基于RT-RAA-CRISPR/Cas13a的寨卡病毒检测方法的建立*

doi:10.3969/j.issn.1671-2587.2025.03.018

临床输血与检验 ›› 2025, Vol. 27 ›› Issue (3): 393-400.DOI: 10.3969/j.issn.1671-2587.2025.03.018

基于RT-RAA-CRISPR/Cas13a的寨卡病毒检测方法的建立^*

张雅路, 石耀强, 刘金胜, 李世林, 陈利民, 杨春晖

中国医学科学院北京协和医学院输血研究所, 四川成都 610052

收稿日期:2025-03-02 发布日期:2025-06-23
通讯作者: 杨春晖,主要从事输血医学研究,（E-mail）yangchunhui@ibt.pumc.edu.cn。
作者简介:张雅路,主要从事输血医学研究,（E-mail）zhangyl@student.pumc.edu.cn。
基金资助:
*本课题受四川省自然科学基金项目（No.2025ZNSFSC0678）资助

A CRISPR/Cas13a-Coupled RT-RAA System for Rapid Zika Virus Diagnosis

ZHANG Yalu, SHI Yaoqiang, LIU Jinsheng, LI Shilin, CHEN Limin, YANG Chunhui

Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, Sichuan 610052

Received:2025-03-02 Published:2025-06-23

摘要/Abstract

摘要： 目的寨卡病毒（Zika virus, ZIKV）是一种经伊蚊传播的新发黄病毒,近期研究提示其可能通过输血途径传播。现有检测方法存在灵敏度不足及覆盖范围有限等问题,亟需开发适用于中小型实验室或现场筛查的便捷检测技术。方法本研究基于CRISPR/Cas13a系统联合逆转录重组酶介导链置换核酸扩增（RT-RAA）技术,建立并优化了一种胶体金试纸条检测法,用于ZIKV RNA的特异性识别。结果所构建的RT-RAA-CRISPR/Cas13a试纸条检测灵敏度达4 copies/μL,特异性为100%,可精准检测梯度浓度标准品及临床样本。对ZIKV阳性样本的检测符合率为100%,阴性符合率亦为100%。血浆样本检测的灵敏度、特异性、阳性预测一致性（positive predictive agreement, PPA）和阴性预测一致性（negative predictive agreement, NPA）均为100%。结论本研究成功开发了一种基于RT-RAA-CRISPR/Cas13a技术的ZIKV快速可视化检测试纸条。该方法操作简便、灵敏度高、结果判读直观,为寨卡感染的现场筛查提供了可替代PCR检测的新型解决方案,特别适用于资源有限场景下的即时诊断需求。

关键词: CRISPR/Cas13a, 逆转录重组酶介导链置换核酸扩增, 寨卡病毒, 即时检测

Abstract: Objective Zika virus (ZIKV) is an emerging arthropod-borne flavivirus transmitted by Aedes mosquitoes. Recent commentaries regarding ZIKV routes of transmission describe a potential transmission by transfusion. The current approaches in use are low in diagnostic efficiency and coverage area of ZIKV infection. Hence, this study intended to drive the ZIKV infection detection to effectively adaptable for any small to medium-sized laboratory or field survey. Methods We established, optimized, and evaluated a colloidal gold test strip for detection of ZIKV RNA based on CRISPR/Cas13a combined with reverse-transcription recombinase-aided amplification assay (RT-RAA) technology. Results The strip detection of ZIKV RNA was established based on RT-RAA-CRISPR-Cas13a technology with a sensitivity of 4 copies/μL and a specificity of 100%. ZIKV RNA gradient concentration standards and clinical samples were effectively identified by this approach. The positive coincidence rate for ZIKV was 100%, while the negative coincidence rate was 100%. The sensitivity, specificity, positive predictive agreement (PPA) and negative predictive agreement (NPA) values of Zika virus liquid plasma detection were 100%, 100%, 100%, and 100%, respectively. Conclusion We have developed rapid and portable RT-RAA-CRISPR/Cas13a-based strip of ZIKV RNA detection for patients. This study provides a visual and faster alternative to current PCR-based diagnosis for ZIKA infection.

