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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 劉旻禕(Min-Yi Liu) | |
dc.contributor.author | Tung-Sing Au Yeung | en |
dc.contributor.author | 歐陽東昇 | zh_TW |
dc.date.accessioned | 2021-06-15T14:02:04Z | - |
dc.date.available | 2026-02-17 | |
dc.date.copyright | 2021-02-23 | |
dc.date.issued | 2021 | |
dc.date.submitted | 2021-02-05 | |
dc.identifier.citation | 1. Abbott (2020). ID NOW COVID-19. 2. Administration, U.S.F.a.D. (2020). Coronavirus (COVID-19) update: FDA issues first emergency use authorization for point of care diagnostic. 3. Ali, Z., Aman, R., Mahas, A., Rao, G.S., Tehseen, M., Marsic, T., Salunke, R., Subudhi, A.K., Hala, S.M., Hamdan, S.M., et al. (2020). iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2. Virus Res 288, 198129. 4. Backer, J.A., Klinkenberg, D., and Wallinga, J. (2020). Incubation period of 2019 novel coronavirus (2019-nCoV) infections among travellers from Wuhan, China, 20-28 January 2020. Euro Surveill 25. 5. Basu, A., Zinger, T., Inglima, K., Woo, K.M., Atie, O., Yurasits, L., See, B., and Aguero-Rosenfeld, M.E. (2020). Performance of Abbott ID Now COVID-19 Rapid Nucleic Acid Amplification Test Using Nasopharyngeal Swabs Transported in Viral Transport Media and Dry Nasal Swabs in a New York City Academic Institution. J Clin Microbiol 58. 6. Brian, D.A., and Baric, R.S. (2005). Coronavirus genome structure and replication. Curr Top Microbiol Immunol 287, 1-30. 7. Cevik, M., Bamford, C.G.G., and Ho, A. (2020). COVID-19 pandemic-a focused review for clinicians. Clin Microbiol Infect 26, 842-847. 8. Cha, R.S., and Thilly, W.G. (1993). Specificity, Efficiency, and Fidelity of Pcr. Pcr Meth Appl 3, S18-S29. 9. Chang, C.K., Sue, S.C., Yu, T.H., Hsieh, C.M., Tsai, C.K., Chiang, Y.C., Lee, S.J., Hsiao, H.H., Wu, W.J., Chang, W.L., et al. (2006). Modular organization of SARS coronavirus nucleocapsid protein. J Biomed Sci 13, 59-72. 10. Chen, Y., Shi, Y., Chen, Y., Yang, Z., Wu, H., Zhou, Z., Li, J., Ping, J., He, L., Shen, H., et al. (2020). Contamination-free visual detection of SARS-CoV-2 with CRISPR/Cas12a: A promising method in the point-of-care detection. Biosens Bioelectron 169, 112642. 11. Color (2020). SARS-CoV-2 LAMP Diagnostic Assay. 12. Corpet, F. (1988). Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16, 10881-10890. 13. Curtis, K.A., Rudolph, D.L., and Owen, S.M. (2009). Sequence-specific detection method for reverse transcription, loop-mediated isothermal amplification of HIV-1. J Med Virol 81, 966-972. 14. Ding, Q., Lu, P., Fan, Y., Xia, Y., and Liu, M. (2020). The clinical characteristics of pneumonia patients coinfected with 2019 novel coronavirus and influenza virus in Wuhan, China. J Med Virol 92, 1549-1555. 15. Francois, P., Tangomo, M., Hibbs, J., Bonetti, E.J., Boehme, C.C., Notomi, T., Perkins, M.D., and Schrenzel, J. (2011). Robustness of a loop-mediated isothermal amplification reaction for diagnostic applications. FEMS Immunol Med Microbiol 62, 41-48. 16. Ganguli, A., Mostafa, A., Berger, J., Aydin, M.Y., Sun, F., Ramirez, S.A.S., Valera, E., Cunningham, B.T., King, W.P., and Bashir, R. (2020). Rapid isothermal amplification and portable detection system for SARS-CoV-2. Proc Natl Acad Sci U S A 117, 22727-22735. 17. Gorbalenya, A.E., Enjuanes, L., Ziebuhr, J., and Snijder, E.J. (2006). Nidovirales: evolving the largest RNA virus genome. Virus Res 117, 17-37. 