分子邏輯閘式A型流感病毒基因分析方法之研究

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Please use this identifier to cite or link to this item: http://140.128.103.80:8080/handle/310901/25983

Title:	分子邏輯閘式A型流感病毒基因分析方法之研究
Other Titles:	Molecular Logic Gate-Based Analytical Method for Viral Genes of Influenza A Virus
Authors:	李俊慶 Lee, Chun-Ching
Contributors:	莊旻傑 Chuang, Min-Chieh 化學系
Keywords:	Influenza virus;diagnostics;Molecular beacon;Dual-sequence;logic gate 流感病毒;診斷;分子探針;雙重目標物;邏輯閘
Date:	2015
Issue Date:	2015-12-15T07:43:07Z (UTC)
Abstract:	由於A型流感病毒經由動物和人類之間交互感染因而產生高致病性的病毒株，因而嚴重危害人體健康，因此於臨床診斷高致病性流感病毒的工具為目前急需的方法，於本研究中發展了具專一性且靈敏的同時偵測血球凝集素(hemagglutinin, HA)和神經氨酸酶(neuraminidase, NA)特定基因片段的方法，利用具莖環結構的核酸分子探針(molecular beacon) 及布林AND邏輯閘機制運算輸出訊號，以降低人為誤判的機會及達到同時偵測雙重基因片段。為了更深入了解同時由雙重基因片段引發分子探針二級結構的變化，本研究使用光學檢測方法針對多個重要因素進行探討，包括序列組成及雜交位置(雜交於分子探針莖部區域的核酸數目)對分子探針構型的影響；參與輔助鏈(assistant strand)幫助霍利迪交叉構型(Holliday junction)的形成；調控霍利迪結構交叉處未配對核酸(unpaired nucleotides)數目造成的影響；以及特定基因片段的選擇。實驗結果顯示，本研究所發展的分子探針能夠專一性的同時偵測流感病毒亞型的雙重基因片段，在H5與N2基因片段同時存在時呈現明顯的陽性結果，且只有同時存在H5和N2基因片段的情況下相較於其他組合(不存在H5和N2基因片段、只有H5基因片段和只有N2基因片段)，得到顯著的螢光訊號。此外，本研究同時探討於雙重基因片段存在之下分子探針解折疊(unfolding)的熱力學和動力學行為，我們發現於不同的溫度和不同未配對核酸數目會影響分子探針解折疊的速率以及熵和焓的變化，且此效應與分子探針熱力學穩定程度及霍利迪結構交叉處鬆緊程度相關。為了達成提高靈敏度的目標，我們進一步利用酵素聚合酶(polymerase)進行鏈取代反應(strand displacement)及產生螢光共振能量轉移效應(Fluorescence Resonance Energy Transfer, FRET)達到訊號擴增的效果，進而提升感測靈敏度，結果顯示此感測機制對於三種陰性組合不易產生偽陽性結果，此感測系統對於同時存在H5和N2基因片段的偵測極限為7 pM。此外，結果顯示此分析方法對於未經純化的聚合酶鏈式反應(polymerase chain reaction, PCR)產物具有良好的感測能力，在H5和N2的PCR產物同時存在下相較於其他三種陰性組合有明顯的訊號區別。此分析技術具有高度應用性，亦開啟以分子探針為核心之雙重目標物分析方法於分子診斷之應用。 Influenza A viruses, pathogens that continuously circulate and mutate among bird, pig, and human hosts, have caused substantial morbidity and mortality globally since the Middle Ages. The emergence of novel virus strains that are capable of causing epidemics or pandemics is even posing tremendous treats to human health. Prior investigation has indicated that the continuous and complex mutation of viruses and concurrent gene expressions of multi-genes were the crucial cuases leading to influenza virus pathogenesis. This underlines the significance of simultaneous diagnostics of multiple genes and necessitates an accurate and rapid subtyping assay to satisfy the urgent need in the clinical diagnosis and disease control of influenza.Motivated by the demand in analysis of multiple sequences, we developed an intelligent strategy based on molecular beacons (MBs), in cooperation with an assistant strand, to logically process dual input genes of hemagglutinin (HA) and neuraminidase (NA) in a AND gate format. Notably, the prerequisite for opening the designed MB in our study was different from conventional configurations reported in “1 input- 1 output fashion”, enabling this strategy suitable for high-fidelity dual-input determination. Systematic insights on the thermodynamic and kinetic parameters were provided to facilitate appropriate designs of MB and the assistant strand. The results concluded a AND gate-most preferable combination of unpaired nucleotide (1,2), in which the greatest output dynamic difference between the simultaneous presence of dual-input and any other scenarios was given, in connection to a MB unzipping rate at 33,235 M-1s-1 and entropy change (S12) at 647 Jmol-1K-1.In addition, the study investigated thermodynamics and kinetics of MB unfolding as functions of unpaired nucleotides and temperature which altered the formation and stability of the Holliday junction. To approach a further lower limit of detection, we utilized polymerase to undergo strand displacement and Fluorescence Resonance Energy Transfer (FRET), attaining signal amplification and sensitivity increase. The signal was low at the three negative combinations in connection to the limit of detection at 7 pM for the simultaneous presence of H5 and N2 genetic fragments.Eventually high-fidelity subtyping of influenza virus was implemented in sample of unpurified amplicons. The screening technology possesses high applicability to expedite the application of molecular diagnostics administering duel target analyzing method which utilizes single molecular probe as the core.
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