Key words: CRISPR/Cas13a, RT-RAA, ZIKV, point of care test

中图分类号:

R373

张雅路, 石耀强, 刘金胜, 李世林, 陈利民, 杨春晖. 基于RT-RAA-CRISPR/Cas13a的寨卡病毒检测方法的建立^*[J]. 临床输血与检验, 2025, 27(3): 393-400.

ZHANG Yalu, SHI Yaoqiang, LIU Jinsheng, LI Shilin, CHEN Limin, YANG Chunhui. A CRISPR/Cas13a-Coupled RT-RAA System for Rapid Zika Virus Diagnosis[J]. JOURNAL OF CLINICAL TRANSFUSION AND LABORATORY MEDICINE, 2025, 27(3): 393-400.

参考文献

[1] MUSSO D,GUBLER D J.Zika virus[J]. Clin Microbiol Rev,2016,29(3):487-524.
[2] MARBÁN-CASTRO E,GONCÉ A,FUMADÓ V,et al. Zika virus infection in pregnant women and their children:a review[J]. Eur J Obstet Gynecol Reprod Biol,2021,265:162-168.
[3] CHRISTIAN K M,SONG H J,MING G L.Pathophysiology and mechanisms of zika virus infection in the nervous system[J]. Annu Rev Neurosci,2019,42: 249-269.
[4] TYSON J,TSAI W Y,TSAI J J,et al.A high-throughput and multiplex microsphere immunoassay based on non-structural protein 1 can discriminate three flavivirus infections[J]. PLoS Negl Trop Dis,2019,13(8):e0007649.
[5] FAHAD A S,TIMM M R,MADAN B,et al.Functional profiling of antibody immune repertoires in convalescent zika virus disease patients[J]. Front Immunol,2021, 12:615102.
[6] WILLYARD C.Screening:in the blood[J]. Nature,2017,549(7673):S19-S21.
[7] ARAUJO R V,FEITOSA-SUNTHEIMER F,GOLD A S, et al.One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples:a protocol to study infection and gene expression during ZIKV infection[J]. Parasit Vectors,2020,13(1):128.
[8] GOODNOUGH L T,MARQUES M B.Zika virus and patient blood management[J]. Anesth Analg,2017, 124(1):282-289.
[9] GOOTENBERG J S,ABUDAYYEH O O,LEE J W,et al.Nucleic acid detection with CRISPR-Cas13a/C2c2[J]. Science,2017,356(6336):438-442.
[10] MYHRVOLD C,FREIJE C A,GOOTENBERG J S, et al.Field-deployable viral diagnostics using CRISPR-Cas13[J]. Science,2018,360(6387):444-448.
[11] TIAN Y,FAN Z H,XU L,et al.CRISPR/Cas13a-assisted rapid and portable HBV DNA detection for low-level viremia patients[J]. Emerg Microbes Infect,2023,12(1): e2177088.
[12] MORALES S V,COELHO G M,RICCIARDI-JORGE T,et al.Development of a quantitative NS1 antigen enzyme-linked immunosorbent assay (ELISA) for Zika virus detection using a novel virus-specific MAb[J]. Sci Rep,2024,14:2544.
[13] ALZATE D,MARÍN E,OROZCO J,et al. Differential detection of zika virus based on PCR[J]. J Virol Methods,2022,301:114459.
[14] MOON J,LIU C C.Asymmetric CRISPR enabling cascade signal amplification for nucleic acid detection by competitive crRNA[J]. Nat Commun,2023,14(1): 7504.
[15] KELLNER M J,KOOB J G,GOOTENBERG J S, et al.SHERLOCK:nucleic acid detection with CRISPR nucleases[J]. Nat Protoc,2019,14(10):2986-3012.

基于RT-RAA-CRISPR/Cas13a的寨卡病毒检测方法的建立^*

A CRISPR/Cas13a-Coupled RT-RAA System for Rapid Zika Virus Diagnosis

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