等温扩增技术检测手足口病病原的研究
发布时间：2018-07-16 18:18
【摘要】：手足口病自2004年以来在国内迅速传播,于2008年引起疫情暴发并在全国范围内大规模流行。据卫生部疫情数据显示,自2009年以来全国手足口病年报告病例人数均为百万以上,每年因该病死亡的儿童数以百计。手足口病已经成为我国的一个重要的公共卫生问题。手足口病是由多种肠道病毒引起的传染病,人肠道病毒71型(HEV71)和柯萨奇病毒A组16型(CVA16)是手足口病的两种主要病原。因此建立针对HEV71病毒和CVA16病毒的特异、快速、简便的检测方法,并建立检测引起手足口病的多种相关肠道病毒的实验方法对控制手足口病疫情具有重要意义。本文内容分为三部分,第一部分是建立检测HEV71病毒和CVA16病毒的逆转录环介导等温扩增方法,对方法进行试点评价；第二部分是建立无需核酸提取的直接检测标本中的HEV71病毒的逆转录环介导等温扩增方法,对方法进行试点评价；第三部分是建立检测多种肠道病毒的逆转录等温扩增方法,应用于肠道病毒感染尤其是手足口病病原的广谱检测。 第一部分检测HEV71病毒和CVA16病毒的逆转录环介导等温扩增方法的建立和评价 建立了应用于HEV71病毒C4亚型和CVA16病毒的逆转录环介导等温扩增方法,方法灵敏度高,反应速度快。检测前在反应体系添加了羟基萘酚蓝(HNB)指示剂,在65℃等温条件下反应60分钟,实验结果可以通过肉眼观察反应溶液混浊度是否增加或者HNB指示剂颜色是否由紫色变为天蓝色来进行判读。使用十倍等比梯度稀释的病毒溶液对检测方法的灵敏度进行测试,实验结果显示检测方法对HEV71病毒的检测限为0.33TCID50,对CVA16病毒的检测限为1.58TICD50。使用了多个血清型的肠道病毒对等温扩增检测方法进行特异性测试,实验结果显示该检测方法特异性高,与其它型别肠道病毒均无交叉反应,包括柯萨奇病毒A组2、4、5、7、9、10、14、24型,柯萨奇病毒B组1、2、3、4、5型和埃可病毒3、6、11、19型。使用47份手足口病病人的粪便标本对该检测方法进行了实验室评价,标本均经过病毒分离或是中和滴定实验鉴定其感染的肠道病毒类型,检测结果显示本研究建立的逆转录环介导等温扩增方法能有效检出临床标本中的HEV71病毒和CVA16病毒,在检测反应中应用HNB染料作为指示剂使得可视化结果判读方式更为有效。 在对RT-LAMP检测方法进行现场试点评估时使用了商业化的等温扩增试剂盒,检测反应条件进行相应优化,其中检测HEV71病毒的反应条件优化为65℃等温条件下反应45分钟,检测CVA16病毒的反应条件优化为65℃等温条件下反应35分钟。使用商业化等温扩增试剂盒后RT-LAMP检测反应的灵敏度和特异性进一步研究结果显示：检测方法灵敏度有一定程度提高,方法对HEV71病毒和CVA16病毒的检测限均为0.1TCID50,同时检测方法的特异性无变化。试点评估的515份手足口病病人临床标本于湖南省2011年手足口病疫情暴发期间收集,所有标本同时进行了实时荧光定量PCR检测和RT-LAMP检测。检测实验结果显示这两个方法的一致性为99.6%(513/515),检测结果不一致的2个标本通过巢式逆转录PCR进一步验证为CVA16病毒。上述实验数据提示检测手足口病主要病原体HEV71病毒和CVA16病毒的RT-LAMP检测方法具有特异性好、灵敏度高等优点,能快速、可视化的检测上述两种手足口病主要病原,具有较好的应用前景,尤其适用于硬件设施有限的城乡医院、基层卫生机构使用。 第二部分直接检测人肠道病毒71型环介导逆转录等温扩增方法建立和评价 建立了一种直接检测手足口病咽拭子标本中人肠道病毒71型的逆转录环介导等温扩增方法,该方法对样品采用热裂解方案进行预处理而无需核酸提取便可对咽拭子标本进行RT-LAMP直接检测。