以反轉錄-迴路媒介恆溫增殖法應用於登革病毒的快速檢測

Rapid Detection of Dengue Virus by Reverse-Transcription Loop-Mediated Isothermal Amplification Assay

鄭千玲(Chien-Ling Cheng)

指導教授 ： 高全良老師

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.01058

摘要

登革病毒屬於黃病毒科（Flaviviridae），依其抗原性不同，可分為四種血清型，即為第一、第二、第三與第四型登革病毒（DENV-1、DENV-2、DENV-3、DENV-4），其媒介是白線斑蚊（Aedes albopictus）及埃及斑蚊（Aedes aegypti）。臨床症狀多元化，可見不顯性症狀、典型登革熱及登革出血熱或登革休克症候群；實驗室常用於登革病毒的測定方法有傳統病毒分離方法、血清學診斷、病毒抗原測定法以及病毒核酸檢測法等，以上四種方法各有其優點及缺點，但均需特殊價昂的研究設備。
迴路媒介恆溫增殖法（Loop-mediated amplification method）為一種快速增殖DNA的方法，其原理是依靠六條辨認八段不同序列的引子和具有strands displacement特性的聚合酵素（Bst DNA polymerase large fragment），經由反覆環狀迴路（loop）的形成，在單一溫度下，可於一小時內快速、連續放大目標序列。由於登革病毒為正股RNA病毒，故加入較耐高溫的反轉錄酵素（AMV Reverse Transcriptase），在恆溫下同時做反轉錄和核酸放大的動作，縮短偵測的反應時間以期達快速檢測的目標。本研究嘗試運用此原理做為登革病毒偵測核酸存在及分型的方法，分別在四型登革病毒3'端未轉譯區（3’-untranslated region）及外套膜區（envelope gene）設計偵測及分型的引子，在恆溫下，即一般水浴槽或加熱器下作用一個小時，再以電泳或是加入SYBR Green I螢光染劑，以肉眼或UV下觀察。結果顯示此方法不但快速且具高專一性，陽性反應只在相對應之引子對中出現，其敏感性最低可至10~0.1pfu/ml。首先以此方法分析32株曾經在台灣地區流行並經由實驗室培養鑑定其血清型的四型登革病毒與一臨床檢體S3119，結果顯示有良好的一致性。
進一步嘗試在此系統加入SYBR Green I螢光染劑，將其應用於即時聚合酶連鎖反應之偵測系統來定量病毒，初步結果顯示，除了可得一標準曲線之外，定量經超高速離心純化的病毒可得到與實際相近之值，或將之序列稀釋帶回標準曲線，亦具有高度相關性，顯示此定量系統的可行性。
最後將此系統應用於三十五個臨床血清檢體，除了與傳統偵測登革病毒核酸的方法比較其定性的結果，並與先前已發表的定量登革病毒方法來比較二者結果之相關性；結果顯示定性結果雖具有100%敏感性，但卻有少部分檢體有偽陽性結果產生，此問題可用限制酶切割來解決；另外，與先前發表的定量法結果相比較，有相當程度的相關性。因此，在後續研究中，希望能進一步改良迴路媒介恆溫增殖法偵測的專一性，期未來能用於登革病毒的快速檢測。

關鍵字

登革病毒 ； 病毒核酸檢測法 ； 迴路媒介恆溫增殖法

並列摘要

Dengue virus belongs to the family Flaviviridae. The virus has a positive single-stranded RNA genome, and has four serologically distinct serotypes (DENV-1, DENV-2, DENV-3, and DENV-4). A number of the genus aedes may act as a vector, including Aedes aegypti and A. albopictus. Dengue virus infection in humans causes a spectrum of illness ranging from inapparent or mild febrile illness to severe and fatal hemorrhagic disease.
Routine laboratory diagnosis of dengue virus infection is commonly based on virus isolation, serodiagnosis, viral antigen detection, and PCR-based detection. However, all of these methods have disadvantages such as time-consuming and tedious work, non-specific outcome or high-cost instrument for amplification. Owing to the problems associated with the current screening systems, the recent invention of loop-mediated isothermal amplification (LAMP) provides a new alternative for molecular diagnosis.
The isothermal nucleic acid amplification method has high specificity, efficiency and rapidity. It uses six kinds of primers to recognize eight different regions and Bst DNA polymerase with strand displacement activity to react at constant temperature. Since the reaction is completed under isothermal condition, on the hand we can obviate the need for technique-specific instruments, hence we reduce the cost of tests, and on the other the efficiency is extremely high because there is no time loss. Therefore, the LAMP assay has emerged as a powerful tool to facilitate point-of-care genetic testing for the field test.
In this study, we sought to establish a easy and rapid reverse transcription loop-mediated isothermal amplification（RT-LAMP）for detection of dengue virus RNA. Just like in a one-step RT-PCR, cDNA synthesis and follow-up amplification can be achieved in a single tube by incubating the viral RNA with a primer mixture in the presence of AMV reverse transcriptase and Bst DNA polymerase. The LAMP products were subjected to electrophoresis or observed with both naked eyes and under UV light after adding SYBR green I. The specificity and applicability of this method were evaluated, and the sensitivity of the assay was estimated to be 0.1pfu/ml to 10pfu/ml.
Using this method, 33 samples（32 Taiwan isolates, and 1 clinical specimen）were demonstrated first to show the good agreement with previous diagnostic results.
Further, we combined the property of SYBR green I and real-time PCR detection system for quantification of the viral load in LAMP detection system. The preliminary data showed that there was a linear correlation between the quantity and the reaction time to reach the threshold. To quantify the viral RNA of four serotypes, we draw standard curves between quantity after logarithm and time to reach the threshold among four serotypes, respectively. After that, using the viral particles obtained from ultrahigh speed centrifugation verifies the feasibility of this quantification system
Last, we examined 15 clinical samples. All the nested RT-PCR-positive samples were positive by RT-LAMP, but few samples may be false-positive results by RT-LAMP after confirmation by restriction enzyme digestion. We also compared the results of quantification with those of the published quantitative RT-PCR. It showed a linear correlation to a certain extent. Our results demonstrate that the RT-LAMP assay is a useful tool for rapid diagnosis of dengue virus.

並列關鍵字

Dengue virus ； loop-mediated isothermal amplification (LAMP)

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