基于CRISPR/Cas13a系统的发热伴血小板减少综合征病毒快速检测方法的建立  

A RAPID CRISPR/CAS13A SYSTEM-BASED METHOD FOR DETECTINGSEVERE FEVER WITH THROMBOCYTOPENIA SYNDROME VIRUS

作　　者：丁康慧 黄俊 范娇 邱少富 刘洪波 刘鸿博 宋宏彬 DING Kang-hui;HUANG Jun;FAN Jiao;QIU Shao-fu;LIU Hong-bo;LIU Hong-bo;SONG Hong-bin(School of Public Health,Anhui Medical University,Hefei 230032,Anhui,China;Center for Disease Control and Prevention,Chinese People's Liberation Army,Beijing 100071,China;Department of Epidemiology,School of Public Health,China Medical University,Shenyang 110000,Liaoning,China)

机构地区：[1]安徽医科大学公共卫生学院,合肥230032 [2]中国人民解放军疾病预防控制中心,北京100070 [3]中国医科大学公共卫生学院流行病学教研室,沈阳110000

出　　处：《寄生虫与医学昆虫学报》2024年第3期153-162,共10页Acta Parasitologica et Medica Entomologica Sinica

摘　　要：目的建立基于CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats,CRISPR)系统的快速、灵敏的现场发热伴血小板减少综合征病毒(severe fever with thrombocytopenia syndrome virus,SFTSV)核酸检测方法。方法首先,经序列比对分析得到SFTSV S基因特异性保守区,设计并筛选多酶恒温扩增(multienzyme isothermal rapid amplification,MIRA)引物和靶标CRISPR RNA(CRISPR RNA,crRNA),经荧光信号和免疫层析试纸读取检测结果。为提升检测敏感度在单反应体系中加入多个检测靶标crRNA。并利用蜱感染模拟样本和蜱源样本验证该方法对蜱样本现场核酸检测的有效性。结果MIRA-CRISPR/Cas13a荧光法检测SFTSV的最低检测限为1 copy/μL,试纸法的最低检测限为10 copies/μL。特异性实验表明SFTSV与其他4种阴性对照病原均无交叉反应。该方法检测蜱感染模拟样本的灵敏度优于RT-PCR法。应用所建立的方法检测29份蜱源SFTSV样本,共检出20份阳性样本和9份阴性样本,与RT-PCR检测结果符合率为100%。结论本文所建立的方法可用于SFTSV的现场快速检测。Objective To develop a rapid and sensitive on-site nucleic acid detection method based on the clustered regularly interspaced short palindromic repeats(CRISPR)system to detect severe fever with thrombocytopenia syndrome virus(SFTSV).Methods Firstly,a specific conserved region of the SFTSV S gene was identified by sequence alignment analysis,followed by the design and selection of multienzyme isothermal rapid amplification(MIRA)primers and target CRISPR RNA(crRNA);detection results were read using fluorescence signals and immunochromatographic paper.Finally,in order to increase sensitivity,multiple detection targets of crRNA were incorporated into a single reaction system.The effectiveness of this method for field nucleic acid detection in tick samples was validated using simulated tick-infected samples and field ticks.Results The minimum detection limit(MDL)of the MIRA-CRISPR/Cas13a fluorescence assay for SFTSV was 1 copy/μL,while that of the stripe-based method was 10 copies/μL.No cross-reactivity was observed between SFTSV and the other four viral controls,indicating the high specificity of the MIRA-CRISPR/Cas13a system.The method demonstrated superior sensitivity for nucleic acid detection in simulated tick infection samples compared to RT-PCR.The analysis of 29 field tick samples revealed that 20 and 9 samples were positive and negative,respectively(100%agreement with RT-PCR results).Conclusions The proposed method could facilitate the rapid detection of SFTSV in the field.

关 键 词：发热伴血小板减少综合征病毒 CRISPR/Cas13a 等温扩增 蜱 核酸检测 

分 类 号：R373.3[医药卫生—病原生物学]