等溫?cái)U(kuò)增技術(shù)檢測(cè)手足口病病原的研究
發(fā)布時(shí)間：2018-07-16 18:18
【摘要】：手足口病自2004年以來在國內(nèi)迅速傳播,于2008年引起疫情暴發(fā)并在全國范圍內(nèi)大規(guī)模流行。據(jù)衛(wèi)生部疫情數(shù)據(jù)顯示,自2009年以來全國手足口病年報(bào)告病例人數(shù)均為百萬以上,每年因該病死亡的兒童數(shù)以百計(jì)。手足口病已經(jīng)成為我國的一個(gè)重要的公共衛(wèi)生問題。手足口病是由多種腸道病毒引起的傳染病,人腸道病毒71型(HEV71)和柯薩奇病毒A組16型(CVA16)是手足口病的兩種主要病原。因此建立針對(duì)HEV71病毒和CVA16病毒的特異、快速、簡(jiǎn)便的檢測(cè)方法,并建立檢測(cè)引起手足口病的多種相關(guān)腸道病毒的實(shí)驗(yàn)方法對(duì)控制手足口病疫情具有重要意義。本文內(nèi)容分為三部分,第一部分是建立檢測(cè)HEV71病毒和CVA16病毒的逆轉(zhuǎn)錄環(huán)介導(dǎo)等溫?cái)U(kuò)增方法,對(duì)方法進(jìn)行試點(diǎn)評(píng)價(jià)；第二部分是建立無需核酸提取的直接檢測(cè)標(biāo)本中的HEV71病毒的逆轉(zhuǎn)錄環(huán)介導(dǎo)等溫?cái)U(kuò)增方法,對(duì)方法進(jìn)行試點(diǎn)評(píng)價(jià)；第三部分是建立檢測(cè)多種腸道病毒的逆轉(zhuǎn)錄等溫?cái)U(kuò)增方法,應(yīng)用于腸道病毒感染尤其是手足口病病原的廣譜檢測(cè)。 第一部分檢測(cè)HEV71病毒和CVA16病毒的逆轉(zhuǎn)錄環(huán)介導(dǎo)等溫?cái)U(kuò)增方法的建立和評(píng)價(jià) 建立了應(yīng)用于HEV71病毒C4亞型和CVA16病毒的逆轉(zhuǎn)錄環(huán)介導(dǎo)等溫?cái)U(kuò)增方法,方法靈敏度高,反應(yīng)速度快。檢測(cè)前在反應(yīng)體系添加了羥基萘酚藍(lán)(HNB)指示劑,在65℃等溫條件下反應(yīng)60分鐘,實(shí)驗(yàn)結(jié)果可以通過肉眼觀察反應(yīng)溶液混濁度是否增加或者HNB指示劑顏色是否由紫色變?yōu)樘焖{(lán)色來進(jìn)行判讀。使用十倍等比梯度稀釋的病毒溶液對(duì)檢測(cè)方法的靈敏度進(jìn)行測(cè)試,實(shí)驗(yàn)結(jié)果顯示檢測(cè)方法對(duì)HEV71病毒的檢測(cè)限為0.33TCID50,對(duì)CVA16病毒的檢測(cè)限為1.58TICD50。使用了多個(gè)血清型的腸道病毒對(duì)等溫?cái)U(kuò)增檢測(cè)方法進(jìn)行特異性測(cè)試,實(shí)驗(yàn)結(jié)果顯示該檢測(cè)方法特異性高,與其它型別腸道病毒均無交叉反應(yīng),包括柯薩奇病毒A組2、4、5、7、9、10、14、24型,柯薩奇病毒B組1、2、3、4、5型和�？刹《�3、6、11、19型。使用47份手足口病病人的糞便標(biāo)本對(duì)該檢測(cè)方法進(jìn)行了實(shí)驗(yàn)室評(píng)價(jià),標(biāo)本均經(jīng)過病毒分離或是中和滴定實(shí)驗(yàn)鑒定其感染的腸道病毒類型,檢測(cè)結(jié)果顯示本研究建立的逆轉(zhuǎn)錄環(huán)介導(dǎo)等溫?cái)U(kuò)增方法能有效檢出臨床標(biāo)本中的HEV71病毒和CVA16病毒,在檢測(cè)反應(yīng)中應(yīng)用HNB染料作為指示劑使得可視化結(jié)果判讀方式更為有效。 在對(duì)RT-LAMP檢測(cè)方法進(jìn)行現(xiàn)場(chǎng)試點(diǎn)評(píng)估時(shí)使用了商業(yè)化的等溫?cái)U(kuò)增試劑盒,檢測(cè)反應(yīng)條件進(jìn)行相應(yīng)優(yōu)化,其中檢測(cè)HEV71病毒的反應(yīng)條件優(yōu)化為65℃等溫條件下反應(yīng)45分鐘,檢測(cè)CVA16病毒的反應(yīng)條件優(yōu)化為65℃等溫條件下反應(yīng)35分鐘。使用商業(yè)化等溫?cái)U(kuò)增試劑盒后RT-LAMP檢測(cè)反應(yīng)的靈敏度和特異性進(jìn)一步研究結(jié)果顯示：檢測(cè)方法靈敏度有一定程度提高,方法對(duì)HEV71病毒和CVA16病毒的檢測(cè)限均為0.1TCID50,同時(shí)檢測(cè)方法的特異性無變化。