中国农业科学2025，Vol.58Issue(12)：2371-2381,11.DOI:10.3864/j.issn.0578-1752.2025.12.008
李属坏死环斑病毒RT-RAA-CRISPR/Cas12a可视化检测方法的建立与应用
Establishment and Application of RT-RAA-CRISPR/Cas12a-Based Visual Detection of Prunus Necrotic Ringspot Virus
摘要
Abstract
[Objective]The study aims to establish a novel visual detection technique for prunus necrotic ringspot virus(PNRSV)by combining reverse transcription recombinase-aided amplification(RT-RAA)with CRISPR/Cas12a system(RT-RAA-CRISPR/Cas12a).[Method]Primers with high amplification efficiency and strong specificity were designed and selected based on the conserved regions of the coat protein(CP)gene of PNRSV.The detection conditions,including primer,probe concentration,temperature,and reaction time were optimized to develop a visual detection method for PNRSV by RT-RAA-CRISPR/Cas12a technology.The specificity of this method was evaluated by detecting PNRSV and common Prunus viruses,including plum pox virus(PPV),apple mosaic virus(ApMV),cucumber mosaic virus(CMV),potato virus X(PVX),and potato virus Y(PVY).The total RNAs from PNRSV-infected fruit were diluted in 10-fold gradients,then RT-PCR,RT-RAA and RT-RAA-CRISPR/Cas12a were performed to compare the sensitivity of the three methods.The RT-RAA-CRISPR/Cas12a and RT-PCR methods were used to detect 31 peach fruit test samples suspected to be infected with the virus collected at the port to verify the practicability of the visual detection method.[Result]The RT-RAA-CRISPR/Cas12a-based visual detection method for PNRSV was successfully established.The optimized working concentrations were as follows:RT-RAA-PNRSV-F2/R2 primers at 0.4 μmol·L-1,fluorescent reporter(FQ)at 800 nmol·L-1,CRISPR-Cas12a at 200 nmol·L-1,and PNRSV-crRNA(CRISPR RNA)at 240 nmol·L-1,the reaction conditions were performed at 41℃for 45 min.This method showed high specificity for PNRSV and had no cross-reaction with other common Prunus viruses.The limit of detection for PNRSV RNA in peach fruit samples reached 3.06 pg·μL-1 and 306 fg·μL-1 using RT-RAA and RT-RAA-CRISPR/Cas12a methods,respectively,showing the sensitivity of RT-RAA-CRISPR/Cas12a was 10 times higher than that of RT-RAA and RT-PCR.Among the 31 tested peach fruit samples at the port,14 positive samples were identified by RT-PCR,while 15 positive samples were found by RT-RAA-CRISPR/Cas12a,indicating a high level of consistency between the two methods.[Conclusion]The RT-RAA-CRISPR/Cas12a visual detection method for PNRSV has been established.It is characterized by simplicity,rapidity,high sensitivity,high specificity,and visual readability,making it well-suited for rapid on-site detection of PNRSV.关键词
李属坏死环斑病毒/RT-RAA/CRISPR/Cas12a/可视化检测Key words
prunus necrotic ringspot virus(PNRSV)/RT-RAA/CRISPR/Cas12a/visual detection引用本文复制引用
张晓琪,沈建国,廖富荣,李为民,金雨洁,沙依旦·吾甫尔,郑璐平..李属坏死环斑病毒RT-RAA-CRISPR/Cas12a可视化检测方法的建立与应用[J].中国农业科学,2025,58(12):2371-2381,11.基金项目
福建省自然科学基金面上项目(2023J01441)、福建省检验检疫技术研究重点实验室开放课题(FJKF2023-02)、福建农林大学科技创新专项基金(KFB23029) （2023J01441）
