摘要
目的 基于CRISPR-Cas13a附带切割荧光检测原理,建立H7亚型禽流感病毒无扩增直接检测方法。方法 从GenBank下载700余条H7亚型禽流感病毒的HA基因核酸序列,比对后针对保守区片段序列确定病毒模板,筛选最佳crRNA并对CRISPR-Cas13a蛋白、H7-crRNA及荧光双标记探针等反应条件进行优化。结果 在优化的反应条件下,60 min内检测敏感度可达100 pmol/L模板ssRNA。特异性良好,与甲型H1N1、H3N2、H9N2流感病毒,以及乙型流感病毒和新城疫病毒疫苗株RNA无交叉反应。结论 建立了针对H7亚型禽流感病毒的无需核酸扩增,直接进行CRISPR-Cas13a附带切割荧光检测的方法,可快速、准确和低成本地即时即地检测。
Objective To establish an amplification-free detection method for H7 subtype avian influenza virus based on principle of CRISPR-Cas13 a collateral cleavage fluorescence determination. Methods More than 700 HA-gene nucleic acid sequences of H7 subtype avian influenza virus were downloaded from GenBank. After comparison,the virus template was determined according to the conserved fragment sequence. The best crRNA was then screened and the reaction conditions of CRISPR-Cas13 a protein,H7 crRNA and fluorescent double-labeled probe were optimized subsequently. Results Under the optimized reaction conditions,the detection sensitivity reached 100 pmol/L H7 template ssRNA within 60 min. The specificity of this method was perfect with no cross reaction between the template RNA of H7 avian influenza virus and other control viruses of A/Shanghaiputuo/1203/2013(H1 N1),A/Shanghaichangning/1507/2012(H3 N2),A/Environmental/Jiangxi/21/2011(H9 N2),B/Beijing/1386/2013(Victoria,BV),B/Chongqingyuzhong/1361/2013(Yamagata,BY) influenza virus strains and vaccine strain LaSota genomic RNA.Conclusion A novel method for direct detection of H7 subtype avian influenza virus without nucleic acid amplification based on CRISPR-Cas13 a collateral cleavage fluorescence detection had been established,which had the potential to enable a fast,accurate and low-cost option for point-of care testing for AIV H7 subtypes.
作者
殷宏
金涌
王玉峰
刘波
邹明强
YIN Hong;JIN Yong;WANG Yu-feng;LIU Bo;ZOU Ming-qiang(Institute of Equipment and Technology,Chinese Academy of Inspection and Quarantine/China Inspection Laboratory Technologies Co.Ltd.,Beijing 100123,China)
出处
《中国国境卫生检疫杂志》
CAS
2022年第1期1-4,共4页
Chinese Journal of Frontier Health and Quarantine
基金
中国检科院基本科研业务费项目(2017JK048)
国家重点研发计划项目(2017YFF0108600)。
