基于RPA-CRISPR/Cas12a的异尖线虫快速可视化检测方法
RPA-CRISPR/Cas12a-based visual and rapid detection of Anisakis
王皓璐 1赵连静 1陈秀琴 1杨亚明 2吴方伟 2刘晓雷 1王洋 1白雪1
作者信息
- 1. 吉林大学动物医学学院人兽共患病研究所人兽共患病研究教育部重点实验室,吉林长春 130062
- 2. 云南省寄生虫病防治所,云南普洱 665000
- 折叠
摘要
为了实现对海产品中异尖线虫的快速、便捷、高效的检测以控制其流行,针对异尖线虫内转录间隔区(ITS)保守序列设计重组酶聚合酶扩增(RPA)引物和CRISPR RNA(crRNA),并通过优化crRNA浓度等反应条件建立RPA-CRISPR/Cas 12a荧光检测体系,并评估该方法的灵敏度、特异性与重复性,通过人工污染样品验证其实际检测能力.结果表明:RPA-CRISPR/Cas12a荧光检测体系可在40 min内完成反应,敏感性为1 copy/μL,该方法仅能检测出异尖线虫,不与宫脂线虫、肠舌状绦虫、带鱼鱼肉组织、小黄花鱼鱼肉组织反应.本实验建立的RPA-CRISPR/Cas12a荧光可视化检测体系快速、灵敏、特异,可应用于海产品中异尖线虫的检测.
Abstract
To achieve rapid,simple,and efficient detection of Anisakis in seafood to control its prevalence,recombinase polymerase amplification(RPA)primers and CRISPR RNA(crRNA)for the conserved sequence of internal transcribed space in Anisakis were designed,the RPA-CRISPR/Cas12a fluorescence detection system was established and reaction conditions such as crRNA concentration were optimized.The detection limit,specificity,and repeatability of this method were evaluated,and its actual detec-tion ability was verified through artificially contaminated samples.The results showed that the RPA-CRISPR/Cas12a fluorescence detection system could complete the reaction within 40 min,and with the sensitivity of 1 copy/μL.This method could only detect Anisakis and not react with Hysterothy-lacium aduncum,Ligula intestinalis,Trichiurus lepturus tissue and Larimichthys polyactis tissue.The RPA-CRISPR/Cas12a fluorescence visualization detection system was fast,sensitive,and specific,and could be applied to the detection of Anisakis in marine products.
关键词
异尖线虫/RPA/CRISPR/Cas12a/可视化检测Key words
Anisakis/RPA/CRISPR/Cas12a/fluorescent detection引用本文复制引用
基金项目
国家重点研发计划(十三五)(2017YFC1601206)
云南省科技人才与平台计划(院士专家工作站)项目(202305AF150167)
出版年
2024
国家科技期刊平台
NSTL
万方数据