環形恆溫核酸擴增法在SARS-CoV-2檢測中的設計與應用於未來新興傳染病的策略

Abstract

2019年末於中國武漢發生SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2)病毒大規模感染事件，該病毒傳播力極強，全球人民的生活型態因而大幅改變，除了造成醫療單位人滿為患，經濟交流、物資流通及觀光產業等的停滯也造成全球的經濟重創。

面臨一個大規模傳染疾病，有效防堵疾病的方法主要為檢測診斷、治療，以及疫苗三者環環相扣，互相配合。本研究著重於診斷測試作為研究與探討主軸，以期能透過適當的檢測方法與防治措施，更有效的減少疾病傳播的風險。COVID-19的主要檢測診斷方法如核酸篩檢、病毒基因定序、抗原快篩與抗體篩檢技術等。現有的篩檢黃金準則為聚合酶連鎖反應(PCR)，以核酸擴增方式檢測與推算病患體內病毒載量，但所需數小時的擴增時間、昂貴熱循環儀器、需要至特定場所由專業訓練人員進行操作等，導致篩檢量不足以應對大量流行時的需求、許多偏遠地區無法使用此技術等缺點。因此，可以藉由一些快速且靈敏度高的新興檢測方法作為輔助PCR診斷的工具。

本研究先使用統計學方法對於SARS-CoV-2通過美國食品藥物管理局(U.S. Food and Drug Administration, FDA)之緊急使用授權(Emergency use authorization, EUA)相關檢測產品進行分析與分類，找出已上市產品所使用的技術與可以施行的場所，進行優缺點分析，探討目前的使用情形，以及是否能有改善的空間，期能找到更有效率或更好的方法，以利及早診斷、及早匡列隔離患者以防堵疾病傳播。探討目前所常用的篩檢方式，以LAMP與PCR、抗原快篩相比，再由價格、效率、適用地點、於不同疾病盛行時期適合應用的時機進行分析與比較，以及該情境下使用該檢測方式之應用優缺點，評斷出適用於不同情境下最適合的篩檢技術以及配套措施。其中通過國際認證並可於居家進行篩檢的恆溫核酸擴增技術：環形核酸擴增法(Loop-Mediated Isothermal Amplification, LAMP)有快速、高準確度的優點而具有極大的潛力。

本研究進而設計了一組LAMP的引物與目標序列，進行凝膠電泳(Gel electrophoresis)以及表面電漿子共振光學系統(Surface plasmon resonance，SPR)，驗證LAMP方法的可行性與檢測時間優勢。實驗中分別進行20分鐘、40分鐘與60分鐘的LAMP反應。實驗結果顯示，LAMP反應可以在20分鐘就在電泳以及SPR顯示出陽性樣本於反應前後的明顯差異。於線性範圍中，擴增時間越久，反應擴增產物越多、訊號量越大。由於SPR的極高靈敏度，將反應產物稀釋3倍後亦可以量測出結果，未來在進行LAMP檢測上的應用可以縮減更多體積，以減少廣泛檢測的成本消耗。此外，由於LAMP單一反應的產物體積較小，難以以折射率計量測出其單位標準量，本研究使用不同鹽度的NaCl在SPR上建立角度與折射率的線性回歸校正曲線，可以對應出反應產物近似的折射率值。

本研究也以時間軸回顧自2019疫情爆發後主要影響台灣疾病盛行率大幅變動的因素，評估不同盛行率時適用的篩檢方法防堵疫情傳播。利用LAMP可以在各種環境下進行快速(20分鐘)核酸檢測，使潛在確診患者都能在家便利的進行最準確、排除假陰性的檢測的優勢，作為一個可行的篩檢補足方案，使診斷流程分流與更加順利。期許得以藉由此研究所統整的LAMP序列選用、引子設計流程、驗證方法與使用建議，為下一次的疾病做好充足準備。當未來再度面臨大規模傳染疾病時，利用LAMP的優勢，快速的以篩檢隔離的防堵對策進行因應。

At the end of 2019, a large-scale infection with the SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) virus occurred in Wuhan, China. The virus is extremely transmissible, leading to significant global impacts on people''s lifestyles, overwhelming healthcare systems, and causing economic disruptions worldwide. Effectively combating a large-scale infectious disease, the main methods are detection, diagnosis, treatment, and vaccines. This study focuses on diagnostic testing, with the aim of reducing the risk of disease transmission through appropriate detection methods in different prevalence rate. The most commonly used diagnosis methods for COVID-19 include nucleic acid screening, viral gene sequencing, antigen rapid screening and antibody screening technology, etc. The current gold standard for screening is polymerase chain reaction (PCR), which uses nucleic acid amplification to detect and estimate the viral load in patients. However, it demands prolonged amplification time, costly equipment, specific location and specialized personnel. These limitations have led to an inadequate screening capacity to meet demands of a large epidemic, and the inability to use this technology in many remote areas. Therefore, novel and highly sensitive diagnostic methods can be used as tools to assist PCR diagnosis shortage. This study employs statistical methods to analyze and classify SARS-CoV-2 related testing products authorized by the U.S. Food and Drug Administration (FDA) under Emergency Use Authorization (EUA). To determine the most suitable screening technology and supporting measures for different situations from the methods and applicable settings of the launched products, this study analyzes the advantages and disadvantages in terms of price, efficiency, applicable locations, suitable timing for application in different disease epidemic periods, explores the current usage situation, and whether there are areas for enhancement, hoping to find more efficient or better methods to facilitate for preventing the spread of the disease by early diagnosis and early isolation of patients. Among these currently commonly used screening methods such as PCR and antigen rapid test, Loop-Mediated Isothermal Amplification (LAMP), a nucleic acid testing method which is with high accuracy and applicable for home testing, exhibits substantial potential. This study outlines the process of selecting target sequences, designing LAMP primers, and validating the feasibility of the LAMP method using gel electrophoresis and Surface Plasmon Resonance (SPR) systems. The experiment involved LAMP reactions conducted for 20, 40, and 60 minutes, with results indicating that LAMP reactions at 20 minutes exhibit noticeable differences in positive samples before and after the reaction, as observed through electrophoresis and SPR. Within the linear range, the longer amplification time, the more reaction products and the greater signal quantities. Due to the high sensitivity of SPR, diluted reaction products can also be measured, reducing testing costs. Calibration curves for angle and refractive index are established on SPR using different concentrations of NaCl, allowing for the approximate refractive index values of reaction products. This study also evaluates factors influenced Taiwan''s disease prevalence significantly since the 2019 pandemic outbreak, and evaluates the screening methods applicable at different prevalence rates to prevent the spread of the epidemic. LAMP, with its ability to conduct rapid nucleic acid testing (20 minutes) in various environments, provides potentially confirmed patients to conveniently conduct the most accurate test at home, eliminating false negatives. This serves as a feasible supplementary solution, streamlining the diagnosis process and improving overall efficiency. The integrated LAMP sequence selection, primer design process, verification methods, and usage recommendations from this study aim to prepare for the next disease outbreak. Leveraging the advantages of LAMP allows for timely screening and isolation strategies, effectively combating the spread of disease in the event of future pandemics.