马铃薯Y病毒RPA-CRISPR/Cas12a检测技术体系的建立与应用

Establishment and Application of RPA-CRISPR/Cas12a Detection System for Potato Virus Y

植物病毒病是制约农作物安全生产的重要因素,病毒检测能够发现病毒并确定病毒的种类,是病害监测预警和防控的关键。该研究以马铃薯Y病毒(PVY)为检测对象,建立了基于RPA-CRISPR/Cas12a的检测体系。结果表明,(1)CRISPR/Cas12a检测体系内Cas12a及各组分为检测顺利进行所必要;(2)crRNA的靶点位置对Cas12a蛋白活性有较大影响,当crRNA的靶点包含部分PAM位点序列时,反应效率最高;(3)RPA-CRISPR/Cas12a检测模板的最低限度为3×10^(2)copies·μL^(-1),灵敏度高于PCR及qPCR检测法;(4)RPA-CRISPR/Cas12a检测体系与核酸粗提及逆转录反应联合,可在非实验室环境下进行PVY检测,整个过程耗时约60分钟。该研究建立了基于RPA-CRISPR/Cas12a的PVY检测技术体系,为在非实验室条件下实时快速检测植物病毒提供了一种有效方法。

Plant virus disease is an important factor restricting the safe production of crops.Virus detection can identify viruses and determine the types of viruses,which is the key to disease monitoring,early warning and prevention in crops production.In this study,a detection system based on Recombinase Polymerase Amplification-Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 12a(RPA-CRISPR/Cas12a)was established for potato virus Y(PVY).The results showed that:(1)Cas12a and other components in the CRISPR/Cas12a detection system were necessary for the detection;(2)The target location of crRNA had a significant effect on Cas12a nuclease activity,and the reaction efficiency was the highest when the target of cr RNA contained part of PAM site sequence;(3)The minimum detection limit of RPA-CRISPR/Cas12a was 3×10^(2) copies·μL^(-1),which was higher than that of PCR and qPCR methods;(4)The combination of RPA-CRISPR/Cas12a system with crude extraction of nucleic acid and reverse transcription could detect PVY in a non-laboratory setting and the whole process took about 60 minutes.The RPA-CRISPR/Cas12a detection system of PVY established in this study provides an effective method for real-time and rapid visual detection of plant viruses under non-laboratory conditions.