本文關鍵詞：流感病毒檢測新技術的建立及初步應用研究　出處：《中國疾病預防控制中心》2010年碩士論文　論文類型：學位論文

  更多相關文章： 新甲型H1N1流感病毒 多重RT-PCR GeXP system 實時定量RT-PCR 環(huán)介導的逆轉錄等溫擴增

【摘要】： 目的：建立和評價兩種基于實驗室的同時檢測新甲型流感病毒和季節(jié)性流感病毒的多重RT-PCR方法；建立和評價一種基于現場的快速診斷新甲型H1N1流感病毒的等溫擴增技術。 方法：(一)利用GeXP system建立多重RT-PCR方法1.從NCBI網站的GenBank數據庫中下載所有甲型流感病毒的Matrix(M)基因、所有甲型流感病毒H1N1亞型的血凝素(HA)基因、季節(jié)性H1N1流感病毒多的血凝素(HA)基因、季節(jié)性H3N2流感病毒的血凝素(HA)基因、2009年流行的新甲型H1N1流感病毒的血凝素(HA)基因的核苷酸序列,利用GeXP eXpress Profiler分別設計針對上述各基因的特異性引物。利用人的RNaseP基因做為內參判斷標本來源和實施質量控制的依據；同時利用Kan(?)RNA做為內參判斷擴增體系是否正常。2.建立8重RT-PCR方法同時檢測新甲型流感病毒和季節(jié)性流感病毒,對其進行方法學評價(包括靈敏度、特異性等),并與WHO推薦的rRT-PCR鑒定結果進行比較。(二)利用CFX96real-time system建立4重rRT-PCR方法1.從NCBI網站的GenBank數據庫中下載季節(jié)性H1N1流感病毒的血凝素(HA)基因、季節(jié)性H3N2流感病毒的血凝素(HA)基因、2009年流行的新甲型H1N1流感病毒的血凝素(HA)基因的核苷酸序列,利用Primer Premier5分別設計針對上述各基因的特異性引物和探針。利用人的RNaseP基因做為內參判斷標本來源和實施質量控制的依據。2.建立4重rRT-PCR方法檢測新甲型流感病毒和季節(jié)性流感病毒,對其進行方法學(包括特異性、靈敏度等)評價,并與WHO推薦的rRT-PCR方法的鑒定結果進行比較。(三)建立環(huán)介導的逆轉錄等溫擴增技術1、選擇人甲型H1N1流感HA基因(GenBank:FJ966974.1)設計相應的RT-LAMP引物。2、建立RT-LAMP特異性檢測新甲型流感病毒的方法,對其進行方法學(包括特異性、靈敏度等)評價,WHO推薦的rRT-PCR方法的鑒定結果進行比較。 結果：1.利用GeXP system建立的8重RT-PCR方法檢測新甲型流感病毒的靈敏度達到10copies RNA。通過對不同來源的流感病毒的驗證,結果表明每對引物均未見交叉反應,特異性較好。通過對29份新甲型流感疑似患者的咽拭子標本進行檢測,GeXP多重RT-PCR的陽性率為97%,明顯高于WHO推薦的rRT-PCR方法(76%)(P0.05)。2.利用CFX96建立的4重rRT-PCR方法檢測新甲型流感病毒的靈敏度達到20copies RNA。通過對不同來源的流感病毒的驗證,結果表明每對引物均未見交叉反應,特異性較好。通過對29份新甲型流感疑似患者的咽拭子標本進行檢測,CFX96的多重rRT-PCR陽性率為93%,高于WHO推薦的rRT-PCR方法(76%)(P0.05)。3.利用RT-LAMP檢測新甲型流感病毒的靈敏度達到60copies RNA。通過對不同來源的流感病毒的驗證,結果表明此法特異性高。通過對29份新甲型流感疑似患者的咽拭子標本進行檢測,RT-LAMP陽性率為90%,高于WHO推薦的rRT-PCR方法(76%)(P0.05)。 結論：成功建立兩種多重RT-PCR同時檢測新甲型流感病毒和季節(jié)性流感病毒的新技術和一種基于現場的快速準確易推廣的檢測新甲型流感病毒的等溫擴增技術,有助于提高我國流感監(jiān)測網絡實驗室的檢測能力。
[Abstract]:Objective: to establish and evaluate two laboratory based multiple RT-PCR methods for simultaneous detection of influenza A and seasonal influenza viruses, and establish and evaluate a field based rapid isothermal amplification technology for diagnosing influenza A (H1N1) virus.
Methods: (a) using GeXP system to establish the multiplex RT-PCR 1. Download all influenza A virus from the website of NCBI GenBank database in Matrix (M) gene, all H1N1 influenza virus hemagglutinin (HA) gene, seasonal H1N1 influenza virus hemagglutinin (HA) gene, H3N2 seasonal influenza virus the hemagglutinin (HA) gene, the 2009 pandemic H1N1 influenza A virus hemagglutinin (HA) gene nucleotide sequence, using GeXP eXpress Profiler specific primers were designed for each of the above genes. Identify the source of the specimen and the implementation of quality control for reference by the human RNaseP gene; at the same time using Kan (?) RNA as a reference to judge whether the normal.2. amplification system established 8 new RT-PCR for simultaneous detection of influenza A virus and seasonal influenza virus, on the methodological evaluation (including sensitivity, specificity, etc.), Compared with WHO and rRT-PCR identification results. (two) recommended by CFX96real-time system 4 rRT-PCR method 1. download H1N1 seasonal influenza virus from the website of NCBI GenBank database (HA) of the hemagglutinin gene, H3N2 seasonal influenza virus hemagglutinin (HA) gene, the 2009 pandemic H1N1 influenza A virus the hemagglutinin (HA) gene nucleotide sequence, using Primer Premier5 were designed according to the gene specific primers and probes. As a reference to judge the source of the specimen and the implementation of quality control based on.2. model is 4 rRT-PCR for the detection of new influenza virus and seasonal influenza virus RNaseP gene by the method of (including its specificity, sensitivity evaluation, etc.) the identification results are compared with rRT-PCR method and recommended by WHO. (three) the establishment of reverse transcription loop mediated isothermal amplification technology 1, choice The influenza A (H1N1) HA gene (GenBank:FJ966974.1) is designed with the corresponding RT-LAMP primer.2, and the method of RT-LAMP specific detection of influenza A virus is established. Its methodology (including specificity, sensitivity, etc.) is evaluated, and the results of rRT-PCR recommended by WHO are compared.
Results: the sensitivity of 1. by 8 RT-PCR method of GeXP system to establish new detection of influenza A virus to 10copies RNA. through verification of different sources of influenza virus, results show that each pair of primers showed no cross reactivity, good specificity. Through testing of 29 samples of the new swine flu suspected patients with throat swab specimens the positive rate of GeXP multiplex RT-PCR was 97%, significantly higher than that of rRT-PCR method recommended by WHO (76%) (P0.05).2. detection sensitivity of new influenza virus using 4 rRT-PCR method established by CFX96 reached 20copies RNA. through verification of different sources of influenza virus, results show that each pair of primers showed no cross reaction specificity. 29. Based on a new influenza A patients with suspected throat swab samples were detected, the positive rate of multiple rRT-PCR CFX96 was 93%, higher than the rRT-PCR method recommended by WHO (76%) (P0.05).3. by RT-LAMP The detection sensitivity of the new influenza virus reached 60copies RNA. through the verification of different sources of influenza virus, the results show that this method has high specificity. The 29 new suspected influenza patients with throat swab specimens were detected, the positive rate of RT-LAMP was 90%, higher than the rRT-PCR method recommended by WHO (76%) (P0.05).
Conclusion: the new technology successfully established two kinds of isothermal RT-PCR multiple simultaneous detection of new influenza virus and seasonal influenza virus and a live fast and accurate easy detection based on the new influenza virus amplification technology, help to improve the detection ability of laboratory influenza surveillance network in China.

