内标多重荧光RT-PCR同时检测肠道病毒71型和柯萨奇病毒A16型

【目的】为对当前爆发的手足口病进行快速准确的检测, 【方法】本研究建立了含内标的同时检测EV71和CA16的多重荧光RT-PCR方法,对该方法的特异性、灵敏度等进行评估,并对400多份临床样品进行了检测。【结果】实验结果表明,该检测方法特异性强,对10株EV71病毒、8株CA16病毒和25株其他人类病毒进行了检测,特异性为100%;该检测方法对EV71和CA16的检测灵敏度分别达到0.1 TCID50和1 TCID50;将0.1-104TCID50/ml EV71和CA16样本进行重复性实验,其变异系数分别为0.9-2.0%和0.9-2.3%。对400多份临床样品分别进行荧光RT-PCR检测和传统方法检测,结果显示,荧光RT-PCR对EV71和CA16的阳性检出率平均为46.1%和14.2%,比传统方法（34.5%和12.8%）的阳性检测率高。另外,实验数据显示,在粪便、直肠拭子、咽喉拭子样本中,PCR抑制物存在的比例为1.8%-3.4%,表明内标对监控PCR抑制物的存在具有重要作用。【结论】本方法能同时对EV71和CA16进行快速检测,并且灵敏度高,特异性好,由于加入了内标,能有效地监控假阴性的出现,适合于手足口病的临床检测。     

肠道病毒71型和柯萨奇病毒A16型多重反转录聚合酶链反应的建立及初步应用

本文旨在建立一种快速、高效的方法检测肠道病毒71型（EV71）和柯萨奇病毒A16型(CA16)的方法,以用于儿童手足口病的病原学监测。通过设计肠道病毒通用引物和CA16与EV71的型特异性引物,建立不同引物浓度配比及两阶段退火温度以提高检测敏感性和特异性的多重反转录聚合酶链反应（RT-PCR）方法,并对首都儿科研究所附属儿童医院2010年3~10月收集的371例手足口病患儿共381份临床标本同时进行病毒分离和核酸检测。结果显示,本研究建立的多重RT-PCR方法对CA16和EV71的最低模板检测浓度分别为5.32 pg/ml和0.64 pg/ml,反应特异度为100%。应用该方法检测381份手足口病临床标本的总阳性率为78.4%,其中CA16与EV71的检测阳性率分别为32.6%和35.8%,二者检测阳性比为1:1.1。以病毒分离为标准,多重RT-PCR对CA16及EV71检测的准确率分别为95.2%和98.6%。因此,本研究新建立的多重RT-PCR方法准确、简便,适用于较大量样本的手足口病病原学监测。2010年引起北京地区儿童手足口病的主要病原为CA16和EV71。     

2007年北京地区儿童手足口病病原的初步筛查

2007年4～6月儿童手足口病流行期间,对北京地区51例皮损症状典型、伴/不伴发热、无重症合并症的手足口病患儿采样,建立RT-PCR方法,以5'非编码区(5'UTR)肠道病毒通用引物、CA16和EV71 VP1区特异性引物直接对82份临床标本进行了初步筛查,肠道病毒阳性率达70.6%。检测病例中CA16阳性25例(25/51)、EV71阳性4例(4/51)、非CA16和EV71的肠道病毒阳性病例7例(7/51),三者比例约为6:1:2。2007年北京地区儿童轻症手足口病主要病原包括CA16和EV71,同时还存在一定比例其它肠道病毒。部分EV71毒株经测序验证及系统进化分析显示为C4基因亚型。     

GeXP多重基因表达遗传分析系统在手足口病病原分型检测中的应用

利用GeXP多重基因表达遗传分析系统,建立一种多重逆转录-聚合酶链反应(RT-PCR)方法,同时检测引起手足口病的9种常见的人肠道病毒—人肠道病毒71型(HEV71)、柯萨奇病毒A组(CVA)16、4、5、9、10型和柯萨奇病毒B组(CVB)1、3、5型。优化多重反应体系中针对5’UTR区的肠道病毒通用引物和11对针对9种血清型人肠道病毒VP1区的特异性引物的浓度比例,分别以病毒细胞培养物和阳性粪便标本来验证多重反应体系的特异性,以TCID50定量的细胞培养物和克隆质粒体外转录的RNA梯度稀释液来检测多重检测体系的灵敏度。结果表明,优化后的多重检测体系,可扩增出人肠道病毒共有的保守片段的和型特异性片段,HEV71和CVA16细胞培养物的检测下限为100.5TCID50/μL,并可在103copies/μL水平同时、特异地检测出9种病毒RNA。该方法灵敏度高、特异性强,可快速对大量临床样本进行高通量检测,用于手足口病的分子流行病学调查。     

