西南农业学报2025，Vol.38Issue(5)：920-926,7.DOI:10.16213/j.cnki.scjas.2025.5.005
RT-RAA-CRISPR/Cas12a试纸条法快速检测苹果坏死花叶病毒方法的建立
Establishment of a rapid detection method for apple necrotic mosaic virus by RT-RAA-CRISPR/Cas12a with alateral flow strip assay
摘要
Abstract
[Objective]Apple mosaic disease was one of the most important diseases in apple trees.Apple necrotic mosaic virus(ApNMV)was the major pathogen causing apple mosaic disease in China.The study aimed to establish a rapid and sensitive detection technique for Ap-NMV to achieve early identification and prevention of the virus.[Method]In the study,the reverse transcription-recombinase aided amplifica-tion(RT-RAA)assay was combined with CRISPR-associated enzyme(Cas)system for ApNMV detection.According to the principles of the CRISPR/Cas12a system,a crRNA was designed to specifically recognize the RT-RAA products of ApNMV.By optimizing the concentration of the ssDNA reporter,the reaction temperature,and the reaction time,the optimal reaction system and conditions were obtained.The detection results were displayed using lateral flow strips.[Result]The detection sensitivity of RT-RAA-CRISPR/Cas12a method was 10 times higher than that of real-time RT-PCR,and the detection limit for RNA of apple samples was 500 fg/μL.Moreover,it showed no cross-reaction with other viruses in apples.The positive rate of field-collected samples detected by the method was higher than that of conventional RT-PCR and real-time RT-PCR.[Conclusion]The ApNMV detection method based on RT-RAA-CRISPR/Cas12a has strong specificity and high sensitivi-ty.The study provides a new tool for the field diagnosis of apple necrotic mosaic virus.关键词
苹果坏死花叶病毒/反转录重组酶等温扩增/CRISPR/Cas12a/现场检测Key words
Apple necrotic mosaic virus/Reverse transcription-recombinase aided amplification/CRISPR/Cas12a/On-site detection分类
农业科技引用本文复制引用
王思元,侯雨萌,董铮,赵振兴,王金国,周涛,张永江..RT-RAA-CRISPR/Cas12a试纸条法快速检测苹果坏死花叶病毒方法的建立[J].西南农业学报,2025,38(5):920-926,7.基金项目
国家重点研发计划资助项目(2023YFF0614400,2023YFF0614402) （2023YFF0614400,2023YFF0614402）