对采用热裂解方案的Direct RT-LAMP方法的灵敏度和特异性进行实验室验证,结果显示检测方法对HEV71型病毒的检测限为1.6TCID50,特异性强,与其它型别的肠道病毒诸如柯萨奇病毒A组2、4、5、7、9、10、14、24型,柯萨奇病毒B组1、2、3、4、5型和埃可病毒3、6、11、19型等均无交叉反应。在试点评价中同时使用三种方法对145份手足口病患者的咽拭子临床标本进行检测,这三种方法分别是不经过标本核酸提取而对标本直接进行检测的Direct RT-LAMP方法,以及提取标本核酸后再进行检测的RT-LAMP方法和实时荧光定量PCR方法。结果显示采用热裂解方案的Direct RT-LAMP方法与提取标本核酸后进行的常规RT-LAMP检测方法相比较,敏感性为90.3%,特异性为100%；采用热裂解方案的Direct RT-LAMP方法与提取标本核酸后进行的实时荧光定量PCR检测方法相比较,敏感性为86.83%,特异性为100%。以上实验数据提示了直接检测标本的Direct RT-LAMP方法具有一定的可行性,检测结果与qPCR等主流方法相比具有相近的一致性,适用于实验条件有限的基层卫生机构对手足口病进行病原初步筛查。 第三部分逆转录等温扩增技术检测人肠道病毒(通用型)方法的建立和评价 肠道病毒感染广泛分布于全球各地,人的一生中往往会经历多次感染。目前已知共18种血清型肠道病毒能引起手足口病和相关疫情。传统的诊断方法由于耗时较长而且敏感性不够高,不能给患者带来及时有效的信息辅助治疗。从NCBI数据库获取了522条肠道病毒全基因组序列信息,通过生物信息学软件进行多序列比对分析,在病毒5'端非翻译区找到数段高同源性保守序列,用来设计一种改良型逆转录等温扩增检测方法的引物。该方法的英文缩写为GEAR,在本研究中应用于肠道病毒通用型检测。检测反应为单管一步法并在配制反应体系时加入羟基萘酚蓝染料,在63℃等温条件下反应60分钟后直接肉眼观察指示剂羟基萘酚蓝的颜色变化进行结果判读。使用了53种肠道病毒血清型和6种引起病毒性腹泻相关的胃肠炎病毒,包括腺病毒、星状病毒、博卡病毒、诺如病毒、轮状病毒、札如病毒等,对肠道病毒通用型等温扩增检测进行特异性验证,结果显示所有型别的肠道病毒RNA均成功的得到扩增而与其它6种病毒均无交叉反应。将改良型逆转录等温扩增检测方法与实时定量荧光PCR检测方法进行了灵敏度平行实验,从人肠道病毒A组、B组、C组各选取2种病毒并对病毒RNA进行十倍等比梯度稀释后用上述两种方法同时进行检测,结果显示这两种检测方法灵敏度基本相当。使用了58份来源于2012年爆发的手足口病疫情临床标本对改良型逆转录等温扩增检测方法进行了进一步评价,标本已经通过实时定量荧光PCR验证其中33份为HEV71病毒感染,25份为CVA16病毒感染,实验结果显示所有标本在肠道病毒通用型逆转录等温扩增方法中均为阳性。本研究为肠道病毒感染,尤其是手足口病的诊断提供一种灵敏、特异、快速、高通量的分子生物学检测方法,将有助于日后手足口病的诊断和疫情的监测。
[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 .
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.5;R440
本文编号：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 .
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.5;R440
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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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