| 王 涌, 曾祥国, 肖桂林, 温 昕, 周鑫鑫, 邓江丽, 韩永超*.草莓白化伴随病毒RT-LAMP检测方法的建立[J].植物保护,2024,50(6):237-245. |
| 草莓白化伴随病毒RT-LAMP检测方法的建立 |
| Establishment of RT-LAMP detection method for strawberry pallidosis-associated virus |
| 投稿时间：2024-03-29 修订日期：2024-04-20 |
| DOI：10.16688/j.zwbh.2024172 |
| 中文关键词: 草莓白化伴随病毒 反转录环介导等温扩增 可视化 快速检测 |
| 英文关键词:strawberry pallidosis-associated virus RT-LAMP visualization rapid detection |
| 基金项目:湖北省农业科技创新中心资助项目（2021-620-000-001-008）；湖北省重点研发计划（2023BBB133，2023BBB041）；武汉市东湖高新区揭榜挂帅项目（2022KJB134） |
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| 中文摘要: |
| 草莓白化伴随病毒(strawberry pallidosis-associated virus,SPaV)能引起草莓白化病,与其他病毒复合侵染后会加重症状。本研究建立了SPaV的反转录环介导等温扩增方法(reverse transcription-loop-mediated isothermal amplification, RT-LAMP),对反应体系的各条件进行评估优化后,检测了RT-LAMP的特异性和灵敏度,且对田间样品进行了测试。结果表明,优化后的反应体系包含2.5 μL 10×Isothermal Amplification Buffer、6 mmol/L Mg2+、1 mmol/L dNTPs、1 μmol/L SPaV-FIP和SPaV-BIP,0.1 μmol/L SPaV-F3和SPaV-B3,0.3 μmol/L SPaV-LB,0.4 mol/L Betaine,0.256 U/μL WarmStart Bst 2.0 DNA Polymerase,0.12 U/μL WarmStart RTx Reverse Transcriptase,1 μL RNA,反应条件为61℃恒温孵育40 min。采用优化后的反应体系,在特异性检测中,只有携带了病毒SPaV的样品才能发生阳性扩增。灵敏度测试中,RT-LAMP比RT-PCR灵敏度高10倍。田间应用中,RT-LAMP和RT-PCR检测结果一致。本研究建立的SPaV RT-LAMP检测方法操作简便、快速、特异,可供实验室及生产实践中应用,助力脱毒种苗的鉴定和田间病毒病调查。 |
| 英文摘要: |
| Strawberry pallidosis-associated virus (SPaV) can cause strawberry pallidosis disease, and the symptoms will worse when SPaV coinfects with other viruses. This study established a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for SPaV detection. After evaluating and optimizing the conditions of the reaction system, the specificity and sensitivity of this RT-LAMP procedure were mensurated and field samples were tested. The results showed that the optimized reaction system contained 2.5 μL 10×Isothermal Amplification Buffer, 6 mmol/L Mg2+, 1 mmol/L dNTPs, 1 μmol/L SPaV-FIP and SPaV-BIP, 0.1 μmol/L SPaV-F3 and SPaV-B3, 0.3 μmol/L SPaV-LB, 0.4 mol/L Betaine, 0.256 U/μL WarmStart Bst 2.0 DNA Polymerase, 0.12 U/μL WarmStart RTx Reverse Transcriptase, 1 μL RNA,and incubated at 61℃ for 40 min. Only the sample containing the SPaV could be positively amplified by the optimized reaction system. The sensitivity of this RT-LAMP was 10 times higher than that of RT-PCR detection. In the field application, the RT-LAMP detection result was consistent with RT-PCR. The RT-LAMP method developed for SPaV detection in this study is simple, rapid and specific, and can be used in laboratory and field to identify virus-free seedlings and investigate virus diseases. |
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