摘要
为建立鸭星状病毒2型(DAstV-2)核酸现场快速检测方法,基于CRISPR/Casl2a切割原理,联合重组酶聚合酶等温扩增(recombinase ploymerase amplification,RPA),设计用于DAstV-2核酸RPA扩增的引物和基于CRISPR/Cas12a反应的向导RNA(crRNA),优化反应条件确立最佳检测体系,并用胶体金横向流动试纸条(lateral flow dipstick,LFD)报告检测结果。结果:该检测体系对DAstV-2重组质粒的检出下限为10 copies/μL,灵敏性强且重复性好;用此检测体系检测鸭常见病毒,除DAstV-2核酸阳性外,其他均为阴性,特异性强;与普通RT-PCR的检测结果相比较,符合率达到100%,且更加方便快捷。研究结果提示,基于RPA-CRISPR/Cas12a-LFD系统的DAstV-2核酸检测方法灵敏性好、特异性强、操作简单,可在37℃恒温下实现DAstV-2的现场快速可视化检测。
To establish a rapid point-of-care detection technology for duck astrovirus type 2(DAstV-2)nucleic acid,based on the cutting principle of CRISPR/Cas12a,combined with recombinase polymerase isothermal amplification(RPA),primers were designed for RPA amplification of DAstV-2 nucleic acid and guide RNA(crRNA)based on the CRISPR/Cas12a reaction.The reaction conditions were optimized to establish the best detection system,and the colloidal gold lateral flow dipstick(LFD)was used to report the detection results.The results showed that the detection limit of the DAstV-2 recombinant plasmid was 10 copies/μL,and the detection system was highly sensitive and reproducible.This detection system was used to detect common duck viruses.Except for DAstV-2 nucleic acid being positive,the other viruses were negative with strong specificity.Compared with the results of the common RT-PCR,the coincidence rate here reached 100%,and the technology was more convenient and faster.The present study suggested that the method for detection of DAstV-2 nucleic acid based on RPA-CRISPR/Cas12a-LFD system possessed high sensitivity,strong specificity and easy operation,and was able to realize rapid visual detection of DAstV-2 at 37℃.
作者
李建柱
赵利琴
王露遥
师睦然
王成才
张梦圆
马超锋
焦凤超
何书海
LI Janzhu;ZHAO Liqin;WANG Luyao;SHI Muran;WANG Chengcai;ZHANG Mengyuan;MA Chaofeng;JIAO Fengchao;HE Shuhai(College of Animal Science and Technology,Xinyang Agriculture and Forestry University,Xinyang 464000,China;Henan Province Waterfowl Resources Development and Utilization and Disease Control Engineering Technology Research Center,Xinyang 464000,China;Xinyang City Animal Husbandry and Veterinary Technology Service Center,Xinyang 464000,China)
出处
《畜牧与兽医》
北大核心
2025年第7期125-130,共6页
Animal Husbandry & Veterinary Medicine
基金
2023年度河南省重点研发与推广专项(科技攻关)(232102111043)
信阳农林学院家禽重大疫病防控科技创新团队项目(2022CXTD06)。