18. Hite, J.M., Eckert, K.A., and Cheng, K.C. (1996). Factors Affecting Fidelity of DNA Synthesis During PCR Amplification of d(C-A)n•d(G-T)n Microsatellite Repeats. Nucleic Acids Research 24, 2429-2434. 19. Hoehl, S., Rabenau, H., Berger, A., Kortenbusch, M., Cinatl, J., Bojkova, D., Behrens, P., Boddinghaus, B., Gotsch, U., Naujoks, F., et al. (2020). Evidence of SARS-CoV-2 Infection in Returning Travelers from Wuhan, China. N Engl J Med 382, 1278-1280. 20. Hoffmann, M., Kleine-Weber, H., Krüger, N., Müller, M., Drosten, C., and Pöhlmann, S. (2020). 21. Horibe, D., Ochiai, T., Shimada, H., Tomonaga, T., Nomura, F., Gun, M., Tanizawa, T., and Hayashi, H. (2007). Rapid detection of metastasis of gastric cancer using reverse transcription loop-mediated isothermal amplification. Int J Cancer 120, 1063-1069. 22. Hsieh, K., Mage, P.L., Csordas, A.T., Eisenstein, M., and Soh, H.T. (2014). Simultaneous elimination of carryover contamination and detection of DNA with uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP). Chem Commun (Camb) 50, 3747-3749. 23. Huang, W.E., Lim, B., Hsu, C.C., Xiong, D., Wu, W., Yu, Y., Jia, H., Wang, Y., Zeng, Y., Ji, M., et al. (2020). RT-LAMP for rapid diagnosis of coronavirus SARS-CoV-2. Microb Biotechnol 13, 950-961. 24. Hui, K.P.Y., Cheung, M.C., Perera, R., Ng, K.C., Bui, C.H.T., Ho, J.C.W., Ng, M.M.T., Kuok, D.I.T., Shih, K.C., Tsao, S.W., et al. (2020). Tropism, replication competence, and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures. Lancet Respir Med 8, 687-695. 25. Jiang, X.Y., Zhang, Z.R., Wang, C.X., Ren, H.G., Gao, L.H., Peng, H.R., Niu, Z.B., Ren, H., Huang, H.Y., and Sun, Q. (2020). Bimodular effects of D614G mutation on the spike glycoprotein of SARS-CoV-2 enhance protein processing, membrane fusion, and viral infectivity. Signal Transduct Tar 5. 26. Kahn, J.S., and McIntosh, K. (2005). History and recent advances in coronavirus discovery. Pediatr Infect Dis J 24, S223-227, discussion S226. 27. Kaneko, H., Kawana, T., Fukushima, E., and Suzutani, T. (2007). Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys Methods 70, 499-501. 28. Kiefer, J.R., Mao, C., Hansen, C.J., Basehore, S.L., Hogrefe, H.H., Braman, J.C., and Beese, L.S. (1997). Crystal structure of a thermostable Bacillus DNA polymerase I large fragment at 2.1 A resolution. Structure 5, 95-108. 29. Kuboki, N., Inoue, N., Sakurai, T., Di Cello, F., Grab, D.J., Suzuki, H., Sugimoto, C., and Igarashi, I. (2003). Loop-mediated isothermal amplification for detection of African trypanosomes. J Clin Microbiol 41, 5517-5524. 30. Laha, S., Chakraborty, J., Das, S., Manna, S.K., Biswas, S., and Chatterjee, R. (2020). Characterizations of SARS-CoV-2 mutational profile, spike protein stability and viral transmission. Infect Genet Evol 85, 104445. 31. Lalli, M.A., Langmade, S.J., Chen, X., Fronick, C.C., Sawyer, C.S., Burcea, L.C., Wilkinson, M.N., Fulton, R.S., Heinz, M., Buchser, W.J., et al. (2020). Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric reverse-transcription loop-mediated isothermal amplification. Clin Chem. 32. Lauer, S.A., Grantz, K.H., Bi, Q., Jones, F.K., Zheng, Q., Meredith, H.R., Azman, A.S., Reich, N.G., and Lessler, J. (2020). The Incubation Period of Coronavirus Disease 2019 (COVID-19) From Publicly Reported Confirmed Cases: Estimation and Application. Ann Intern Med 172, 577-582. 33. Lee, D., La Mura, M., Allnutt, T.R., and Powell, W. (2009). Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences. BMC Biotechnol 9, 7. 34. Long, Q.X., Tang, X.J., Shi, Q.L., Li, Q., Deng, H.J., Yuan, J., Hu, J.L., Xu, W., Zhang, Y., Lv, F.J., et al. (2020). Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections. Nat Med 26, 1200-1204. 35. Lu, R., Zhao, X., Li, J., Niu, P., Yang, B., Wu, H., Wang, W., Song, H., Huang, B., Zhu, N., et al. (2020). Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 395, 565-574. 36. Ma, C., Wang, F., Wang, X., Han, L., Jing, H., Zhang, H., and Shi, C. (2017). A novel method to control carryover contamination in isothermal nucleic acid amplification. Chem Commun (Camb) 53, 10696-10699. 37. Ma, Y., Wu, L., Shaw, N., Gao, Y., Wang, J., Sun, Y., Lou, Z., Yan, L., Zhang, R., and Rao, Z. (2015). Structural basis and functional analysis of the SARS coronavirus nsp14-nsp10 complex. Proc Natl Acad Sci U S A 112, 9436-9441. 38. Masters, P.S. (2006). The molecular biology of coronaviruses. Adv Virus Res 66, 193-292. 39. Mayboroda, O., Katakis, I., and O'Sullivan, C.K. (2018). Multiplexed isothermal nucleic acid amplification. Anal Biochem 545, 20-30. 40. McBride, R., van Zyl, M., and Fielding, B.C. (2014). The Coronavirus Nucleocapsid Is a Multifunctional Protein. Viruses-Basel 6, 2991-3018. 41. Mohon, A.N., Oberding, L., Hundt, J., van Marle, G., Pabbaraju, K., Berenger, B.M., Lisboa, L., Griener, T., Czub, M., Doolan, C., et al. (2020). Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2. J Virol Methods 286, 113972. 42. Mori, Y., Kitao, M., Tomita, N., and Notomi, T. (2004). Real-time turbidimetry of LAMP reaction for quantifying template DNA. J Biochem Biophys Methods 59, 145-157. 43. Mori, Y., Nagamine, K., Tomita, N., and Notomi, T. (2001). Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 289, 150-154. 44. Munster, V.J., Koopmans, M., van Doremalen, N., van Riel, D., and de Wit, E. (2020). A Novel Coronavirus Emerging in China - Key Questions for Impact Assessment. N Engl J Med 382, 692-694. 45. Nagamine, K., Hase, T., and Notomi, T. (2002). Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16, 223-229. 46. Nguyen, T., Duong Bang, D., and Wolff, A. (2020). 2019 Novel Coronavirus Disease (COVID-19): Paving the Road for Rapid Detection and Point-of-Care Diagnostics. Micromachines (Basel) 11. 47. Niessen, L., Luo, J., Denschlag, C., and Vogel, R.F. (2013). The application of loop-mediated isothermal amplification (LAMP) in food testing for bacterial pathogens and fungal contaminants. Food Microbiol 36, 191-206. 48. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., and Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28, E63. 49. Ott, I.M., Strine, M.S., Watkins, A.E., Boot, M., Kalinich, C.C., Harden, C.A., Vogels, C.B.F., Casanovas-Massana, A., Moore, A.J., Muenker, M.C., et al. (2020). Simply saliva: stability of SARS-CoV-2 detection negates the need for expensive collection devices. medRxiv. 50. Pachetti, M., Marini, B., Benedetti, F., Giudici, F., Mauro, E., Storici, P., Masciovecchio, C., Angeletti, S., Ciccozzi, M., Gallo, R.C., et al. (2020). Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant. J Transl Med 18. 51. Pang, B., Xu, J., Liu, Y., Peng, H., Feng, W., Cao, Y., Wu, J., Xiao, H., Pabbaraju, K., Tipples, G., et al. (2020). Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology. Anal Chem 92, 16204-16212. 52. Pinto, D., Park, Y.J., Beltramello, M., Walls, A.C., Tortorici, M.A., Bianchi, S., Jaconi, S., Culap, K., Zatta, F., De Marco, A., et al. (2020). Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody. Nature 583, 290-295. 53. Poole, C.B., Tanner, N.A., Zhang, Y., Evans, T.C., Jr., and Carlow, C.K. (2012). Diagnosis of brugian filariasis by loop-mediated isothermal amplification. PLoS Negl Trop Dis 6, e1948. 54. Poon, L.L., Leung, C.S., Chan, K.H., Lee, J.H., Yuen, K.Y., Guan, Y., and Peiris, J.S. (2005). Detection of human influenza A viruses by loop-mediated isothermal amplification. J Clin Microbiol 43, 427-430. 55. Ranney, M.L., Griffeth, V., and Jha, A.K. (2020). Critical Supply Shortages - The Need for Ventilators and Personal Protective Equipment during the Covid-19 Pandemic. N Engl J Med 382, e41. 56. Satyanarayana, M. (2020). Shortage of RNA extraction kits hampers efforts to ramp up COVID-19 coronavirus testing (Chemical Engineering News). 57. Sawicki, S.G., Sawicki, D.L., and Siddell, S.G. (2007). A contemporary view of coronavirus transcription. J Virol 81, 20-29. 58. Schermer, B., Fabretti, F., Damagnez, M., Di Cristanziano, V., Heger, E., Arjune, S., Tanner, N.A., Imhof, T., Koch, M., Ladha, A., et al. (2020). Rapid SARS-CoV-2 testing in primary material based on a novel multiplex RT-LAMP assay. PLoS One 15, e0238612. 59. Slabodkin, G. (2020). FDA chief warns of supply ‘pressure’ on reagents for coronavirus tests. 60. Sola, I., Almazan, F., Zuniga, S., and Enjuanes, L. (2015). Continuous and Discontinuous RNA Synthesis in Coronaviruses. Annu Rev Virol 2, 265-288. 61. Taki, K., Yokota, I., Fukumoto, T., Iwasaki, S., Fujisawa, S., Takahashi, M., Negishi, S., Hayasaka, K., Sato, K., Oguri, S., et al. (2020). SARS-CoV-2 detection by fluorescence loop-mediated isothermal amplification with and without RNA extraction. J Infect Chemother. 62. Tanner, N.A., and Evans, T.C., Jr. (2014). Loop-mediated isothermal amplification for detection of nucleic acids. Curr Protoc Mol Biol 105, Unit 15 14. 63. Tanner, N.A., Zhang, Y., and Evans, T.C., Jr. (2015). Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes. Biotechniques 58, 59-68. 64. Tomita, N., Mori, Y., Kanda, H., and Notomi, T. (2008). Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3, 877-882. 65. van Dorp, L., Acman, M., Richard, D., Shaw, L.P., Ford, C.E., Ormond, L., Owen, C.J., Pang, J., Tan, C.C.S., Boshier, F.A.T., et al. (2020). Emergence of genomic diversity and recurrent mutations in SARS-CoV-2. Infect Genet Evol 83, 104351. 66. Wang, D.G., Brewster, J.D., Paul, M., and Tomasula, P.M. (2015). Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification. Molecules 20, 6048-6059. 67. Whiting, S.H., and Champoux, J.J. (1998). Properties of strand displacement synthesis by Moloney murine leukemia virus reverse transcriptase: mechanistic implications. J Mol Biol 278, 559-577. 68. WHO (2020). Laboratory testing for coronavirus disease (COVID-19) in suspected human cases. WHO/COVID-19/laboratory/2020.2025. 69. WHO (2021). WHO Coronavirus Disease (COVID-19) Dashboard. 70. Wise, J. (2020). Covid-19: New coronavirus variant is identified in UK. BMJ 371, m4857. 71. Woo, P.C., Lau, S.K., Lam, C.S., Lau, C.C., Tsang, A.K., Lau, J.H., Bai, R., Teng, J.L., Tsang, C.C., Wang, M., et al. (2012). Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J Virol 86, 3995-4008. 72. Wrapp, D., Wang, N., Corbett, K.S., Goldsmith, J.A., Hsieh, C.L., Abiona, O., Graham, B.S., and McLellan, J.S. (2020). Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367, 1260-1263. 