試點(diǎn)評(píng)估的515份手足口病病人臨床標(biāo)本于湖南省2011年手足口病疫情暴發(fā)期間收集,所有標(biāo)本同時(shí)進(jìn)行了實(shí)時(shí)熒光定量PCR檢測(cè)和RT-LAMP檢測(cè)。檢測(cè)實(shí)驗(yàn)結(jié)果顯示這兩個(gè)方法的一致性為99.6%(513/515),檢測(cè)結(jié)果不一致的2個(gè)標(biāo)本通過巢式逆轉(zhuǎn)錄PCR進(jìn)一步驗(yàn)證為CVA16病毒。上述實(shí)驗(yàn)數(shù)據(jù)提示檢測(cè)手足口病主要病原體HEV71病毒和CVA16病毒的RT-LAMP檢測(cè)方法具有特異性好、靈敏度高等優(yōu)點(diǎn),能快速、可視化的檢測(cè)上述兩種手足口病主要病原,具有較好的應(yīng)用前景,尤其適用于硬件設(shè)施有限的城鄉(xiāng)醫(yī)院、基層衛(wèi)生機(jī)構(gòu)使用。 第二部分直接檢測(cè)人腸道病毒71型環(huán)介導(dǎo)逆轉(zhuǎn)錄等溫?cái)U(kuò)增方法建立和評(píng)價(jià) 建立了一種直接檢測(cè)手足口病咽拭子標(biāo)本中人腸道病毒71型的逆轉(zhuǎn)錄環(huán)介導(dǎo)等溫?cái)U(kuò)增方法,該方法對(duì)樣品采用熱裂解方案進(jìn)行預(yù)處理而無需核酸提取便可對(duì)咽拭子標(biāo)本進(jìn)行RT-LAMP直接檢測(cè)。對(duì)采用熱裂解方案的Direct RT-LAMP方法的靈敏度和特異性進(jìn)行實(shí)驗(yàn)室驗(yàn)證,結(jié)果顯示檢測(cè)方法對(duì)HEV71型病毒的檢測(cè)限為1.6TCID50,特異性強(qiáng),與其它型別的腸道病毒諸如柯薩奇病毒A組2、4、5、7、9、10、14、24型,柯薩奇病毒B組1、2、3、4、5型和�？刹《�3、6、11、19型等均無交叉反應(yīng)。在試點(diǎn)評(píng)價(jià)中同時(shí)使用三種方法對(duì)145份手足口病患者的咽拭子臨床標(biāo)本進(jìn)行檢測(cè),這三種方法分別是不經(jīng)過標(biāo)本核酸提取而對(duì)標(biāo)本直接進(jìn)行檢測(cè)的Direct RT-LAMP方法,以及提取標(biāo)本核酸后再進(jìn)行檢測(cè)的RT-LAMP方法和實(shí)時(shí)熒光定量PCR方法。結(jié)果顯示采用熱裂解方案的Direct RT-LAMP方法與提取標(biāo)本核酸后進(jìn)行的常規(guī)RT-LAMP檢測(cè)方法相比較,敏感性為90.3%,特異性為100%；采用熱裂解方案的Direct RT-LAMP方法與提取標(biāo)本核酸后進(jìn)行的實(shí)時(shí)熒光定量PCR檢測(cè)方法相比較,敏感性為86.83%,特異性為100%。以上實(shí)驗(yàn)數(shù)據(jù)提示了直接檢測(cè)標(biāo)本的Direct RT-LAMP方法具有一定的可行性,檢測(cè)結(jié)果與qPCR等主流方法相比具有相近的一致性,適用于實(shí)驗(yàn)條件有限的基層衛(wèi)生機(jī)構(gòu)對(duì)手足口病進(jìn)行病原初步篩查。 第三部分逆轉(zhuǎn)錄等溫?cái)U(kuò)增技術(shù)檢測(cè)人腸道病毒(通用型)方法的建立和評(píng)價(jià) 腸道病毒感染廣泛分布于全球各地,人的一生中往往會(huì)經(jīng)歷多次感染。目前已知共18種血清型腸道病毒能引起手足口病和相關(guān)疫情。傳統(tǒng)的診斷方法由于耗時(shí)較長(zhǎng)而且敏感性不夠高,不能給患者帶來及時(shí)有效的信息輔助治療。從NCBI數(shù)據(jù)庫獲取了522條腸道病毒全基因組序列信息,通過生物信息學(xué)軟件進(jìn)行多序列比對(duì)分析,在病毒5'端非翻譯區(qū)找到數(shù)段高同源性保守序列,用來設(shè)計(jì)一種改良型逆轉(zhuǎn)錄等溫?cái)U(kuò)增檢測(cè)方法的引物。該方法的英文縮寫為GEAR,在本研究中應(yīng)用于腸道病毒通用型檢測(cè)。檢測(cè)反應(yīng)為單管一步法并在配制反應(yīng)體系時(shí)加入羥基萘酚藍(lán)染料,在63℃等溫條件下反應(yīng)60分鐘后直接肉眼觀察指示劑羥基萘酚藍(lán)的顏色變化進(jìn)行結(jié)果判讀。使用了53種腸道病毒血清型和6種引起病毒性腹瀉相關(guān)的胃腸炎病毒,包括腺病毒、星狀病毒、博卡病毒、諾如病毒、輪狀病毒、札如病毒等,對(duì)腸道病毒通用型等溫?cái)U(kuò)增檢測(cè)進(jìn)行特異性驗(yàn)證,結(jié)果顯示所有型別的腸道病毒RNA均成功的得到擴(kuò)增而與其它6種病毒均無交叉反應(yīng)。