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

作　　者：尹新颖 曹际娟 李鑫 杨莉莉 朴永哲 YIN Xinying;CAO Jijuan;LI Xin;YANG Lili;PIAO Yongzhe(Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education,Dalian Minzu University,Dalian 116600,China;Dalian Customs District,Dalian 116001,China)

机构地区：[1]大连民族大学,生物技术与资源利用教育部重点实验室,大连116600 [2]大连海关技术中心,大连116001

出　　处：《植物保护》2023年第2期264-271,共8页Plant Protection

基　　金：大连市高层次人才创新支持计划(2019CT09)。

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4.21×10^(3) 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。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 4.21×10^(3) 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.

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 

分 类 号：S436.421[农业科学—农业昆虫与害虫防治]