Jia Li, Xiaojun Wang, Liujie Chen, Lili Duan, Fenghua Tan, Kai Li, Zheng Hu
{"title":"用基于 RT-RPA -CRISPR/Cas13a 的方法在室温下检测 SARS-CoV-2 及其 Omicron 变体","authors":"Jia Li, Xiaojun Wang, Liujie Chen, Lili Duan, Fenghua Tan, Kai Li, Zheng Hu","doi":"10.61186/rbmb.12.3.425","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>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.</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>RT-RPA, combined with CRISPR/Cas13a, can detect the SARS-CoV-2 with a minimum content of 10<sup>2</sup> copies/μL, and can effectively distinguish between the original strain and the Omicron variant with a minimum limit of detection (LOD) of 10<sup>3</sup> copies/μL.</p><p><strong>Conclusions: </strong>The method developed in this study has potential application in clinical detection of SARS-CoV-2 and its omicron variants.</p>","PeriodicalId":45319,"journal":{"name":"Reports of Biochemistry and Molecular Biology","volume":null,"pages":null},"PeriodicalIF":1.6000,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11015926/pdf/","citationCount":"0","resultStr":"{\"title\":\"SARS-CoV-2 and Its Omicron Variants Detection with RT-RPA -CRISPR/Cas13a-Based Method at Room Temperature.\",\"authors\":\"Jia Li, Xiaojun Wang, Liujie Chen, Lili Duan, Fenghua Tan, Kai Li, Zheng Hu\",\"doi\":\"10.61186/rbmb.12.3.425\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>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.</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>RT-RPA, combined with CRISPR/Cas13a, can detect the SARS-CoV-2 with a minimum content of 10<sup>2</sup> copies/μL, and can effectively distinguish between the original strain and the Omicron variant with a minimum limit of detection (LOD) of 10<sup>3</sup> copies/μL.</p><p><strong>Conclusions: </strong>The method developed in this study has potential application in clinical detection of SARS-CoV-2 and its omicron variants.</p>\",\"PeriodicalId\":45319,\"journal\":{\"name\":\"Reports of Biochemistry and Molecular Biology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.6000,\"publicationDate\":\"2023-10-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11015926/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Reports of Biochemistry and Molecular Biology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.61186/rbmb.12.3.425\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Reports of Biochemistry and Molecular Biology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.61186/rbmb.12.3.425","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
SARS-CoV-2 and Its Omicron Variants Detection with RT-RPA -CRISPR/Cas13a-Based Method at Room Temperature.
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.
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.