基于RT-RPA-CRISPR/Cas13a方法在室温下检测严重急性呼吸综合征冠状病毒2（SARS-CoV-2）及其奥密克戎变种
SARS-CoV-2 and Its Omicron Variants Detection with RT-RPA -CRISPR/Cas13a-Based Method at Room Temperature.
作者信息
Li Jia, Wang Xiaojun, Chen Liujie, Duan Lili, Tan Fenghua, Li Kai, Hu Zheng
机构信息
These authors contributed equally to this work.
The First Clinical College of Xiangnan University, the First People's Hospital of Chenzhou Affiliated Xiangnan University, Chenzhou 423000, Hunan, China.
出版信息
Rep Biochem Mol Biol. 2023 Oct;12(3):425-437. doi: 10.61186/rbmb.12.3.425.
BACKGROUND
The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a global health crisis, with genetic mutations and evolution further creating uncertainty about epidemic risk. It is imperative to rapidly determine the nucleic acid sequence of SARS-CoV-2 and its variants to combat the coronavirus pandemic. Our goal was to develop a rapid, room-temperature, point-of-care (POC) detection system to determine the nucleic acid sequences of SARS-CoV-2 isolates, especially omicron variants.
METHODS
Based on the conserved nucleotide sequence of SARS-CoV-2, bioinformatics software was used to analyze, design, and screen optimal enzymatic isothermal amplification primers and efficient CRISPR RNAs (crRNAs) of CRISPR/Cas13a to the target sequences. Reverse transcription-recombinase polymerase amplification (RT-RPA) was used to amplify the virus, and CRISPR/Cas13a-crRNA was used to cleave the SARS-CoV-2 target sequence. The sensitivity of nucleic acid detection was assessed by serial dilution of plasmid templates. All reactions were performed at room temperature.
RESULTS
RT-RPA, combined with CRISPR/Cas13a, can detect the SARS-CoV-2 with a minimum content of 10 copies/μL, and can effectively distinguish between the original strain and the Omicron variant with a minimum limit of detection (LOD) of 10 copies/μL.
CONCLUSIONS
The method developed in this study has potential application in clinical detection of SARS-CoV-2 and its omicron variants.
背景
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的爆发引发了全球健康危机，其基因突变和进化进一步增加了疫情风险的不确定性。迅速确定SARS-CoV-2及其变体的核酸序列对于抗击新冠疫情至关重要。我们的目标是开发一种快速、室温、即时检测（POC）系统，以确定SARS-CoV-2分离株，特别是奥密克戎变体的核酸序列。
方法
基于SARS-CoV-2的保守核苷酸序列，利用生物信息学软件分析、设计和筛选针对目标序列的最佳酶促等温扩增引物和CRISPR/Cas13a的高效CRISPR RNA（crRNA）。采用逆转录-重组酶聚合酶扩增（RT-RPA）对病毒进行扩增，并用CRISPR/Cas13a-crRNA切割SARS-CoV-2目标序列。通过对质粒模板进行系列稀释来评估核酸检测的灵敏度。所有反应均在室温下进行。
结果
RT-RPA与CRISPR/Cas13a相结合，能够检测最低含量为10拷贝/μL的SARS-CoV-2，并且能够有效区分原始毒株和奥密克戎变体，最低检测限（LOD）为10拷贝/μL。
结论
本研究开发的方法在SARS-CoV-2及其奥密克戎变体的临床检测中具有潜在应用价值。
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