摘要
目的建立一种基于反转录重组酶介导的等温扩增(reverse transcription recombinase-aid amplification,RT-RAA)联合CRISPR/Cas12a反应的人副流感病毒(human parainfluenza virus,HPIV)核酸检测方法。方法根据HPIV核衣壳蛋白(nucleocapsid protein,N)基因保守区序列,设计RT-RAA所需型特异性引物及crRNA(CRISPR RNA,crRNA),应用Cas12a切割荧光标记探针产生的荧光强度筛选高敏感性HPIV1-4型特异性crRNA并优化crRNA、Cas12a和探针浓度。利用体外转录RNA及临床样本,评价RT-RAA联合CRISPR/Cas12a检测方法的检测下限及特异性。结果从设计的crRNA中筛选出高敏感性的HPIV1-4型特异性crRNA,并建立基于荧光强度测量的RT-RAA联合CRISPR/Cas12a的HPIV核酸检测方法,该方法对各型HPIV的检测下限均可达到10拷贝/反应。采用该方法检测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。结论建立的HPIV 1-4型荧光法CRISPR核酸检测方法快速、特异,且可无需专业核酸检测设备。
Objective To establish a nucleic acid detection method for human parainfluenza virus(HPIV)based on reverse transcription recombinase-aid amplification(RT-RAA)combined with CRISPR/Cas12a.Methods The type-specific primer pairs of RT-RAA and CRISPR RNA(crRNA)targeted on conserved sequence of nucleocapsid protein(N)gene of HPIV were designed.Fluorescence intensities from cleavage of fluorophore labeled probes mediated by Cas12a were measured for screening of crRNA and concentration optimization of crRNA,Cas12a as well as the probe.With the RNA transcribed in vitro and clinical specimen,the lower limit of detection and specificity of RT-RAA combined CRISPR/Cas12a detection were evaluated.Results The crRNA specific to each type of HPIV 1-4,with strongest cleavage activity,were screened out.With the optimal concentration of crRNA,Cas12a as well as the probe,the lower limit of detection could reach 10 copies of target gene per reaction on fluorescence intensity measurement.No cross-reaction was found in clinical samples of eight other respiratory viruses detected by this method.Conclusions The established HPIV1-4 fluorescence CRISPR nucleic acid detection method is rapid,specific,and does not require professional nucleic acid detection equipment.
作者
张政涛
游丽斌
翁育伟
Zhang Zhengtao;You Libin;Weng Yuwei(The School of Public Health,Fujian Medical University,Fuzhou 350122,China;Fujian Provincial Center for Disease Control and Prevention,Fujian Provincial Key Laboratory of Zoonosis Research,Fuzhou 350012,China)
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
2023年第5期524-529,共6页
Chinese Journal of Experimental and Clinical Virology
基金
福建省卫生健康中青年科研重大项目(2021ZQNZD006)
福建省科技创新平台建设项目(2019Y2001)。
作者简介
通信作者:翁育伟,Email:wengywfjcdc@aliyun.com。
