Ⅰ型脊髓灰质炎野病毒实时荧光RT-PCR快速检测方法的评价

目的 评价和探讨Ⅰ型脊髓灰质炎(脊灰)野病毒快速检测策略.方法 采集671名来自脊灰野病毒流行疫区的在京学生粪便标本,采用一组实时荧光RT-PCR代替标准方法分别进行肠道病毒通用型检测、脊灰病毒分型检测、Ⅰ型脊灰疫苗株和野毒株鉴别检测.并利用Ⅰ、Ⅱ和Ⅲ型脊灰病毒标准株(Sabin株)和Ⅰ型野毒株对实时荧光RT-PCR和标准方法的灵敏度进行比较和评价.结果 (1)新脊灰病毒检测流程(肠道病毒通用型检测+脊灰病毒分型检测+脊灰野毒鉴别检测)检出非脊灰肠道病毒33例;脊灰病毒疫苗株16例,其中Ⅰ型5例、Ⅱ型1例、Ⅲ型3例、Ⅰ+Ⅱ型1例、Ⅰ+Ⅲ型4例、Ⅰ+Ⅱ+Ⅲ型2例;脊灰病毒Ⅰ型野毒株3例.(2)3种实时荧光RT-PCR检测脊灰病毒比标准方法敏感1～ 100倍.其中肠道通用型检测和脊灰病毒分型检测2种方法对Ⅱ型脊灰病毒疫苗株的检测,比标准方法敏感100倍;脊灰病毒分型检测能准确区分Ⅰ、Ⅱ和Ⅲ型脊灰疫苗株,脊灰野毒株鉴别检测对Ⅰ型疫苗株的检测比标准方法敏感10倍;3种实时荧光RT-PCR均能有效检测出Ⅰ型脊灰野毒株,比标准方法敏感10倍.结论 新的脊灰病毒检测流程,可大幅缩短检测时间,提高检测通量,且灵敏度高于标准方法,适合于脊灰应急检测应对突发疫情.     

脊髓灰质炎病毒实时荧光定量RT-PCR检测方法的 建立及应用

摘要:目的　在省级脊髓灰质炎（脊灰）实验室建立一种快速、灵敏、准确的脊灰病毒（Poliovirus,PV）型内鉴定（Intratypic Differentiation,ITD）及基因型鉴定的方法。方法　以PV衣壳蛋白（Capsid Protein）VP1编码区基因序列为目标,设计并合成引物和 Taqman 探针,建立实时荧光定量RT-PCR（Real-time RT-PCR,rRT-PCR）检测体系,并考察该方法的重复性、灵敏性和特异性。结果　该方法能快速、灵敏地鉴定出PV血清型及毒株类型,在1.0×108 copies/μl～1.0×103 copies/μl检测范围之间有良好的线性关系,相关系数为0.993,最低检测限为103.5CCID50/0.1 ml。结论　成功在省级实验室建立了PV的r RT-PCR检测技术,该技术特异性强,敏感性高,操作简便快速,适用于PV的型内鉴定和基因型鉴定,可应用于实验室诊断,为免疫策略快速提供依据。     

马尔堡病毒的实时荧光RT-PCR检测方法研究

目的建立马尔堡病毒的实时荧光RT-PCR检测方法。方法人工合成马尔堡病毒特异性核酸序列作为阳性对照模板,设计实时荧光RT-PCR引物、探针并构建反应体系,对反应条件进行优化,验证该方法的特异性、灵敏度。结果建立的马尔堡病毒实时荧光RT-PCR检测方法对马尔堡病毒核酸检测有高度特异性,与1型～4型登革病毒、日本脑炎病毒和基孔肯雅病毒均无交叉反应,检测灵敏度为102拷贝/反应。结论该方法灵敏度高、特异性强,适用于对马尔堡病毒的快速检验。     

微流控实时荧光定量RT-PCR和实时荧光定量RT-PCR检测两种出血热病毒的比较

目的 比较微流控实时荧光定量RT-PCR和实时荧光定量RT-PCR检测两种出血热病毒的情况。方法 以汉城病毒(Seoul virus, SEOV)和汗滩病毒(Hantaan virus, HTNV)作为靶序列设计特异性的引物探针,制备体外转录RNA参考品,对其灵敏性进行评价,通过SEOV和HTNV的假病毒或者病毒培养物制备模拟阳性样本,分别通过微流控和荧光定量RT-PCR检测。结果 SEOV、HTNV体外转录RNA参考品拷贝数浓度分别是2.54×10¹²、1.26×10¹² copies/μl。SEOV、HTNV微流控实时荧光定量RT-PCR检测最低检出限值分别是119.5、123.8 copies/PCR,实时荧光定量RT-PCR检测分别是120.4、128.9 copies/PCR。两种方法检测不同浓度SEOV、HTNV的变异系数均<2%,检出率均为100%。结论 微流控实时荧光定量RT-PCR检测具有很好的特异性、稳定性和灵敏性,适用于现场实验室检测。     

