發(fā)布時(shí)間：2018-01-01 14:14

  本文關(guān)鍵詞：麻疹病毒核酸等溫?cái)U(kuò)增試紙條快速檢測(cè)技術(shù)的研究與應(yīng)用　出處：《浙江理工大學(xué)》2016年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 麻疹病毒 快速檢測(cè) 逆轉(zhuǎn)錄環(huán)介導(dǎo)等溫?cái)U(kuò)增 橫向流動(dòng)試紙條

【摘要】：麻疹是由麻疹病毒引起的以發(fā)熱、出疹為特征的急性呼吸道傳染病,該病傳染性強(qiáng),致死率高,是目前導(dǎo)致發(fā)展中國(guó)家兒童死亡的重要原因。20世紀(jì)60年代以來(lái),麻疹疫苗開(kāi)始廣泛使用,使麻疹的發(fā)病率、死亡率大幅度降低,但據(jù)世界衛(wèi)生組織(World Health Organization,WHO)統(tǒng)計(jì),全球每年仍有超過(guò)2千萬(wàn)麻疹病例。WHO制定的全球控制與消除麻疹計(jì)劃中,麻疹的實(shí)驗(yàn)室檢測(cè)是麻疹監(jiān)測(cè)的重要組成部分。本研究將逆轉(zhuǎn)錄環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)(Reverse-transcriptase loop-mediated isothermal amplification,RT-LAMP)與帶防污染裝置的橫向流動(dòng)試紙條檢測(cè)技術(shù)(lateral flow dipstick,LFD)相結(jié)合,建立了一種麻疹病毒核酸快速檢測(cè)的新方法。研究針對(duì)麻疹病毒血凝素基因設(shè)計(jì)6條RT-LAMP擴(kuò)增引物與1條特異性檢測(cè)探針,其中上游內(nèi)引物與探針的5’端分別標(biāo)記生物素(biotin)與異硫氰酸熒光素(Fluorescein isothiocyanate,FITC),進(jìn)行帶biotin標(biāo)記的RT-LAMP反應(yīng),同時(shí)進(jìn)行與FITC-探針的雜交,最后利用LFD對(duì)等溫?cái)U(kuò)增產(chǎn)物完成可視化核酸試紙條檢測(cè)。通過(guò)對(duì)檢測(cè)體系的優(yōu)化,建立的麻疹病毒RT-LAMP-LFD快速檢測(cè)技術(shù)能在1 h內(nèi)完成對(duì)樣本的檢測(cè)(包括核酸提取),方法檢測(cè)敏感性與熒光RT-PCR方法相同,達(dá)到10個(gè)RNA copies/μL,同時(shí)與其他常見(jiàn)呼吸道傳染病病毒無(wú)交叉反應(yīng),具有良好的檢測(cè)特異性。收集494份疑似麻疹患者咽拭子標(biāo)本,用本研究建立的RT-LAMP-LFD快速方法進(jìn)行檢測(cè),同時(shí)與熒光RT-PCR法進(jìn)行比較,結(jié)果顯示兩種方法對(duì)494份臨床樣本的檢測(cè)結(jié)果無(wú)顯著性差異。結(jié)果表明,本研究研發(fā)的麻疹病毒核酸RT-LAMP-LFD檢測(cè)技術(shù)對(duì)疑似麻疹患者臨床樣本的檢測(cè)速度快,而且靈敏度高、特異性好、操作簡(jiǎn)單、儀器依賴程度低,有望發(fā)展成為麻疹病毒核酸快速檢測(cè)的有效手段。
[Abstract]:Measles is an acute respiratory infectious disease caused by measles virus, characterized by fever and rash. It is an important cause of child mortality in developing countries. Since 60s of the 20th century, measles vaccine has been widely used, which has greatly reduced the incidence and mortality of measles. But according to WHO World Health Organization. There are still more than 20 million measles cases in the world every year. Laboratory detection of measles is an important part of measles surveillance. Reverse-transcriptase loop-mediated isothermal amplification. RT-LAMP) was combined with the lateral flow stickle-LFDs with anti-pollution devices. A new method for rapid detection of measles virus nucleic acid was established. Six RT-LAMP amplified primers and one specific probe were designed for measles virus hemagglutinin gene. The upstream primer and probe were labeled with biotin and Fluorescein isothiocyanate, respectively. Biotin labeled RT-LAMP reaction and hybridization with FITC- probe were performed. Finally, LFD was used to detect the isothermal products of nucleic acid strips. The detection system was optimized. The established rapid detection technique for measles virus RT-LAMP-LFD can complete the detection of samples within 1 h (including nucleic acid extraction). The sensitivity of the method is the same as that of fluorescent RT-PCR method. It reached 10 RNA copias / 渭 L, and had no cross reaction with other common respiratory infectious diseases virus, and had good detection specificity. 494 specimens of throat swabs of suspected measles patients were collected. The rapid RT-LAMP-LFD method was established in this study and compared with the fluorescent RT-PCR method. The results showed that there was no significant difference between the two methods in 494 clinical samples. The measles virus nucleic acid RT-LAMP-LFD detection technology developed in this study for suspected measles patients clinical samples detection speed, and high sensitivity, specificity, simple operation, low degree of instrument dependence. It is expected to be an effective method for rapid detection of measles virus nucleic acid.
【學(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R440;R511.1

【相似文獻(xiàn)】

相關(guān)期刊論文 前5條

1 馬麗敏;盧亦愚;徐昌平;馮燕;;依賴解旋酶恒溫?cái)U(kuò)增技術(shù)快速檢測(cè)麻疹病毒核酸[J];中國(guó)疫苗和免疫;2012年06期

2 姜明杰;甘云;梁勇;張振國(guó);盧國(guó)際;王翠華;李彩云;王蔣麗;;熒光定量-聚合酶鏈反應(yīng)法快速檢測(cè)麻疹病毒核酸[J];中國(guó)衛(wèi)生檢驗(yàn)雜志;2007年04期

3 盧亦愚,嚴(yán)菊英,馮燕,史雯,茅海燕;實(shí)時(shí)熒光定量PCR技術(shù)快速檢測(cè)麻疹病毒核酸的研究[J];浙江預(yù)防醫(yī)學(xué);2004年11期

4 盧亦愚,嚴(yán)菊英,馮燕,史雯,茅海燕;Taq Man熒光定量逆轉(zhuǎn)錄聚合酶鏈反應(yīng)快速檢測(cè)麻疹病毒核酸[J];中國(guó)計(jì)劃免疫;2005年01期

5 ;[J];;年期

相關(guān)碩士學(xué)位論文 前1條

1 徐昌平;麻疹病毒核酸等溫?cái)U(kuò)增試紙條快速檢測(cè)技術(shù)的研究與應(yīng)用[D];浙江理工大學(xué);2016年

，

本文編號(hào)：1364851