传染性皮下和造血器官坏死病毒RPA与CRISPR-Cas12a可视化快速检测方法的建立
Establishment of rapid visual detection methods for infectious hypodermal and hematopoietic necrosis virus based on RPA and CRISPR-Cas12a
周傲白雪 1徐博文 1沙才华 1邵建宏 1赵福振 1廖秀云 1黄海超 1蒋晓霞 1罗宝正 1陈轩1
作者信息
- 1. 拱北海关技术中心,广东 珠海 519015
- 折叠
摘要
根据传染性皮下和造血器官坏死病毒(IHHNV)基因序列的保守区域设计并合成多组RPA引物和特异性crRNA,经过筛选与优化,建立RPA-Cas12a系统荧光检测法和试纸条法 2 种可视化快速检测IHHNV的方法.结果显示,这 2 种可视化检测方法在 37℃恒温反应 40 min可检测出IHHNV;与对虾白斑综合征病毒、虾肝肠胞虫和十足目虹彩病毒 1 无交叉反应;该方法灵敏度较高,最低检测浓度为 10 copies/μL;应用该方法对 10 份IHHNV阳性临床样本进行检测,阳性检出率为 100%.综合结果表明,建立的荧光法和试纸条 2 种可视化检测IHHNV的方法操作简单,反应灵敏,适用于对虾养殖场或基层实验室对IHHNV的定期监测和初筛.
Abstract
After screening and optimization,two visual rapid detection methods,the RPA-Cas12a fluorescent detection and lateral flow dipstick,for infectious hypodermal and hematopoietic necrosis virus(IHHNV)were established by designing and synthesizing multiple sets of recombinase polymerase amplification primers and specific CRISPR RNA based on the conserved regions of the IHHNV gene se-quences.The results demonstrated that both visual detection methods could detect IHHNV after a con-stant temperature reaction at 37℃for 40 min.There was no cross-reactivity with white spot syndrome virus,Enterocytozoon hepatopenaei and decapod iridescent virus 1.The method exhibited high sensiti-vity,with a detection limit of 10 copies/μL.When applied to test 10 positive clinical samples of IHH-NV,the conformity rate was 100%.Overall,the established fluorescence and paper strip visual detec-tion methods are simple to operate,highly sensitive,and suitable for regular monitoring and prelimi-nary screening of IHHNV in shrimp farming facilities or basic laboratories.
关键词
传染性皮下和造血器官坏死病毒/重组酶聚合酶扩增/CRISPR-Cas12a/检测方法Key words
infectious hypodermal and hematopoietic necrosis virus/recombinase polymerase am-plification/CRISPR-Cas12a/detection method引用本文复制引用
基金项目
广东省重点领域研发计划(2022B1111030001)
拱北海关科研项目(2023GK009)
出版年
2024
国家科技期刊平台
NSTL
万方数据