新疆出血热病毒的实时荧光RT-PCR检测方法研究

目的:建立新疆出血热病毒的实时荧光RT-PCR检测方法。方法:分析新疆出血热病毒核酸S片段序列的保守性,设计新疆出血热病毒实时荧光RT-PCR引物和探针。以体外转录后的RNA作为阳性对照模板,摸索出实时荧光RT-PCR检测的最佳引物浓度、探针浓度和反应体系条件,并进行灵敏度和特异性分析。结果:建立的新疆出血热病毒的实时荧光RT-PCR检测方法最佳反应体系为:正向引物CCHFV-FP终浓度250 nM,反向引物CCHFV-RP终浓度500 nM,探针CCHFV-Probe终浓度500 nM;扩增程序为:50℃10 min,95℃10 min;95℃5 s,57℃20 s 45次循环。该方法最低检测限约为2 copies病毒/反应,与森林脑炎病毒和汉坦病毒无交叉反应。结论:该方法灵敏度高、特异性强,适用于新疆出血热病毒的快速检测。     

病毒分离、RT-PCR和实时荧光RT-PCR法检测肠道病毒71型比较

评价病毒分离培养、RT-PCR及实时荧光RT-PCR法对于肠道病毒7l型的检测效果。方法分别用疖毒分离培养、RT-PCR、实时荧光RT-PCR方法对2009年龙岩市手足口病哨点监测医院送检的153份手足口病患者咽捌子样本进行EV71检测，并对检测结果进行配对卡方检验。结果3种方法总体符合率为81．0％，3种检测方法对样本呻EV71阳性检出率差异无统计学意义（P〉0．05）。结论病毒分离、RT-PCR和实时荧光RT-PCR检测方法均适用于EV71的检测，可以作为EV71的日常临床诊断方法。     

实时荧光RT-PCR及RT-PCR快速检测肠道病毒71型和柯萨奇A16病毒

肠道病毒71型(Enterovirus 71,简称EV71)和柯萨奇病毒A组16型(Coxsackievirus A16,简称CA16)是引起手足口病的两种主要的病原体,常相伴造成手足口病的暴发流行,每次流行中两种病毒中所占的比例有所不同[1].EV71感染引起HFMD常有严重的神经系统并发症发生,CA16感染引起自限性的手足口病,极少发生严重并发症和死亡[2～6].2008年安徽、广东(包括深圳市)、浙江等28个省市出现手足口病暴发流行,因此,亟须一种特异性强,灵敏度高的方法对这两种病毒进行快速区分检测.     

普马拉病毒实时荧光定量RT-PCR方法的建立

目的建立一种适应国境口岸地区特定鼠种中普马拉病毒实时荧光定量RT-PCR检测方法。方法利用Beacon Designer7.0软件设计引物和探针,以人工合成普马拉病毒S基因的片段作为模板,进行实时荧光定量RT-PCR检测,并验证该方法的灵敏度及特异性。结果模板的Ct值与模板稀释浓度的对数存在良好的线性关系,标准曲线y=-3.122x＋38.605,R2=0.995,PCR扩增效率为109.1%,其最低检出限为31.6copies/μl。结论建立的实时荧光定量RT-PCR方法特异性好、灵敏度高,适合于普马拉病毒的快速检测。     

实时荧光RT-PCR检测20份流感考核盲样

目的将实时荧光RT-PCR法应用于流感病毒盲样考核中,以便及时找出问题。方法设计高度特异性的引物,采用实时荧光RT-PCR法对2011年和2012年国家下发的共20份流感考核盲样进行检测。结果经实时荧光RT-PCR法检测,2011年10份盲样中B型2份,新甲型H1N1 2份,季节性H1亚型1份,季节性H3亚型2份,禽流感H5亚型1份,阴性2份。2012年10份考核盲样10份盲样中B型流感1份,新甲型H1N1 1份,季节性H1亚型1份,季节性H3亚型2份,禽流感H5亚型2份,阴性3份。根据国家流感中心的反馈,各年度正确率100%。结论将实时荧光RT-PCR法应用于流感盲样考核中,保证了检测结果的准确性。     

