猪圆环病毒2型LAMP-CRISPR/Cas12a检测方法的建立与应用
Establishment and application of a method for the detection of porcine circovirus type 2 LAMP-CRISPR/Cas12a
毛首会 1杜国玉 2刘晓波 3吴锦艳 2兰喜 2何继军 2尚佑军 2王桂琴 3刘永杰2
作者信息
- 1. 中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州 730046;宁夏大学农学院,宁夏银川 750021
- 2. 中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州 730046
- 3. 宁夏大学农学院,宁夏银川 750021
- 折叠
摘要
本研究旨在建立一种快速、准确且易于操作的猪圆环病毒2型(porcine circovirus 2,PCV2)检测方法.首先以PCV2的ORF2基因作为靶基因,设计4条LAMP与5条CRISPR特异性引物,再分别优化获得LAMP反应体系和CRISPR检测体系中各成分最佳配比浓度,经过特异性、灵敏度、重复性以及符合性验证,最终建立了快速检测PCV2的LAMP-CRISPR/Cas12a方法.结果显示,该方法最小检测量接近1 copy质粒DNA,灵敏度远高于本实验室常用的PCR方法,且在相同条件下只有PCV2呈阳性结果,其他均呈现阴性,表明特异性良好.同时该方法具有良好的重复性,批内变异系数小于5％,批间变异系数小于10％,该方法与qPCR之间的符合率较高,可通过肉眼观察在蓝光下的反应产物判断结果.结果表明,建立的LAMP-CRISPR/Cas12a体系适用于PCV2的快速检测.
Abstract
This study aimed to establish a rapid,accurate,and user-friendly detection method for porcine circovirus 2(PCV2).Initially,targeting the ORF2 gene of PCV2,four LAMP and five CRISPR-specific primers were designed and the optimal concentrations of components in the LAMP reaction system and CRISPR detection system were optimized.Following validation of specificity,sensitivity,repeatabili-ty,and compliance,the rapid detection method for PCV2,LAMP-CRISPR/Cas12a,was successfully devel-oped.The results demonstrated that the method's minimum detection threshold was close to 1 copy of plasmid DNA,showing significantly higher sensitivity compared to the PCR method commonly used in our laboratory.Under the same conditions,only PCV2 yielded positive results,while all others tested nega-tive,indicating excellent specificity.The method exhibited good repeatability,with an intra-batch coefficient of variation below 5％and an inter-batch coefficient of variation below 10％.It showed high agreement with qPCR and allowed for visual observation of reaction products under blue light.These findings suggest that the established LAMP-CRISPR/Cas12a system is suitable for the rapid detec-tion of PCV2.
关键词
猪圆环病毒2型/CRISPR/Cas12a检测/环介导等温扩增Key words
porcine circovirus type 2/CRISPR/Cas12a detection/loop-mediated isothermal amplification引用本文复制引用
基金项目
国家生猪技术创新中心先导科技项目(NCTIP-XD/C03)
出版年
2024
国家科技期刊平台
NSTL
万方数据