摘要
【目的】建立甘蔗线条花叶病毒(Sugarcane streak mosaic virus,SCSMV)的RT-LAMP检测方法,为SCSMV的检测提供技术支持。【方法】根据甘蔗线条花叶病毒外壳蛋白基因的保守序列设计特异引物,优化RT-LAMP的反应条件,对比分析RT-LAMP的检测灵敏度和特异性;并在RT-LAMP反应产物中加入SYBR GREEN I进行可视化检测,方便快速判定结果。【结果】成功建立了一种用于检测SCSMV的RT-LAMP方法,可在60~63℃下60 min完成对SCSMV的检测,且具有较高的灵敏度,比传统RT-PCR高100倍,最低检测RNA浓度为167.9×10-5 ng。此外,该方法具有较高的特异性,可特异检测SCSMV。【结论】建立了一种灵敏、简便、快速的SCSMV RT-LAMP检测方法并用于田间甘蔗样品的检测。
【Objective】In order to provide technical support for the detection of sugarcane streak mosaic virus(SCSMV),a reverse transcription loop-mediated isothermal amplification(RT-LAMP)assay was established.【Method】A set of specific primers were designed from the conserved regions of coat protein gene sequences of SCSMV.The reaction conditions for RT-LAMP were optimized and the detection sensibility and specificity of RT-LAMP were analyzed and compared.For fluorescence detection,the RT-LAMP reaction products were also visually detected by adding SYBR Green I dye to identify the results in a convenient and rapid way.【Result】A RT-LAMP assay was developed for the rapid detection of SCSMV,and the whole detection process could be completed in 60 minutes at 60-63℃.The RT-LAMP established in this study had high sensitivity,which was 100 times higher than that of the traditional RT-PCR,and the lowest detection concentration was 167.9×10-5 ng.In addition,the method had high specificity and could be used for the specific detection of SCSMV.【Conclusion】A sensitive,convenient and rapid RT-LAMP assay was established and used for field detection of SCSMV samples.
作者
李战彪
谢慧婷
崔丽贤
蔡健和
秦碧霞
LI Zhanbiao;XIE Huiting;CUI Lixian;CAI Jianhe;QIN Bixia(Institute of Plant Protection,Guangxi Academy of Agricultural Sciences,Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pests,Nanning 530007,China)
出处
《广东农业科学》
CAS
2020年第4期92-98,共7页
Guangdong Agricultural Sciences
基金
Major Science and Technology Project of Guangxi Province(Guike AA17202042-12)
Basic Research Project of Guangxi Academy of Agricultural Sciences(2015YT42)。
