以反轉錄恆溫環型核酸擴增法建立新型冠狀病毒之檢測

Development of RT-LAMP For SARS-CoV-2 Detection

歐陽東昇(Tung-Sing Au Yeung)

指導教授 ： 劉旻禕

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202100585

摘要

新型冠狀病毒(Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)從2019年12月 起在武漢引發「嚴重特殊傳染性肺炎」 (Coronavirus Disease-2019, COVID-19)。截至2021年1月底，全球確診感染人數已突破1億人，並確定200萬人因此死亡。新型冠狀病毒是一種高傳染性病毒，其主要傳播途徑為飛沫傳染。目前，除了將感染的病人隔離外，並沒有能有效治療COVID-19的藥物。但是，由於感染COVID-19 的病人在早期大多呈現與一般感冒相似的症狀，並且新型冠狀病毒能在人體潛伏14天或以上，因此很難在一般理學檢查中被篩檢出來。對於SARS-CoV-2病毒核酸，定量反轉錄聚合酶連鎖反應(Quantitative reverse-transcription polymerase chain reaction, qRT-PCR)是目前的黃金標準檢測方法。 然而，這個方法在執行上需要特殊的精密儀器進行檢測分析，也需要專業訓練的醫檢人員操作。這些條件都限制了qRT-PCR的使用範圍及可篩檢的樣本數目。因此，我們的目標是建立一個能夠適用於定點照護(point of care)且快速簡單從病人中檢驗新型冠狀病毒的方法。我們透過反轉錄恆溫環型核酸擴增法(Reverse transcription loop-mediated isothermal amplification, RT-LAMP)，建立一個能在恆溫下反應將目標核酸快速增幅的方式。為了方便判讀結果，加入了酚紅酸鹼指示劑，使RT-LAMP的增幅結果可以簡單地以肉眼判斷其顏色變化。由於新型冠狀病毒的核蛋白(Nucleoprotein, N)基因在不同地區的新型冠狀病毒分離株中都呈現高度的保留性，因此我們針對該基因設計了RT-LAMP的引子。根據我們的實驗結果，確認我們的引子能專一且靈敏地篩檢新型冠狀病毒核酸，並且能在30分鐘內得到檢測結果。概括而論，我們建立了一個快速篩檢SARS-COV-2 病毒的方法，而檢驗結果僅需肉眼即可觀察，我們期望將這個具潛力的核酸檢測方法應用於定點照護檢驗(point-of-care testing, POCT)。

關鍵字

反轉錄恆溫環型核酸擴增法 ； 新型冠狀病毒 ； 定點照護檢驗 ； 比色法反轉錄恆溫環型核酸擴增法 ； 核蛋白

並列摘要

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a serious outbreak of coronavirus disease COVID-19 pandemic since Dec 2019. As of Jan 31, 2021, more than 100 million confirmed cases and 2 million deaths have been reported worldwide. SARS-CoV-2 is a highly contagious virus which can be spreaded by droplet transmission. Currently, there are no effective therapies or vaccines against COVID-19, and the only way to prevent transmission of SARS-CoV-2 is to quarantine infected patients. However, it is difficult to detect SARS-CoV-2 in the early infection stage since most of the patients display flu-like symptoms or even asymptomatic. Also, the incubation period of COVID-19 can be up to 14 days. Currently, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is the most common and gold standard detection method for SARS-CoV-2 RNA. However, this method has several limitations, such as specialized equipment and skilled technicians. These strict requirements may limit the number that can be detected per period of time. Our goal is to develop a quick and simple SARS-CoV-2 screening test which can be delivered at the point of care. We utilize the reverse transcription loop-mediated isothermal amplification (RT-LAMP), which is based on an isothermal nucleic acid amplification method, to develop the test. The RT-LAMP results with colorimetric changes can be determined easily by nude eye. Due to SARS-CoV-2 nucleoprotein (N) gene is highly conserved among the isolates, we designed a set of primers which specific bind to the SARS-CoV-2 N genes. Our RT-LAMP showed high specificity and sensitivity in detecting SARS-CoV-2 but not other coronaviruses nor common human respiratory disease-causing viruses within 30 minutes. Overall, we developed a RT-LAMP-based SARS-CoV-2 detection method, which delivered fast amplification and easy readout, with the potential to be applied to the point of care.

並列關鍵字

Reverse Transcription Loop-Mediated Isothermal Amplification ； Severe acute respiratory syndrome coronavirus 2 ； point-of-care testing ； colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification ； Nucleoprotein

參考文獻

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