等温扩增联合CRISPR系统检测人副流感病毒方法的建立  

Isothermal amplification combined with a CRISPR system for detection of human parainfluenza virus

作　　者：张政涛 郑达锋 刘子榕 游丽斌 翁育伟[2,3] Zheng-tao ZHANG;Da-feng ZHENG;Zi-rong LIU;Li-bin YOU;Yu-wei WENG(Nanping First Hospital Affiliated to Fujian Medical University,Nanping 353000,China;The School of Public Health,Fujian Medical University,Fuzhou 350108,China;Fujian Provincial Center for Disease Control and Prevention,Fujian Provincial Key Laboratory of Zoonosis Research,Fuzhou 350012,China)

机构地区：[1]福建医科大学附属南平第一医院,南平353000 [2]福建医科大学公共卫生学院,福州350108 [3]福建省疾病预防控制中心,福建省人兽共患病研究重点实验室,福州350012

出　　处：《中国人兽共患病学报》2024年第12期1122-1127,共6页Chinese Journal of Zoonoses

基　　金：福建省卫生健康中青年科研重大项目(No.2021ZQNZD006);福建省科技创新平台建设项目(No.2019Y2001)联合资助。

摘　　要：目的应用反转录重组酶介导等温扩增(reverse transcription recombinase-aid amplification,RT-RAA)联合CRISPR体系,建立人副流感病毒(human parainfluenza virus,HPIV)核酸检测方法。方法根据HPIV-1核衣壳蛋白(nucleocapsid,N)基因保守区序列,设计RT-RAA所需型特异性引物及crRNA(CRISPR RNA),应用Cas13a切割荧光标记探针产生的荧光强度优化crRNA和Cas13a浓度。利用体外转录RNA及临床样本,评价RT-RAA联合CRISPR/Cas13a检测方法的检测下限、敏感性和特异性。结果RT-RAA联合CRISPR/Cas13a方法对HPIV-1的检测下限可达到1拷贝/反应,该方法检测HPIV-2~4及8种其他呼吸道病毒感染的临床样本,均未显示有交叉反应;该检测方法的灵敏度(100%)和特异度(97.8%)高,与商业化试剂盒比较,具有较高的检测一致率(Kappa=0.88)。结论联合应用RT-RAA和CRISPR/Cas13a技术,成功建立HPIV-1核酸检测方法,该方法具有快速、灵敏和特异的特点,且无需专业核酸检测设备。This study was aimed at establishing a nucleic acid detection method for human parainfluenza viruses(HPIV)based on reverse transcription recombinase-aided amplification(RT-RAA)combined with a CRISPR system.Type-specific primer pairs for RT-RAA and CRISPR RNA(crRNA)targeting conserved sequences of the nucleocapsid(N)gene of HPIV-1 were designed.Fluorescence intensities from the cleavage of fluorophore labeled probes mediated by Cas13a were measured to optimize the crRNA and Cas13a concentrations.We evaluated the lower limit of detection,sensitivity,and specificity of RT-RAA combined with CRISPR/Cas13a detection in both in vitro transcribed RNAs and clinical specimens.The limit of detection of RT-RAA combined with CRISPR/Cas13a for HPIV-1 reached 1 copy/reaction.The method was subsequently used to detect clinical samples containing HPIV-2~4 and eight other respiratory viruses,and no cross-reaction was observed.This method had high sensitivity(100%)and specificity(97.8%),and high detection consistency with commercial kits(Kappa=0.88).Thus,our HPIV-1 nucleic acid detection method based on RT-RAA combined with the CRISPR/Cas13a system was successfully established.This method is rapid,sensitive,and specific,and does not require specialized nucleic acid detection equipment.

关 键 词：人副流感病毒 RT-RAA CRISPR Cas12a Cas13a 

分 类 号：R373.1[医药卫生—病原生物学]