73. Wu, J., Li, J., Zhu, G., Zhang, Y., Bi, Z., Yu, Y., Huang, B., Fu, S., Tan, Y., Sun, J., et al. (2020a). Clinical Features of Maintenance Hemodialysis Patients with 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China. Clin J Am Soc Nephrol 15, 1139-1145. 74. Wu, J.T., Leung, K., and Leung, G.M. (2020b). Nowcasting and forecasting the potential domestic and international spread of the 2019-nCoV outbreak originating in Wuhan, China: a modelling study. Lancet 395, 689-697. 75. Xu, R., Cui, B., Duan, X., Zhang, P., Zhou, X., and Yuan, Q. (2020). Saliva: potential diagnostic value and transmission of 2019-nCoV. Int J Oral Sci 12, 11. 76. Yan, R., Zhang, Y., Li, Y., Xia, L., Guo, Y., and Zhou, Q. (2020). Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 367, 1444-1448. 77. Zhang, X., Lowe, S.B., and Gooding, J.J. (2014). Brief review of monitoring methods for loop-mediated isothermal amplification (LAMP). Biosens Bioelectron 61, 491-499. 78. Zhang, Y., Odiwuor, N., Xiong, J., Sun, L., Nyaruaba, R.O., Wei, H., and Tanner, N.A. (2020). Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP. medRxiv, 2020.2002.2026.20028373. 79. Zhou, P., Yang, X.L., Wang, X.G., Hu, B., Zhang, L., Zhang, W., Si, H.R., Zhu, Y., Li, B., Huang, C.L., et al. (2020). A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273. 80. Zhu, N., Zhang, D., Wang, W., Li, X., Yang, B., Song, J., Zhao, X., Huang, B., Shi, W., Lu, R., et al. (2020). A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med 382, 727-733. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51997 | - |
dc.description.abstract | 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)。 | zh_TW |
dc.description.abstract | 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. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T14:02:04Z (GMT). No. of bitstreams: 1 U0001-0502202114375400.pdf: 4116549 bytes, checksum: 5344b6e3910067bbe103412c141294c6 (MD5) Previous issue date: 2021 | en |
dc.description.tableofcontents | Table of Contents
誌謝 i 中文摘要 ii Abstract iv Table of Contents vi List of Figures ix List of Tables xi Chapter 1 : Introduction 1 1.1 Overview of the principle of Loop-mediated Isothermal Amplification (LAMP) 1 1.2 Mechanism of Loop-mediated Isothermal Amplification (LAMP) 5 1.3 Introductions to SARS-CoV-2 7 1.4 Introduction clinical workflow from sample collection to the analysis 12 1.5 The urgent demand of an alternative nucleic acid tests at point of care during SARS-CoV-2 outbreak 13 1.6 The LAMP/RT-LAMP assay for point-of-care testing 17 1.7 Specific aim 21 Chapter 2 : Materials and Methods 22 2.1 Materials: 22 2.1.1 Cells 22 2.1.2 Competent cells 22 2.1.3 Primer 22 2.1.4 Plasmid 22 2.1.5 Reagent 23 2.1.6 Commercial kit 23 2.2 Methods 24 2.2.1 RT-LAMP primer design 24 2.2.2 RT-LAMP assay 25 2.2.3 RNA extraction by TRIzol 26 2.2.4 RNA extraction by GeneaidTM viral nucleic acid extraction kit 27 2.2.5 PCR amplification and sub-cloning 28 2.2.6 In vitro transcription with T7 RNA polymerase 29 2.2.7 Two step qRT-PCR 30 2.2.8 One step qRT-PCR 31 Chapter 3 : Results 32 3.1 LAMP primer design and validation 32 3.2 The range of amplification temperature of the RT-LAMP assay 35 3.3 Specificity of the SARS-CoV-2 N-13 primer set 36 3.4 Sensitivity evaluation of the RT-LAMP assay with N-13 primer by the synthetic DNA and RNA samples 38 3.5 Validation of RT-LAMP assay by RNA from SARS-CoV-2 cell culture samples 41 3.6 