2013～2018年辽宁省手足口病病原学监测及其分子特征分析

辽宁省是手足口病(Hand,foot and mouth disease,HFMD)的高发省份,自2013年开始病原构成发生了较大的变化。本研究对辽宁省2013～2018年手足口病的病原学监测进行分析,以了解辽宁省肠道病毒病原构成情况及其流行特点,为下一步的预防控制工作提供科学依据。2013～2018年辽宁省14个市累计送检手足口病临床病例阳性标本18 018份,标本的核酸检测由辽宁省各市完成,对其中3 669份手足口病例标本进行了病毒分离,分离到毒株1 003株,对分离到的阳性毒株进行VP1区基因扩增和序列测定,并进行分子特征分析。在送检的核酸阳性的标本中,肠道病毒71型(Enterovirous 71,EV71)的阳性标本构成比是18.95%,柯萨奇病毒A组16型(Coxsachievirus A16,CA16)阳性标本构成比是28.71%,其它肠道病毒阳性标本构成比是占52.34%。重症病例标本数345例,其中EV71占32.80%;CA16占8.07%;其他肠道病毒占59.13%。2013年以其他肠道病毒为主,占53.76%;2014以CA16为主,占39.05%;2015以其...     

以主要流行EV71 VP1基因高度保守区为靶点的TaqMan荧光定量PCR检测方法的建立

在我国,肠道病毒71型(enterovirus 71,EV71)C4型是引起手足口病的主要流行基因型。为建立EV71C4型TaqMan荧光定量PCR检测方法,在C4型EV71VP1基因的高保守区,设计合成引物和TaqMan探针,将包含此目的区段的基因片段克隆到pcDNA3.1载体中,通过体外转录获得标准品,并以梯度稀释的标准品为模板建立工作曲线,进而在优化反应条件的基础上建立TaqMan荧光实时定量PCR检测方法。实验中,所设计引物、探针的高度保守性保证了C4型EV71的高效扩增。经反应条件优化,引物和探针的最佳工作浓度分别为300 nmol/L和200 nmol/L,在1×10~301×10~3拷贝数检测范围内具有良好的线性关系(R~2=1),灵敏度可达到10~2 copies/μL。通过对该方法进行检验发现,批间和组间重复实验的变异系数均小于0.5%,且该方法对柯萨奇A16(coxsackievirus A16,CA16)柯萨奇B1(coxsackievirus B1,CB1)人轮状病毒(human rotavirus,HRV)单纯疱疹病毒2型(herpes Simplex virus type 2,HSV-2)均无交叉反应,对6份EV71阳性样本检出率为100%。以上数据表明,文中建立的TaqMan荧光定量PCR方法可为我国主要流行C4型EV71感染的快速诊断及疾病监控提供有效途径。     

EV71病毒核酸快速检测方法的比较

PCR方法检测肠道病毒71型(EV71)的分子诊断技术十分快速敏感。目前已有多篇文献报道了不同的检测EV71的PCR方法,但对不同方法检测结果的比较还少有报道。本研究将检测肠道病毒通用5’UTR基因的3种定量PCR(rRT-PCR)方法和1种普通PCR(cRT-PCR)方法和特异性检测EV71的2种rRT-PCR和1种cRT-PCR方法进行特异性和敏感性比较评估。所有试验方法都有较好的特异性,无交叉反应。核酸最低检测限值为从8.19×101～8.19×105个拷贝,rRT-PCR比cRT-PCR更敏感。所有的rRT-PCR方法检测50份临床标本时都较敏感。     

