| 尹新颖1, 曹际娟1*, 李 鑫2, 杨莉莉1, 朴永哲1.基于免核酸提取可视化RT-LAMP快速检测黄瓜绿斑驳花叶病毒[J].植物保护,2023,49(2):264-271. |
| 基于免核酸提取可视化RT-LAMP快速检测黄瓜绿斑驳花叶病毒 |
| Rapid detection of cucumber green mottle mosaic virus based on visual RT-LAMP without nucleic acid extraction |
| 投稿时间：2021-11-26 修订日期：2022-02-22 |
| DOI：10.16688/j.zwbh.2021663 |
| 中文关键词: 黄瓜绿斑驳花叶病毒 逆转录环介导恒温扩增(RT-LAMP) 可视化 免核酸提取 |
| 英文关键词:cucumber green mottle mosaic virus (CGMMV) RT-LAMP visualization free nucleic acid extraction |
| 基金项目:大连市高层次人才创新支持计划(2019CT09) |
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| 中文摘要: |
| 本文基于逆转录环介导恒温扩增 (RT-LAMP) 技术建立葫芦科作物中黄瓜绿斑驳花叶病毒 (CGMMV) 免核酸提取的可视化快速检测方法?根据CGMMV全基因组序列, 筛选保守区域并设计特异性RT-LAMP检测引物?通过荧光扩增方法 (加SYTO-9) 和可视化方法 (加钙黄绿素), 开展RT-LMAP特异性?灵敏度和重现性试验分析?结果表明, 优化筛选的引物组可以特异性地检测CGMMV, 检测灵敏度达到4.21×103 fg/μL, 组内和组间变异系数均小于5%, 重现性好?对瓜类作物田间种苗?植株叶片及市售种子等103份样品进行检测及一致性分析, 表明该方法与基于RNA提取RT-LAMP?荧光RT-qPCR及免疫测试条检测结果之间的Kappa值均为1.0 (1~1, 95%置信区间), 具有很好的一致性?基于免核酸提取的可视化RT-LAMP快检方法成为适合现场快速检测的理想选择, 对于CGMMV危害葫芦科作物的田间监测以及疫情防控具有广泛的推广应用前景? |
| 英文摘要: |
| To realize the rapid detection of cucumber green mottle mosaic virus (CGMMV), a visual detection method of CGMMV in Cucurbitaceae crops was established based on RT-LAMP without nucleic acid extraction. According to the complete genome sequence of CGMMV, the conserved regions were screened and specific RT-LAMP detection primers were designed. Specificity, sensitivity and repeatability were analyzed by RT-LAMP fluorescence amplification (SYTO-9) and visual RT-LAMP method (calcein). Results showed that the optimized primers for this method can detect CGMMV specifically, and the detection sensitivity is 4.21×103 fg/μL total RNA. The coefficient of variation (CV) within and between groups were less 5%, with good repeatability. The 103 samples of field seedlings, plant leaves and commercial seeds of melon crops were monitored, detected and analyzed. The results showed that the Kappa value between this method and RT-LAMP based on RNA extraction, fluorescent RT-qPCR and immunoassay strip were 1.0 (1-1, 95% confidence interval), and indicated an excellent test agreement. The visual RT-LAMP method has become an ideal choice for field rapid detection. It has a wide application prospect for field monitoring, preventing and controlling the epidemic of CGMMV. |
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