等溫擴增技術檢測手足口病病原的研究
發(fā)布時間：2018-07-16 18:18
【摘要】：手足口病自2004年以來在國內迅速傳播,于2008年引起疫情暴發(fā)并在全國范圍內大規(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。手足口病已經(jīng)成為我國的一個重要的公共衛(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HEV71病毒和CVA16病毒的特異、快速、簡便的檢測方法,并建立檢測引起手足口病的多種相關腸道病毒的實驗方法對控制手足口病疫情具有重要意義。本文內容分為三部分,第一部分是建立檢測HEV71病毒和CVA16病毒的逆轉錄環(huán)介導等溫擴增方法,對方法進行試點評價；第二部分是建立無需核酸提取的直接檢測標本中的HEV71病毒的逆轉錄環(huán)介導等溫擴增方法,對方法進行試點評價；第三部分是建立檢測多種腸道病毒的逆轉錄等溫擴增方法,應用于腸道病毒感染尤其是手足口病病原的廣譜檢測。 第一部分檢測HEV71病毒和CVA16病毒的逆轉錄環(huán)介導等溫擴增方法的建立和評價 建立了應用于HEV71病毒C4亞型和CVA16病毒的逆轉錄環(huán)介導等溫擴增方法,方法靈敏度高,反應速度快。檢測前在反應體系添加了羥基萘酚藍(HNB)指示劑,在65℃等溫條件下反應60分鐘,實驗結果可以通過肉眼觀察反應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{色來進行判讀。使用十倍等比梯度稀釋的病毒溶液對檢測方法的靈敏度進行測試,實驗結果顯示檢測方法對HEV71病毒的檢測限為0.33TCID50,對CVA16病毒的檢測限為1.58TICD50。使用了多個血清型的腸道病毒對等溫擴增檢測方法進行特異性測試,實驗結果顯示該檢測方法特異性高,與其它型別腸道病毒均無交叉反應,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本對該檢測方法進行了實驗室評價,標本均經(jīng)過病毒分離或是中和滴定實驗鑒定其感染的腸道病毒類型,檢測結果顯示本研究建立的逆轉錄環(huán)介導等溫擴增方法能有效檢出臨床標本中的HEV71病毒和CVA16病毒,在檢測反應中應用HNB染料作為指示劑使得可視化結果判讀方式更為有效。 在對RT-LAMP檢測方法進行現(xiàn)場試點評估時使用了商業(yè)化的等溫擴增試劑盒,檢測反應條件進行相應優(yōu)化,其中檢測HEV71病毒的反應條件優(yōu)化為65℃等溫條件下反應45分鐘,檢測CVA16病毒的反應條件優(yōu)化為65℃等溫條件下反應35分鐘。使用商業(yè)化等溫擴增試劑盒后RT-LAMP檢測反應的靈敏度和特異性進一步研究結果顯示：檢測方法靈敏度有一定程度提高,方法對HEV71病毒和CVA16病毒的檢測限均為0.1TCID50,同時檢測方法的特異性無變化。試點評估的515份手足口病病人臨床標本于湖南省2011年手足口病疫情暴發(fā)期間收集,所有標本同時進行了實時熒光定量PCR檢測和RT-LAMP檢測。檢測實驗結果顯示這兩個方法的一致性為99.6%(513/515),檢測結果不一致的2個標本通過巢式逆轉錄PCR進一步驗證為CVA16病毒。上述實驗數(shù)據(jù)提示檢測手足口病主要病原體HEV71病毒和CVA16病毒的RT-LAMP檢測方法具有特異性好、靈敏度高等優(yōu)點,能快速、可視化的檢測上述兩種手足口病主要病原,具有較好的應用前景,尤其適用于硬件設施有限的城鄉(xiāng)醫(yī)院、基層衛(wèi)生機構使用。 第二部分直接檢測人腸道病毒71型環(huán)介導逆轉錄等溫擴增方法建立和評價 建立了一種直接檢測手足口病咽拭子標本中人腸道病毒71型的逆轉錄環(huán)介導等溫擴增方法,該方法對樣品采用熱裂解方案進行預處理而無需核酸提取便可對咽拭子標本進行RT-LAMP直接檢測。對采用熱裂解方案的Direct RT-LAMP方法的靈敏度和特異性進行實驗室驗證,結果顯示檢測方法對HEV71型病毒的檢測限為1.6TCID50,特異性強,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型和埃可病毒3、6、11、19型等均無交叉反應。