2013年大连市手足口病病原监测结果分析

目的通过对2013年大连市手足口病（HFMD）的病原进行鉴定,以了解其型别分布。方法采用realtime-PCR方法对754份标本进行肠道病毒（EV）通用引物和肠道病毒71型（EV71）、柯萨奇病毒A组16型（CVA16）、柯萨奇病毒A组6型（CVA6）型特异性引物检测,对EV通用引物检测结果为阳性,但EV71、CVA16和CVA6检测结果均为阴性的标本采用巢氏PCR进行肠道病毒VP1基因部分序列的扩增、测序和生物信息学分析以鉴定其型别。结果 2013年引起大连市HFMD的主要病原CVA6占44.30%（334/754）、CVA16占19.12%（114/754）、EV71占11.54%（87/754）,另有少数病例由CVA2、4、5、8型,CVB2、4型,ECHO9型及EV的其他型别引起。结论 CVA6取代EV71和CVA16成为2013年大连市HFMD病原的主要流行型别,后续应加强对HFMD病原的监测,全面了解其型别分布及毒株变异情况以更有效地控制疾病流行。     

2014-2018年辽宁省手足口病病原构成及其他肠道病毒基因进化特征

目的 了解辽宁省2014-2018年手足口病病原构成以及其他(非EV71和非CA16的肠道病毒)肠道病毒基因进化特征。 方法 采用2014-2018年辽宁省14个市送检的手足口病确诊病例的阳性标本,通过细胞培养法分离肠道病毒,提取病毒RNA,采用RT PCR法对肠道病毒VP1区基因进行扩增和序列测定。利用BLAST对测序结果比对后确定病毒基因型别,并构建系统进化树。 结果 手足口病阳性标本病原构成以其他肠道病毒为主,构成比为51.96%,在重症病例中阳性标本构成比是59.29%。优势流行病原在2014和2016年以CA16为主,其他年份以其他肠道病毒为主。CA10、CA6和CA4肠道病毒的分离株与相应的原型株的亲缘关系较远,与国内的分离株的亲缘关系较近。 结论 EV71和CA16感染的构成比总体呈下降趋势,其他肠道病毒的构成比缓慢上升,是本地手足口病的优势病原,要加强对其他肠道病毒的检测和分型工作,有效预防和控制手足口病。     

115例手足口病原学监测结果分析

目的:探讨长沙地区手足口病的病原学特征,为当地的疾病预防和控制提供科学依据.方法:使用肠道通用和EV71、CA16特异性引物,对2009年长沙市的115例手足口病患者的疱疹液、粪便、咽拭子标本,进行RT-PCR鉴定,并对手足口病疫情开展监测,采用流行病学方法对结果进行统计分析.结果:在采集到标本的115例患者中,84例患者检测结果为阳性,其中19例检测为EV71病毒核酸阳性(22.62%);30例检测CA16阳性(35.71%);35例检测到其他肠道病毒(41.67%).阳性标本中男女性别比为(1.27:1),发病年龄主要集中1-5岁儿童(90.47%),尤其是在3岁组(34.52%);发病季节主要集中于4-8月.结论:在长沙地区手足口病易发于1-5岁儿童,应在春夏季重点加强这一人群的预防和控制措施.     

A One-Step,Triplex, Real-Time RT-PCR Assay for the Simultaneous Detection of Enterovirus 71, Coxsackie A16 and Pan-Enterovirus in a Single Tube

The recent, ongoing epidemic of hand, foot, and mouth disease (HFMD), which is caused by enterovirus infection, has affected millions of children and resulted in thousands of deaths in China. Enterovirus 71 (EV71) and coxsackie A16 (CA16) are the two major distinct pathogens for HFMD. However, EV71 is more commonly associated with neurologic complications and even fatalities. Therefore, simultaneously detecting and differentiating EV71 and CA16 specifically from other enteroviruses for diagnosing HFMD is important. Here, we developed a one-step, triplex, real-time RT-PCR assay for the simultaneous detection of EV71, CA16, and pan-enterovirus (EVs) in a single tube with an internal amplification control. The detection results for the serially diluted viruses indicate that the lower limit of detection for this assay is 0.001–0.04 TCID₅₀/ml, 0.02 TCID₅₀/ml, and 0.001 TCID₅₀/ml for EVs, EV71, and CA16, respectively. After evaluating known HFMD virus stocks of 17 strains of 16 different serotypes, this assay showed a favorable detection spectrum and no obvious cross-reactivity. The results for 141 clinical throat swabs from HFMD-suspected patients demonstrated sensitivities of 98.4%, 98.7%, and 100% for EVs, EV71, and CA16, respectively, and 100% specificity for each virus. The application of this one-step, triplex, real-time RT-PCR assay in clinical units will contribute to HFMD surveillance and help to identify causative pathogen in patients with suspected HFMD.     

