樱桃小果病毒1 RT-LAMP检测方法的建立与应用

Establishment and application of RT-LAMP method on Little cherry virus 1

樱桃小果病毒1(LChV-1)已成为严重影响樱桃产量与质量的重要因素之一。本研究基于环介导的等温扩增技术建立LChV-1的快速检测体系。依据NCBI数据库中LChV-1外壳蛋白基因序列设计了3组特异性引物,经优化,筛选获得1组特异性引物。以感染LChV-1的甜樱桃叶片总RNA为模板,构建RT-LAMP检测体系为:6.0 mM Mg^(2+),0.2μM的外引物和1.2μM的内引物,在57℃条件下反应60 min。使用RT-LAMP方法对35个疑似樱桃小果病的甜樱桃样品进行检测,发现有13个样品感染了LChV-1,检测结果与RT-PCR法一致。RT-LAMP法具有特异性强、快速等特点,适合对LChV-1田间样品的快速检测与鉴定。

Little cherry virus 1(LChV-1)is one of the most pathogenic virus which seriously affects cherry yield and quality.In this study,a rapid detection system for LChV-1 was established based on a loop-mediated isothermal amplification(LAMP)method.Three sets of DNA primers were designed and synthesized based on the nucleotide sequence of the coat protein of LChV-1 for one step RT-LAMP detection system,and we screened one optimal pair of primers for this system.Total RNA extracted from LChV-1-infected cherry leaves was used as the template.The RT-LAMP was performed at temperature of 57℃for 60 minutes,and the reaction system contained 6.0 mM Mg^(2+),0.2μM and 1.2μM of the outer and inner primers,respectively.Results indicated that the RT-LAMP method was specific and rapid in detecting LChV-1.We used the RT-LAMP method to detect 35 field samples from Shandong Province,and discovered that 13 samples were infected by LChV-1,and the detection results were consistent with the RT-PCR method.The RT-LAMP method was highly specific,sensitive,practicable and efficient and suitable for the rapid detection and identification of LChV-1 samples.