發(fā)布時(shí)間：2018-01-10 07:20

  本文關(guān)鍵詞：流感病毒檢測(cè)新技術(shù)的建立及初步應(yīng)用研究　出處：《中國(guó)疾病預(yù)防控制中心》2010年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 新甲型H1N1流感病毒 多重RT-PCR GeXP system 實(shí)時(shí)定量RT-PCR 環(huán)介導(dǎo)的逆轉(zhuǎn)錄等溫?cái)U(kuò)增

【摘要】： 目的：建立和評(píng)價(jià)兩種基于實(shí)驗(yàn)室的同時(shí)檢測(cè)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(píng)價(jià)一種基于現(xiàn)場(chǎng)的快速診斷新甲型H1N1流感病毒的等溫?cái)U(kuò)增技術(shù)。 方法：(一)利用GeXP system建立多重RT-PCR方法1.從NCBI網(wǎng)站的GenBank數(shù)據(jù)庫(kù)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(shè)計(jì)針對(duì)上述各基因的特異性引物。利用人的RNaseP基因做為內(nèi)參判斷標(biāo)本來(lái)源和實(shí)施質(zhì)量控制的依據(jù)；同時(shí)利用Kan(?)RNA做為內(nèi)參判斷擴(kuò)增體系是否正常。2.建立8重RT-PCR方法同時(shí)檢測(cè)新甲型流感病毒和季節(jié)性流感病毒,對(duì)其進(jìn)行方法學(xué)評(píng)價(jià)(包括靈敏度、特異性等),并與WHO推薦的rRT-PCR鑒定結(jié)果進(jìn)行比較。(二)利用CFX96real-time system建立4重rRT-PCR方法1.從NCBI網(wǎng)站的GenBank數(shù)據(jù)庫(kù)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(shè)計(jì)針對(duì)上述各基因的特異性引物和探針。利用人的RNaseP基因做為內(nèi)參判斷標(biāo)本來(lái)源和實(shí)施質(zhì)量控制的依據(jù)。2.建立4重rRT-PCR方法檢測(cè)新甲型流感病毒和季節(jié)性流感病毒,對(duì)其進(jìn)行方法學(xué)(包括特異性、靈敏度等)評(píng)價(jià),并與WHO推薦的rRT-PCR方法的鑒定結(jié)果進(jìn)行比較。(三)建立環(huán)介導(dǎo)的逆轉(zhuǎn)錄等溫?cái)U(kuò)增技術(shù)1、選擇人甲型H1N1流感HA基因(GenBank:FJ966974.1)設(shè)計(jì)相應(yīng)的RT-LAMP引物。2、建立RT-LAMP特異性檢測(cè)新甲型流感病毒的方法,對(duì)其進(jìn)行方法學(xué)(包括特異性、靈敏度等)評(píng)價(jià),WHO推薦的rRT-PCR方法的鑒定結(jié)果進(jìn)行比較。 結(jié)果：1.利用GeXP system建立的8重RT-PCR方法檢測(cè)新甲型流感病毒的靈敏度達(dá)到10copies RNA。通過(guò)對(duì)不同來(lái)源的流感病毒的驗(yàn)證,結(jié)果表明每對(duì)引物均未見(jiàn)交叉反應(yīng),特異性較好。通過(guò)對(duì)29份新甲型流感疑似患者的咽拭子標(biāo)本進(jìn)行檢測(cè),GeXP多重RT-PCR的陽(yáng)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(cè)新甲型流感病毒的靈敏度達(dá)到20copies RNA。通過(guò)對(duì)不同來(lái)源的流感病毒的驗(yàn)證,結(jié)果表明每對(duì)引物均未見(jiàn)交叉反應(yīng),特異性較好。通過(guò)對(duì)29份新甲型流感疑似患者的咽拭子標(biāo)本進(jìn)行檢測(cè),CFX96的多重rRT-PCR陽(yáng)性率為93%,高于WHO推薦的rRT-PCR方法(76%)(P0.05)。3.利用RT-LAMP檢測(cè)新甲型流感病毒的靈敏度達(dá)到60copies RNA。通過(guò)對(duì)不同來(lái)源的流感病毒的驗(yàn)證,結(jié)果表明此法特異性高。通過(guò)對(duì)29份新甲型流感疑似患者的咽拭子標(biāo)本進(jìn)行檢測(cè),RT-LAMP陽(yáng)性率為90%,高于WHO推薦的rRT-PCR方法(76%)(P0.05)。 結(jié)論：成功建立兩種多重RT-PCR同時(shí)檢測(cè)新甲型流感病毒和季節(jié)性流感病毒的新技術(shù)和一種基于現(xiàn)場(chǎng)的快速準(zhǔn)確易推廣的檢測(cè)新甲型流感病毒的等溫?cái)U(kuò)增技術(shù),有助于提高我國(guó)流感監(jiān)測(cè)網(wǎng)絡(luò)實(shí)驗(yàn)室的檢測(cè)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(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R373

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