1005-2755.2025.03.004.
基金信息:
国家重点研发计划项目（2022YFC2601500;2022YFC2601503）; 三亚崖州湾科技城管理局资助项目（SYND-2021-03）