【學位授予單位】：中國疾病預防控制中心
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R373

【參考文獻】

相關期刊論文 前5條

1 李啟明;馬學軍;周蕊;彭夫望;高寒春;匡治州;侯云德;;環(huán)介導逆轉錄等溫擴增技術(RT-LAMP)在丙型肝炎病毒基因檢測中的應用[J];病毒學報;2006年05期

2 李啟明;馬學軍;高寒春;周蕊;匡治州;侯云德;;逆轉錄環(huán)介導等溫核酸擴增技術(RT-LAMP)在H5N1禽流感病毒基因檢測中的應用[J];病毒學報;2008年03期

3 王大燕;高榮保;李曉丹;王偉;溫樂英;鄒淑梅;藍雨;楊磊;郭俊峰;李希研;趙翔;李梓;曾玉紅;成艷輝;譚敏菊;李鑫婉;郭元吉;李德新;舒躍龍;;甲型H1N1流感病毒快速核酸檢測技術的建立[J];病毒學報;2009年S1期

4 秦萌;王大燕;黃芳;聶凱;曲梅;王淼;韓峰;趙翔;成艷輝;舒躍龍;馬學軍;;同時檢測新甲型H1N1及人季節(jié)性H1N1和H3N2流感病毒的單管多重熒光定量PCR方法[J];病毒學報;2010年02期

5 魏民,梁浩,陳健平,陳釗,關琪,邢輝,馮毅,洪坤學,邵一鳴;使用多重巢式PCR對我國HIV-1主要流行株亞型鑒定方法的建立[J];中華實驗和臨床病毒學雜志;2004年01期

，

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