將改良型逆轉(zhuǎn)錄等溫?cái)U(kuò)增檢測(cè)方法與實(shí)時(shí)定量熒光PCR檢測(cè)方法進(jìn)行了靈敏度平行實(shí)驗(yàn),從人腸道病毒A組、B組、C組各選取2種病毒并對(duì)病毒RNA進(jìn)行十倍等比梯度稀釋后用上述兩種方法同時(shí)進(jìn)行檢測(cè),結(jié)果顯示這兩種檢測(cè)方法靈敏度基本相當(dāng)。使用了58份來源于2012年爆發(fā)的手足口病疫情臨床標(biāo)本對(duì)改良型逆轉(zhuǎn)錄等溫?cái)U(kuò)增檢測(cè)方法進(jìn)行了進(jìn)一步評(píng)價(jià),標(biāo)本已經(jīng)通過實(shí)時(shí)定量熒光PCR驗(yàn)證其中33份為HEV71病毒感染,25份為CVA16病毒感染,實(shí)驗(yàn)結(jié)果顯示所有標(biāo)本在腸道病毒通用型逆轉(zhuǎn)錄等溫?cái)U(kuò)增方法中均為陽性。本研究為腸道病毒感染,尤其是手足口病的診斷提供一種靈敏、特異、快速、高通量的分子生物學(xué)檢測(cè)方法,將有助于日后手足口病的診斷和疫情的監(jiān)測(cè)。
[Abstract]:Hand - foot - mouth disease has been rapidly spread in China since 2004 . It has caused the outbreak of epidemic situation in 2008 . It has become an important public health problem in China since 2009 . The hand - foot - mouth disease has become an important public health problem in China .
the second part is to establish a retrovirus - mediated isothermal amplification method for directly detecting HEV71 virus in a specimen without nucleic acid extraction , and carrying out pilot evaluation on the method ;
the third part is to establish a reverse transcription isothermal amplification method for detecting a plurality of enteroviruses , and is applied to the broad - spectrum detection of the enteroviruses infection , in particular the hand - foot - mouth disease pathogen .
Establishment and Evaluation of Loop - mediated Isothermal Amplification Method for Detecting HEV71 Virus and CVA16 Virus in Part I
A reverse transcription loop - mediated isothermal amplification ( RT - PCR ) method was established for the detection of CVA16 virus . The results showed that the detection limit was 0.33TCID , and the detection limit of CVA16 virus was 1.58TICD50 . The results showed that the detection method has no cross - reaction to the detection method . The results show that the detection method has no cross - reaction to the detection method of HEV . The results show that the RT - PCR method established in this study can effectively detect the HEV71 virus and CVA16 virus in clinical specimens .