Immunity to avirulent enterovirus 71 and coxsackie A16 virus protects against enterovirus 71 infection in mice

In this study, we sought to determine whether intratypic and intertypic cross-reactivity protected against enterovirus 71 (EV71) infection in a murine infection model. We demonstrate that active immunization of 1-day-old mice with avirulent EV71 strain or coxsackie A16 virus (CA16) by the oral route developed anti-EV71 antibodies with neutralizing activity (1:16 and 1:2, respectively). Splenocytes from both EV71- and CA16-immunized mice proliferated upon EV71 or CA16, but not coxsackie B3 virus (CB3), antigen stimulation. Immunized mice became more resistant to virulent EV71 strain challenge than nonimmunized mice. There was an increase in the percentage of activated splenic T cells and B cells in the immunized mice 2 days after EV71 challenge. The CA16 immune serum reacted with EV71 antigens in an enzyme-linked immunosorbent assay and neutralized EV71 but not CB3 or poliovirus at a titer of 1:4. Passive immunization with the CA16 immune serum reduced the clinical score, diminished the organ viral load, and increased the survival rate of mice upon EV71 challenge. CB3 neither shared in vitro cross-reactivity with EV71 nor provided in vivo protection after both active and passive immunization. These results illustrated that live vaccine is feasible for EV71 and that intertypic cross-reactivity of enteroviruses may provide a way to determine the prevalence of EV71.     

肠道病毒71型安徽、河南株的分离与VP1序列进化分析

旨在研究手足口病患者中肠道病毒71型分离株的病毒基因型特征。采集手足口病患者的粪便标本,进行病毒分离和逆转录-聚合酶链式反应(RT-PCR)特异性扩增进行鉴定,同时选取其中9株EV71分离株,对其抗原决定簇部位VP1区进行核酸序列测定,并参考EV71 A、B、C各基因型的参考株和以往中国EV71的分离株进行同源分析和构建系统发生树。结果显示,所分析的9株病毒株均为C4亚型,3株安徽株H7、H8和H9的VP1序列相似度很高(≥98.8%,其中H7、H9的相似度为100.0%),4株河南株H3、H4、H5和H6相似度较高(≥98.4%,H3、H4和H5≥99.6%,其中H3、H4的相似度为100.0%),它们同河南株H1、H5的相似度也较高(≥97.2%),河南株H2虽然与其他河南株具有较高的序列相似度,但进化分析表明,其与安徽株同源性较高。结果表明,安徽株H7、H8和H9株变异速率明显加快,这可能导致了手足口病在安徽省的率先爆发与大流行,河南株H2最初可能由安徽传入河南。     

Evaluation of a Viral Microarray Based on Simultaneous Extraction and Amplification of Viral Nucleotide Acid for Detecting Human Herpesviruses and Enteroviruses

In this study, a viral microarray based assay was developed to detect the human herpesviruses and enteroviruses associated with central nervous system infections, including herpes simplex virus type 1, type 2 (HSV1 and HSV2), Epstein-Barr virus (EBV), cytomegalovirus (CMV), enterovirus 71 (EV71), coxsackievirus A 16 (CA16) and B 5(CB5). The DNA polymerase gene of human herpesviruses and 5’-untranslated region of enteroviruses were selected as the targets to design primers and probes. Human herpesviruses DNA and enteroviruses RNA were extracted simultaneously by using a guanidinium thiocyanate acid buffer, and were subsequently amplified through a biotinylated asymmetry multiplex RT-PCR with the specific primer of enteroviruses. In total, 90 blood samples and 49 cerebrospinal fluids samples with suspected systemic or neurological virus infections were investigated. Out of 139 samples, 66 were identified as positive. The specificities of this multiplex RT-PCR microarray assay were over 96% but the sensitivities were various from 100% for HSV1, HSV2, EV71 and CB5, 95.83% for CMV, 80% for EBV to 71.43% for CA16 in comparison with reference standards of TaqMan qPCR/qRT-PCR. The high Kappa values (>0.90) from HSV1, HSV2, CMV, EV71 and CB5 were obtained, indicating almost perfect agreement in term of the 5 viruses detection. But lower Kappa values for EBV (0.63) and CA16 (0.74) displayed a moderate to substantial agreement. This study provides an innovation of simultaneous extraction, amplification, hybridization and detection of DNA viruses and RNA viruses with simplicity and specificity, and demonstrates a potential clinical utility for a variety of viruses’ detection.     

