以重組酶聚合酶擴增(RPA)新型冠狀病毒(SARS-CoV-2)之定性與定量感測晶片技術開發
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2022
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從2019年，由嚴重急性呼吸道症候群冠狀病毒2型(severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)引起2019新型冠狀病毒疾病(Coronavirus Disease 2019, COVID-19)已迅速傳播至全球，各國急需要高效、快速、具特異性和低價的診斷方法，以早期檢測到受感染的人，對抗SARS-CoV-2。其中，逆轉錄聚合酶鏈反應(reverse transcription-polymerase chain reaction, RT-PCR)是標準常用和準確度較高的方法檢測SARS-CoV-2，但缺點是檢測耗時、需要昂貴的設備與專業人員在生物安全第二等級(biosafety level-2, BSL-2)實驗室操作。本研究利用具有快速、高效、高靈敏的重組酶聚合酶擴增法(recombinase polymerase amplification, RPA)來檢測SARS-CoV-2高度保守N基因序列，並且結合規律間隔重複短迴文序列簇(clustered regularly interspaced short palindromic repeats, CRISPR)的Cas12a酶做專一性檢測，以減少恆溫擴增的假陽性結果。透過開發RPA-CRISPR/Cas12a方法以檢測 SARS-CoV-2，使用側向流分析(lateral flow assay, LFA)和螢光強度(fluorescence intensity, FI)做定性分析。此外，定量RPA的方法較少，本研究使用表面電漿子共振(surface plasmon resonance, SPR)和電化學阻抗譜(electrochemical impedance spectroscopy, EIS)做SARS-CoV-2的 RPA 定量分析與專一性檢測，檢測極限(limit of detection, LOD)為每微升(μL) 1個SARS-CoV-2的去氧核醣核酸(deoxyribonucleic acid, DNA)拷貝數。本研究開發定性與定量專一性檢測SARS-CoV-2方法，具有快速和準確高的特性，可作為COVID-19早期診斷的有力工具。
Since 2019, Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly around the world. Therefore, many countries urgently need high-efficiency, fast, specific and low-cost diagnostic methods, which can mainly detect infected people at an early stage and fight against SARS-CoV-2. One of them, reverse transcription-polymerase chain reaction (RT-PCR) was the standard and high accuracy method for detecting SARS-CoV-2, but it had the disadvantages of time-consuming detection, expensive equipment and professionals required in biosafety level-2(BSL-2) laboratories for operating. In this study, the highly conserved N gene sequence of SARS-CoV-2 was amplified by recombinase polymerase amplification (RPA), which had the advantages of rapidity, efficiency and high sensitivity. And the Cas12a detection of clustered regularly interspaced short palindromic repeats (CRISPR) was used for specific detection to reduce false positives and misjudgments of isothermal amplification results. The RPA-CRISPR/Cas12a method was developed to detect SARS-CoV-2 by using lateral flow assay (LFA) and fluorescence intensity (FI) for qualitative analysis. In addition, there were few methods for quantitative RPA, such as surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS), were used for quantitative analysis and specific detection of SARS-CoV-2. The assay’s limit of detection (LOD) was 1 copy/μL of SARS-CoV-2 DNA. This study develops the qualitative and quantitative specific detection method for SARS-CoV-2, which is fast and accurate to be used as a powerful tool for the early diagnosis of COVID-19.
Since 2019, Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly around the world. Therefore, many countries urgently need high-efficiency, fast, specific and low-cost diagnostic methods, which can mainly detect infected people at an early stage and fight against SARS-CoV-2. One of them, reverse transcription-polymerase chain reaction (RT-PCR) was the standard and high accuracy method for detecting SARS-CoV-2, but it had the disadvantages of time-consuming detection, expensive equipment and professionals required in biosafety level-2(BSL-2) laboratories for operating. In this study, the highly conserved N gene sequence of SARS-CoV-2 was amplified by recombinase polymerase amplification (RPA), which had the advantages of rapidity, efficiency and high sensitivity. And the Cas12a detection of clustered regularly interspaced short palindromic repeats (CRISPR) was used for specific detection to reduce false positives and misjudgments of isothermal amplification results. The RPA-CRISPR/Cas12a method was developed to detect SARS-CoV-2 by using lateral flow assay (LFA) and fluorescence intensity (FI) for qualitative analysis. In addition, there were few methods for quantitative RPA, such as surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS), were used for quantitative analysis and specific detection of SARS-CoV-2. The assay’s limit of detection (LOD) was 1 copy/μL of SARS-CoV-2 DNA. This study develops the qualitative and quantitative specific detection method for SARS-CoV-2, which is fast and accurate to be used as a powerful tool for the early diagnosis of COVID-19.
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嚴重急性呼吸道症候群冠狀病毒2型, 2019新型冠狀病毒疾病, 規律間隔重複短迴文序列簇, 重組酶聚合酶擴增, 表面電漿子共振, 電化學阻抗譜, 側向流分析, 螢光強度, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coronavirus disease 2019 (COVID-19), clustered regularly interspaced short palindromic repeats (CRISPR), recombinase polymerase amplification (RPA), surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), lateral flow assay (LFA), fluorescence intensity (FI)