A commercial isothermal amplification kit was used in the field trial assessment of the RT - LAMP detection method , and the reaction conditions were optimized accordingly . The results showed that the sensitivity and specificity of the detection method were improved . The results showed that the sensitivity of the two methods was 99.6 % ( 513 / 515 ) .
The second part directly detects the establishment and evaluation of the RT - isothermal amplification method of human intestinal virus 71 loop - mediated isothermal amplification .
A reverse transcription loop - mediated isothermal amplification ( RT - LAMP ) method for detecting human reovirus 71 in hand - foot - mouth disease was established .
Direct RT - LAMP method was used to detect the direct RT - LAMP method . The results showed that the direct RT - LAMP method was feasible . The results showed that the direct RT - LAMP method was similar to the mainstream method such as qPCR and was suitable for the primary screening of hand - foot - mouth disease with limited experimental conditions .
Establishment and Evaluation of Human Enterovirus ( General ) Method by Reverse Transcription Isothermal Amplification
An improved RT - PCR method was used to detect enteroviruses , including adenovirus , star virus , Boca virus , noe virus , rotavirus , and so on . The results showed that all the samples were positive in RT - PCR .
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R512.5;R440
本文編號(hào)：2127234
[Abstract]:Hand - foot - mouth disease has been rapidly spread in China since 2004 . It has caused the outbreak of epidemic situation in 2008 . It has become an important public health problem in China since 2009 . The hand - foot - mouth disease has become an important public health problem in China .
the second part is to establish a retrovirus - mediated isothermal amplification method for directly detecting HEV71 virus in a specimen without nucleic acid extraction , and carrying out pilot evaluation on the method ;
the third part is to establish a reverse transcription isothermal amplification method for detecting a plurality of enteroviruses , and is applied to the broad - spectrum detection of the enteroviruses infection , in particular the hand - foot - mouth disease pathogen .
Establishment and Evaluation of Loop - mediated Isothermal Amplification Method for Detecting HEV71 Virus and CVA16 Virus in Part I
A reverse transcription loop - mediated isothermal amplification ( RT - PCR ) method was established for the detection of CVA16 virus . The results showed that the detection limit was 0.33TCID , and the detection limit of CVA16 virus was 1.58TICD50 . The results showed that the detection method has no cross - reaction to the detection method . The results show that the detection method has no cross - reaction to the detection method of HEV . The results show that the RT - PCR method established in this study can effectively detect the HEV71 virus and CVA16 virus in clinical specimens .
A commercial isothermal amplification kit was used in the field trial assessment of the RT - LAMP detection method , and the reaction conditions were optimized accordingly . The results showed that the sensitivity and specificity of the detection method were improved . The results showed that the sensitivity of the two methods was 99.6 % ( 513 / 515 ) .
The second part directly detects the establishment and evaluation of the RT - isothermal amplification method of human intestinal virus 71 loop - mediated isothermal amplification .
A reverse transcription loop - mediated isothermal amplification ( RT - LAMP ) method for detecting human reovirus 71 in hand - foot - mouth disease was established .
Direct RT - LAMP method was used to detect the direct RT - LAMP method . The results showed that the direct RT - LAMP method was feasible . The results showed that the direct RT - LAMP method was similar to the mainstream method such as qPCR and was suitable for the primary screening of hand - foot - mouth disease with limited experimental conditions .
Establishment and Evaluation of Human Enterovirus ( General ) Method by Reverse Transcription Isothermal Amplification
An improved RT - PCR method was used to detect enteroviruses , including adenovirus , star virus , Boca virus , noe virus , rotavirus , and so on . The results showed that all the samples were positive in RT - PCR .
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R512.5;R440
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
1 李啟明;馬學(xué)軍;高寒春;周蕊;匡治州;侯云德;;逆轉(zhuǎn)錄環(huán)介導(dǎo)等溫核酸擴(kuò)增技術(shù)(RT-LAMP)在H5N1禽流感病毒基因檢測(cè)中的應(yīng)用[J];病毒學(xué)報(bào);2008年03期
2 查磊;蔡欣;應(yīng)曉敏;徐東剛;曹源;李伍舉;;BioSunLAMP:一個(gè)用于環(huán)介導(dǎo)等溫?cái)U(kuò)增的引物設(shè)計(jì)軟件[J];軍事醫(yī)學(xué);2012年03期
3 ;Molecular Epidemiological Analysis of Echovirus 19 Isolated From an Outbreak Associated With Hand, Foot, and Mouth Disease (HFMD) in Shandong Province of China[J];Biomedical and Environmental Sciences;2007年04期
，本文編號(hào)：2127234
本文鏈接：http://www.sikaile.net/yixuelunwen/chuanranbingxuelunwen/2127234.html
最近更新
教材專著
