{"title":"用于快速检测 SARS-CoV-2 的高灵敏度 RT-qLAMP 分析法的开发与验证:方法学方面。","authors":"Faezeh Mahmoudi, Davod Jafari, Seyedeh Mona Mousavi Esfahani, Arshad Hoseini, Mahmood Barati, Neda Saraygord-Afshari","doi":"10.1007/s12033-024-01275-7","DOIUrl":null,"url":null,"abstract":"<p><p>Specific and reliable diagnostic methods are becoming increasingly essential to identify patients in light of the high transmission rate and the recent appearance of the new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For the specific detection of SARS-CoV-2, our quantitative reverse transcription loop-mediated isothermal amplification (RT-qLAMP) assay implementation demonstrates how flexible it can be with two readouts: visualized colorimetric and real-time fluorescence. Different factors were optimized to improve the reaction conditions, including temperature (60 °C), assay runtime (60 min), primers, MgSO<sub>4</sub> (6 mM), dNTPs (1 mM), LAMP Buffer (1.2 mM Tris-HCl), KCl (50 mM), pH (8), and phenol red (10 mM) concentrations. Regarding analytical sensitivity, the colorimetric RT-LAMP method detected samples with C<sub>t</sub> values up to 29, while the RT-qLAMP assay identified up to C<sub>t</sub> = 31. RT-qLAMP was evaluated on 40 clinical samples (25 positives and 15 negatives) for viral RNA detection. All negative samples were found negative through fluorescent reading in RT-qLAMP and quantitative reverse transcription PCR (RT-qPCR) assays. Twenty-three clinically positive samples demonstrated a positive RT-qLAMP reaction (up to C<sub>t</sub> ≤ 31) with 92% clinical sensitivity, 100% clinical specificity, 100% positive predictive value (PPV), 88.24% negative predictive values (NPV), and 95% accuracy.</p>","PeriodicalId":18865,"journal":{"name":"Molecular Biotechnology","volume":null,"pages":null},"PeriodicalIF":2.4000,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Development and Validation of a Highly Sensitive RT-qLAMP Assay for Rapid Detection of SARS-CoV-2: Methodological Aspects.\",\"authors\":\"Faezeh Mahmoudi, Davod Jafari, Seyedeh Mona Mousavi Esfahani, Arshad Hoseini, Mahmood Barati, Neda Saraygord-Afshari\",\"doi\":\"10.1007/s12033-024-01275-7\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Specific and reliable diagnostic methods are becoming increasingly essential to identify patients in light of the high transmission rate and the recent appearance of the new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For the specific detection of SARS-CoV-2, our quantitative reverse transcription loop-mediated isothermal amplification (RT-qLAMP) assay implementation demonstrates how flexible it can be with two readouts: visualized colorimetric and real-time fluorescence. Different factors were optimized to improve the reaction conditions, including temperature (60 °C), assay runtime (60 min), primers, MgSO<sub>4</sub> (6 mM), dNTPs (1 mM), LAMP Buffer (1.2 mM Tris-HCl), KCl (50 mM), pH (8), and phenol red (10 mM) concentrations. Regarding analytical sensitivity, the colorimetric RT-LAMP method detected samples with C<sub>t</sub> values up to 29, while the RT-qLAMP assay identified up to C<sub>t</sub> = 31. RT-qLAMP was evaluated on 40 clinical samples (25 positives and 15 negatives) for viral RNA detection. All negative samples were found negative through fluorescent reading in RT-qLAMP and quantitative reverse transcription PCR (RT-qPCR) assays. Twenty-three clinically positive samples demonstrated a positive RT-qLAMP reaction (up to C<sub>t</sub> ≤ 31) with 92% clinical sensitivity, 100% clinical specificity, 100% positive predictive value (PPV), 88.24% negative predictive values (NPV), and 95% accuracy.</p>\",\"PeriodicalId\":18865,\"journal\":{\"name\":\"Molecular Biotechnology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.4000,\"publicationDate\":\"2024-09-24\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Molecular Biotechnology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1007/s12033-024-01275-7\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Biotechnology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1007/s12033-024-01275-7","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Development and Validation of a Highly Sensitive RT-qLAMP Assay for Rapid Detection of SARS-CoV-2: Methodological Aspects.
Specific and reliable diagnostic methods are becoming increasingly essential to identify patients in light of the high transmission rate and the recent appearance of the new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For the specific detection of SARS-CoV-2, our quantitative reverse transcription loop-mediated isothermal amplification (RT-qLAMP) assay implementation demonstrates how flexible it can be with two readouts: visualized colorimetric and real-time fluorescence. Different factors were optimized to improve the reaction conditions, including temperature (60 °C), assay runtime (60 min), primers, MgSO4 (6 mM), dNTPs (1 mM), LAMP Buffer (1.2 mM Tris-HCl), KCl (50 mM), pH (8), and phenol red (10 mM) concentrations. Regarding analytical sensitivity, the colorimetric RT-LAMP method detected samples with Ct values up to 29, while the RT-qLAMP assay identified up to Ct = 31. RT-qLAMP was evaluated on 40 clinical samples (25 positives and 15 negatives) for viral RNA detection. All negative samples were found negative through fluorescent reading in RT-qLAMP and quantitative reverse transcription PCR (RT-qPCR) assays. Twenty-three clinically positive samples demonstrated a positive RT-qLAMP reaction (up to Ct ≤ 31) with 92% clinical sensitivity, 100% clinical specificity, 100% positive predictive value (PPV), 88.24% negative predictive values (NPV), and 95% accuracy.
期刊介绍:
Molecular Biotechnology publishes original research papers on the application of molecular biology to both basic and applied research in the field of biotechnology. Particular areas of interest include the following: stability and expression of cloned gene products, cell transformation, gene cloning systems and the production of recombinant proteins, protein purification and analysis, transgenic species, developmental biology, mutation analysis, the applications of DNA fingerprinting, RNA interference, and PCR technology, microarray technology, proteomics, mass spectrometry, bioinformatics, plant molecular biology, microbial genetics, gene probes and the diagnosis of disease, pharmaceutical and health care products, therapeutic agents, vaccines, gene targeting, gene therapy, stem cell technology and tissue engineering, antisense technology, protein engineering and enzyme technology, monoclonal antibodies, glycobiology and glycomics, and agricultural biotechnology.