基于CRISPR均相电化学生物传感器检测SARS-CoV-2和HIV-1方法的建立

A homogeneous electrochemical biosensor based on CRISPR for SARS CoV-2 and HIV-1 detection

目的建立基于CRISPR-Cas13a系统的均相电化学生物传感器并用于病原微生物的核酸检测。方法通过逆转录-重组酶介导链替换核酸扩增技术(RT-RAA)对目标核酸进行扩增,经过体外转录后,激活Cas13a蛋白高效切割单链RNA的活性并对标记有亚甲基蓝(MB)的报告RNA进行剪切,构建CRISPR均相电化学生物传感器;通过遴选报告RNA上寡核苷酸的长度优化该传感器;利用该传感器检测新型冠状病毒(SARS-CoV-2)RNA和Ⅰ型人类免疫缺陷病毒(HIV-1)RNA,并对其检测性能进行评价。结果建立了一种基于CRISPR-Cas13a系统和RT-RAA等温扩增技术的均相电化学生物传感器,利该方法使用相对应的扩增引物和CRISPR RNA(crRNA)后能稳定检测出低至10^(2)拷贝/μl的SARS-CoV-2 RNA和HIV-1 RNA,并与其他多种病原体未发生交叉反应。结论该基于CRISPR-Cas13a系统的均相电化学生物传感器具有制备简单、灵敏度高、特异性好和通用性强等优点,为病原微生物的现场快速检测提供了新的可行方案。

Objective To establish a homogeneous electrochemical biosensor based on the CRISPR-Cas13a system for detection of pathogenic microorganism nucleic acid.Methods The target nucleic acid was amplified by reverse transcription recombinase-aided amplification(RT-RAA)technology.After in vitro transcription(IVT),A CRISPR homogeneous electrochemical biosensor was constructed by activating the trans-cleavage activity of Cas13a protein for single-stranded RNA and cleaving the reporter RNA labeled with methylene blue(MB).The biosensor was optimized by selecting the length of oligonucleotides on the reporter RNA before the biosensor′s detection performance was verified by detecting nucleic acid substances of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and human immunodeficiency virus type 1(HIV-1).Results A homogeneous electrochemical biosensor based on the CRISPR-Cas13a system and RT-RAA isothermal amplification technology was established.Using the corresponding amplification primers and crRNA,the method could stably detect 10^(2) copies/μl of SARS-CoV-2 RNA and HIV-1 RNA,but did not cross-react with other pathogens.Conclusion The homogeneous electrochemical biosensor based on the CRISPR-Cas13a system has the advantages of simple preparation,high sensitivity,good specificity and universality,and provides a new feasible scheme for pointof-care testing(POCT)of pathogenic microorganisms.