SARS-CoV-2 and Its Omicron Variants Detection with RT-RPA -CRISPR/Cas13a-Based Method at Room Temperature.

IF 1.6 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Reports of Biochemistry and Molecular Biology Pub Date : 2023-10-01 DOI:10.61186/rbmb.12.3.425
Jia Li, Xiaojun Wang, Liujie Chen, Lili Duan, Fenghua Tan, Kai Li, Zheng Hu
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Abstract

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 102 copies/μL, and can effectively distinguish between the original strain and the Omicron variant with a minimum limit of detection (LOD) of 103 copies/μL.

Conclusions: The method developed in this study has potential application in clinical detection of SARS-CoV-2 and its omicron variants.

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用基于 RT-RPA -CRISPR/Cas13a 的方法在室温下检测 SARS-CoV-2 及其 Omicron 变体
背景:严重急性呼吸系统综合征冠状病毒 2(SARS-CoV-2)的爆发引发了全球健康危机,基因突变和进化进一步造成了流行风险的不确定性。当务之急是迅速确定 SARS-CoV-2 及其变种的核酸序列,以应对冠状病毒大流行。我们的目标是开发一种快速、室温、床旁(POC)检测系统,以确定 SARS-CoV-2 分离株的核酸序列,尤其是奥米克变异株:方法:根据SARS-CoV-2的保守核苷酸序列,使用生物信息学软件分析、设计和筛选出最佳的酶等温扩增引物和CRISPR/Cas13a的高效CRISPR RNA(crRNA)。反转录-重组聚合酶扩增(RT-RPA)用于扩增病毒,CRISPR/Cas13a-crRNA用于裂解SARS-CoV-2靶序列。核酸检测的灵敏度通过质粒模板的系列稀释进行评估。所有反应均在室温下进行:结果:RT-RPA结合CRISPR/Cas13a可检测出SARS-CoV-2,最低含量为102拷贝/μL,并能有效区分原始菌株和Omicron变异株,最低检测限(LOD)为103拷贝/μL:结论:本研究开发的方法可用于临床检测 SARS-CoV-2 及其奥米克龙变异株。
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来源期刊
Reports of Biochemistry and Molecular Biology
Reports of Biochemistry and Molecular Biology BIOCHEMISTRY & MOLECULAR BIOLOGY-
CiteScore
2.80
自引率
23.50%
发文量
60
审稿时长
10 weeks
期刊介绍: The Reports of Biochemistry & Molecular Biology (RBMB) is the official journal of the Varastegan Institute for Medical Sciences and is dedicated to furthering international exchange of medical and biomedical science experience and opinion and a platform for worldwide dissemination. The RBMB is a medical journal that gives special emphasis to biochemical research and molecular biology studies. The Journal invites original and review articles, short communications, reports on experiments and clinical cases, and case reports containing new insights into any aspect of biochemistry and molecular biology that are not published or being considered for publication elsewhere. Publications are accepted in the form of reports of original research, brief communications, case reports, structured reviews, editorials, commentaries, views and perspectives, letters to authors, book reviews, resources, news, and event agenda.
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