QCMD blind test of in-house RT-LAMP assay 42 3.7 Validation of internal control primers targeting host genes 44 3.8 Validation of the performance of RT-LAMP assay under the background of human RNA 45 Chapter 4 : Discussion 46 4.1 Point of care test is critical for controlling the SARS-CoV-2 pandemic 46 4.2 Colorimetric RT-LAMP assay has shown high potentials in point-of-care test 46 4.3 Difficulties and limitation of RT-LAMP to be improved 48 4.3.1 Aerosol transmission contamination 48 4.3.2 The selection of pH indicator 49 4.3.3 Mutations accumulated in the SARS-CoV-2 genome over time may affect primer efficiency 49 Chapter 5 : Reference 50 Appendix 87 List of Figures Figure 1. LAMP amplification mechanism 59 Figure 2. Discontinuous transcription of SARS-CoV-2 subgenomic mRNA 61 Figure 3. Alignment of the N gene sequences of seven coronaviruses 62 Figure 4. Alignment of N gene from SARS-CoV-2 isolate 64 Figure 5. N-1, N-13 and N-17 primers target regions in the SARS-CoV-2 N gene 65 Figure 6. N-1 and N-13 primers could successfully amplify the N gene cDNA in the colorimetric LAMP assay. 66 Figure 7. N-13 primer set amplified the SARS-CoV-2 N gene at 57.5 -70°C 67 Figure 8. N-13 primer set could not amplify the SARS-CoV-2 N gene at 55°C 68 Figure 9. N-13 primer sets specifically amplified N gene of SARS-CoV-2 but not HCoV-229E nor HCoV-OC43 69 Figure 10. N-13 primer sets specifically amplified N gene of SARS-CoV-2 but not SARS-CoV nor MERS-CoV 70 Figure 11. The respiratory disease-causing viruses were not detected by N-13 primer 71 Figure 12. Sensitivity evaluation of the LAMP assay in detecting commercially available N gene cDNA 72 Figure 13. Two-step qPCR result of Figure 12 73 Figure 14. In vitro transcribed SARS-CoV-2 N gene RNA was validated by electrophoresis in agarose gel 74 Figure 15. RT-LAMP assay with N-primer set was able to detect down to 10-10 dilution of RNA per reaction 75 Figure 16. One-step qPCR result of Figure 15 76 Figure 17. RT-LAMP assay was able to detect down to 0.02 fg RNA per reaction 77 Figure 18. RT-LAMP assay with the N-13 primer set has a detection limit in 20 fg 78 Figure 19. RT-LAMP assay could detect sample whose Ct value is below 33 79 Figure 20. QCMD samples were detected by RT-LAMP 81 Figure 21. Validation of internal control primers targeting host genes 82 Figure 22. Validation of the performance of RT-LAMP assay under the background of human RNA 83 List of Tables Table 1. Primers used in (RT-)LAMP 84 Table 2. Primers used in cloning 85 Table 3. Primers used in qRT-PCR 85 Table 4. The RT-LAMP and qPCR result of Figure 19 86 | |
dc.language.iso | zh-TW | |
dc.title | 以反轉錄恆溫環型核酸擴增法建立新型冠狀病毒之檢測 | zh_TW |
dc.title | Development of RT-LAMP For SARS-CoV-2 Detection | en |
dc.type | Thesis | |
dc.date.schoolyear | 109-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張淑媛(Sui-Yuan Chang),盧彥文(Yen-Wen Lu) | |
dc.subject.keyword | 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, | zh_TW |
dc.subject.keyword | Reverse Transcription Loop-Mediated Isothermal Amplification,Severe acute respiratory syndrome coronavirus 2,point-of-care testing,colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification,Nucleoprotein, | en |
dc.relation.page | 87 | |
dc.identifier.doi | 10.6342/NTU202100585 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2021-02-08 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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