在試點評價中同時使用三種方法對145份手足口病患者的咽拭子臨床標本進行檢測,這三種方法分別是不經(jīng)過標本核酸提取而對標本直接進行檢測的Direct RT-LAMP方法,以及提取標本核酸后再進行檢測的RT-LAMP方法和實時熒光定量PCR方法。結果顯示采用熱裂解方案的Direct RT-LAMP方法與提取標本核酸后進行的常規(guī)RT-LAMP檢測方法相比較,敏感性為90.3%,特異性為100%；采用熱裂解方案的Direct RT-LAMP方法與提取標本核酸后進行的實時熒光定量PCR檢測方法相比較,敏感性為86.83%,特異性為100%。以上實驗數(shù)據(jù)提示了直接檢測標本的Direct RT-LAMP方法具有一定的可行性,檢測結果與qPCR等主流方法相比具有相近的一致性,適用于實驗條件有限的基層衛(wèi)生機構對手足口病進行病原初步篩查。 第三部分逆轉錄等溫擴增技術檢測人腸道病毒(通用型)方法的建立和評價 腸道病毒感染廣泛分布于全球各地,人的一生中往往會經(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疫情。傳統(tǒng)的診斷方法由于耗時較長而且敏感性不夠高,不能給患者帶來及時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,通過生物信息學軟件進行多序列比對分析,在病毒5'端非翻譯區(qū)找到數(shù)段高同源性保守序列,用來設計一種改良型逆轉錄等溫擴增檢測方法的引物。該方法的英文縮寫為GEAR,在本研究中應用于腸道病毒通用型檢測。檢測反應為單管一步法并在配制反應體系時加入羥基萘酚藍染料,在63℃等溫條件下反應60分鐘后直接肉眼觀察指示劑羥基萘酚藍的顏色變化進行結果判讀。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,對腸道病毒通用型等溫擴增檢測進行特異性驗證,結果顯示所有型別的腸道病毒RNA均成功的得到擴增而與其它6種病毒均無交叉反應。將改良型逆轉錄等溫擴增檢測方法與實時定量熒光PCR檢測方法進行了靈敏度平行實驗,從人腸道病毒A組、B組、C組各選取2種病毒并對病毒RNA進行十倍等比梯度稀釋后用上述兩種方法同時進行檢測,結果顯示這兩種檢測方法靈敏度基本相當。使用了58份來源于2012年爆發(fā)的手足口病疫情臨床標本對改良型逆轉錄等溫擴增檢測方法進行了進一步評價,標本已經(jīng)通過實時定量熒光PCR驗證其中33份為HEV71病毒感染,25份為CVA16病毒感染,實驗結果顯示所有標本在腸道病毒通用型逆轉錄等溫擴增方法中均為陽性。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檢測方法,將有助于日后手足口病的診斷和疫情的監(jiān)測。
[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 .
【學位授予單位】：中國疾病預防控制中心
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R512.5;R440
本文編號：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 .
【學位授予單位】：中國疾病預防控制中心
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R512.5;R440
【參考文獻】
相關期刊論文 前3條
1 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
2 查磊;蔡欣;應曉敏;徐東剛;曹源;李伍舉;;BioSunLAMP:一個用于環(huán)介導等溫擴增的引物設計軟件[J];軍事醫(yī)學;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
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