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/Cas12a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。
To achieve rapid,simple,and efficient detection of Anisakis in seafood to control its prevalence,recombinase polymerase amplification(RPA)primers and CRISPR RNA(cr RNA)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 cr RNA concentration were optimized.The detection limit,specificity,and repeatability of this method were evaluated,and its actual detection 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 Hysterothylacium 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.
作者
王皓璐
赵连静
陈秀琴
杨亚明
吴方伟
刘晓雷
王洋
白雪
WANG Haolu;ZHAO Lianjing;CHEN Xiuqin;YANG Yaming;WU Fangwei;LIU Xiaolei;WANG Yang;BAI Xue(Key Laboratory of Zoonosis Research,Ministry of Education/Institute of Zoonosis,College of Veterinary Medicine,Jilin University,Changchun 130062,China;Yunnan Institute of Parasitic Diseases,Puer 665000,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2024年第6期735-741,共7页
Chinese Veterinary Science
基金
“十三五”国家重点研发计划项目(2017YFC1601206)
云南省科技人才与平台计划(院士专家工作站)项目(202305AF150167)。
