應用恆溫環形核酸增幅法於致病原偵測之效益探討

本篇論文回顧了應用恆溫環型核酸增幅法(loop-mediated isothermal amplification, LAMP)於致病原檢測的文獻，並比較其與聚合酶鍊鎖反應(PCR)、RT-PCR、nested PCR及real-time PCR於致病原檢測的專一性及靈敏度。LAMP反應只需利用特殊設計的引子與核酸聚合酶在恆溫(60-65℃)的狀態下，即可快速地擴增出標的核酸。LAMP反應具有非常高的專一性，因為啟始反應需要兩對引子辨認模板核酸的六段序列，才能進行後續反應。另外，LAMP反應不需循環升降溫步驟，故不但所需時間較PCR短且只需簡單的設備例如水浴槽或乾浴槽即可操作。在LAMP反應過程中，會有大量副產物焦磷酸(pyrophosphate)產生，與鎂離子反應後產生白色的焦磷酸鎂(magnesium pyrophosphate)沈澱，可直接肉眼辨識混濁度或以簡單的濁度計測量。若加入螢光染劑如SYBR Green等，目測之靈敏度可與電泳凝膠法相當。自從西元2000年第一篇LAMP技術發表後，已有越來越多應用LAMP來檢測引起人類、動植物、魚類等疾病的病原菌或病毒的報告被發表。在回顧過論文中列出的這些文獻後，我結論出LAMP具有高度的專一性，且其靈敏度優於PCR並與RT-PCR或nested PCR或real-time PCR相當。

關鍵字

恆溫環形增幅；恆溫環型核酸增幅法；致病原偵測

並列摘要

This thesis reviewed the articles that applied loop-mediated isothermal amplification (LAMP) to the detection of pathogens, and compared the specificities and sensitivities of LAMP to PCR, reverse transcription PCR, nested PCR, as well as real-time PCR. The LAMP reaction requires a set of four specially designed primers and a DNA polymerase, under isothermal conditions (60-65℃), can rapidly amplify target DNA. The specificity of LAMP is extremely high because the set of four specially designed primers recognize six distinct target DNA sequences which play the role of initiating the follow-up reactions. Comparing to PCR, LAMP has short reaction time because it doesn’t need the thermal cycling steps. Furthermore, simple equipments such as a water bath or heat block are sufficient for the DNA amplification. LAMP reaction yields a large amount of the by-product pyrophosphate ion, which reacts with magnesium ions in the reaction to form white magnesium pyrophosphate precipitate. As a result, real-time monitoring of the LAMP reaction can be achieved by the naked eye or by real-time measurement of turbidity. In addition, the sensitivity of staining LAMP amplicons with fluorescent intercalating dye such as SYBR green equals that of gel electrophoresis method. Since the first report of LAMP in 2000, applications of LAMP for the detection of pathogens of human, animals, agriculture and fishing industry diseases were reported by more and more publications. After reviewing selected articles, I conclude that LAMP is highly specific and the sensitivity of LAMP is superior to PCR, and is comparable to nested PCR and real-time PCR.

並列關鍵字

loop-mediated isothermal ； LAMP ； pathogens detection

參考文獻

1. Mullis, K., F. Faloona, and S. Scharf. (1986). Specific enzymatic amplification of DNA in vitro. Cold Spring Harb. Symp. Quant. Biol. 51:263–273.

2. Notomi, T., H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe, N. Amino, and T. Hase. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28:E63.

3. Tang, Y. W., P. S. Mitchell, M. J. Espy, T. F. Smith, and D. H. Persing. (1999). Molecular diagnosis of herpes simplex virus infections in the central nervous system. J. Clin. Microbiol. 37:2127–2136.

4. Gyllensten, U. B., and H. A. Erlich. (1989). Ancient roots for polymorphism at the HLA-DQ alpha locus in primates. Proc. Natl. Acad. Sci. U S A. 86:9986–9990.

5. Boehm, C. D. (1989). Use of polymerase chain reaction for diagnosis of inherited disorders. Clin. Chem. 35:1843–1848.

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應用恆溫環形核酸增幅法於致病原偵測之效益探討

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