Development and Evaluation of a Pseudovirus-Luciferase Assay for Rapid and Quantitative Detection of Neutralizing Antibodies against Enterovirus 71

The level of neutralizing antibodies (NtAb) induced by vaccine inoculation is an important endpoint to evaluate the efficacy of EV71 vaccine. In order to evaluate the efficacy of EV71 vaccine, here, we reported the development of a novel pseudovirus system expression firefly luciferase (PVLA) for the quantitative measurement of NtAb. We first evaluated and validated the sensitivity and specificity of the PVLA method. A total of 326 serum samples from an epidemiological survey and 144 serum specimens from 3 clinical trials of EV71 vaccines were used, and the level of each specimen''s neutralizing antibodies (NtAb) was measured in parallel using both the conventional CPE-based and PVLA-based assay. Against the standard neutralization assay based on the inhibition of the cytopathic effect (CPE), the sensitivity and specificity of the PVLA method are 98% and 96%, respectively. Then, we tested the potential interference of NtAb against hepatitis A virus, Polio-I, Polio-II, and Polio-III standard antisera (WHO) and goat anti-G10/CA16 serum, the PVLA based assay showed no cross-reactivity with NtAb against other specific sera. Importantly, unlike CPE based method, no live replication-competent EV71 is used during the measurement. Taken together, PVLA is a rapid and specific assay with higher sensitivity and accuracy. It could serve as a valuable tool in assessing the efficacy of EV71 vaccines in clinical trials and disease surveillance in epidemiology studies.     

Genotypes of the Enterovirus Causing Hand Foot and Mouth Disease in Shanghai,China, 2012-2013

Sporadic HFMD (hand foot and mouth disease, HFMD) cases and outbreaks caused by etiologic agents other than EV71 and CA16 have increased globally. We conducted this study to investigate the prevalence and genetic characteristics of enteroviruses, especially the non-EV71 and non-CA16 enteroviruses, causing HFMD in Shanghai. Clinical specimens were collected from patients with a diagnosis of HFMD. A partial length of VP1 was amplified with RT-PCR and subjected to direct sequencing. Phylogenetic analyses were performed using MEGA 5.0. The ages of the HFMD cases ranged from 3 to 96 months, and the male/female ratio was 1.41. The median hospital stay was 2.96 days. Up to 18.0% of patients had neurologic system complications such as encephalitis, meningoencephalitis or meningitis. Of the 480 samples, 417 were positive for enterovirus (86.9%) with RT-PCR. A total of 13 enterovirus genotypes were identified. The most frequent genotypes were CA6 (31.9%), EV71 (30.6%), CA16 (8.8%) and CA10 (7.5%). Infections with CA6, EV71, CA16 and CA10 were prevalent throughout the years of study, while the proportion of CA6 notably increased from Sep. 2012 to Dec. 2013. Phylogenetic analyses showed that EV71 strains belonged to the C4a subgenogroup and CA16 was identified as B1b subgenogroup. The CA6 strains were assigned to genogroup F, whereas the CA10 strains were assigned to genogroup D. Patients infected with CA6 were typically younger, had a shorter hospital stay and had a lower incidence of neurologic system complications when compared to patients infected with EV71. Our study demonstrates that the enterovirus genotypes causing HFMD were diversified, and there was an increasing prevalence of the non-EV71 and non-CA16 enteroviruses from 2012 to 2013. CA6 was the most predominant pathogen causing HFMD from Sep. 2012 to Dec. 2013, and it often caused relatively mild HFMD symptoms. Most severe HFMD cases were associated with EV71 infection.