实时荧光RT-PCR技术在黄热病快速检测中的应用

[目的]建立黄热病病毒的实时荧光RT-PCR检测方法并用于口岸黄热病的快速检测. [方法]体外转录黄热病病毒5-UTR的部分序列核酸作为阳性对照模板,设计实时荧光RT-PCR不同的反应程序和引物/探针浓度的组合,摸索最佳反应体系条件并用于黄热病疫苗和入境发热患者标本的检测.[结果]建立的黄热病病毒的实时荧光RT-PCR检测方法最佳反应体系为:2xRT-PCR缓冲液10 μl,正向引物5UTR-FP终浓度750 nM,反向引物5UTR-RP终浓度750 nM,探针5UTR-Probe终浓度500 nM,25xRT-PCR Enzyme Mix 0.8μl,RNA 5μl,用DEPC H2O补足到反应总体积20μl;Roche lightcycler仪器适用的扩增程序为:45℃10 min,95℃10 min;95℃5 s,56℃10 s(single),72℃15 s,45次循环.该方法最低检测限约为20copies病毒/反应,与登革病毒、西尼罗病毒、日本脑炎病毒、基孔肯雅病毒等蚊媒病毒无交叉反应.应用该方法对黄热病疫苗、媒介蚊虫和入境发热患者的血清标本进行检测,结果为黄热病疫苗核酸阳性,其他标本为阴性.[结论]该实时荧光RT-PER方法灵敏度高,特异性强,适用于黄热病病毒的快速检验,防止该病在国境口岸的传入.     

Mucosal immunity and poliovirus vaccines: impact on wild poliovirus infection and transmission

Since the resolution of the World Assembly in 1988 to eradicate polio globally, substantial progress toward this target has been achieved, but the final goal remains elusive. India and other tropical developing countries present a unique challenge because of the much lower oral poliovirus vaccine (OPV) immunogenicity compared to industrialized countries, both in terms of humoral and mucosal immunity. To overcome this challenge, further research is needed to elucidate the causes for the suboptimal OPV immunogenicity, better defining the optimal vaccine schedules and delivery strategies, developing and evaluating adjuvants to boost OPV immunogenicity, and improving the methods for directly measuring mucosal immunity.     

贵州省2004年一起疫苗衍生株脊髓灰质炎病毒流行的调查分析

目的证实2004年贵州省贞丰县发生的Ⅰ型疫苗衍生株脊髓灰质炎（脊灰）病毒循环（cVDPVs），分析cVDPVs发生原因，及时发现脊灰野病毒（wild—poliovirus）、疫苗衍生株脊灰病毒（VDPVs）和临床相似症状的其他脊灰疫苗相关株病毒。方法对疫区进行流行病学现场调查，采集急性弛缓性麻痹（AFP）病例及密切接触者粪便标本进行脊灰病毒（PV）分离鉴定与基因序列测定，并对贞丰县近几年报告AFP病例及接触者粪便标本病毒学监测结果进行分析。结果从贞丰县挽澜乡2例AFP病例和3名密切接触者粪便标本中分离到Ⅰ型VDPVs；发生Ⅰ型cVDPVs事件后，5例诊断为临床符合脊灰的AFP病例中有3例分离到Ⅰ型或Ⅱ型脊灰疫苗相关株病毒；病毒学监测结果显示，贞丰县人群中肠道病毒阳性检出率（55．1％）明显高于贵州全省水平（23．2％），2004年PV分离率（36．8％）明显高于往年，16株PV中Ⅰ型所占比例（43．8％）明显高于贵州全省平均水平（18．3％）。结论脊灰Ⅰ型VDPVs已经在贞丰县引起了循环（cVDPVs）；人群中PV和非脊灰肠道病毒带毒率明显增高及疫苗接种率严重低下，是该次Ⅰ型cVDPVs发生的原因；应加强无脊灰状态下疫苗接种率的评估和早期疫情预测工作。     

Effect of substituting IPV for tOPV on immunity to poliovirus in Bangladeshi infants: An open-label randomized controlled trial

BackgroundThe Polio Endgame strategy includes phased withdrawal of oral poliovirus vaccines (OPV) coordinated with introduction of inactivated poliovirus vaccine (IPV) to ensure population immunity. The impact of IPV introduction into a primary OPV series of immunizations in a developing country is uncertain.MethodsBetween May 2011 and November 2012, we enrolled 700 Bangladeshi infant-mother dyads from Dhaka slums into an open-label randomized controlled trial to test whether substituting an injected IPV dose for the standard Expanded Program on Immunization (EPI) fourth tOPV dose at infant age 39 weeks would reduce fecal shedding and enhance systemic immunity. The primary endpoint was mucosal immunity to poliovirus at age one year, measured by fecal excretion of any Sabin virus at five time points up to 25 days post-52 week tOPV challenge, analyzed by the intention to treat principle.FindingsWe randomized 350 families to the tOPV and IPV vaccination arms. Neither study arm resulted in superior intestinal protection at 52 weeks measured by the prevalence of infants shedding any of three poliovirus serotypes, but the IPV dose induced significantly higher seroprevalence and seroconversion rates. This result was identical for poliovirus detection by cell culture or RT-qPCR. The non-significant estimated culture-based shedding risk difference was −3% favoring IPV, and the two vaccination schedules were inferred to be equivalent within a 95% confidence margin of −10% to +4%. Results for shedding analyses stratified by poliovirus type were similar.ConclusionsNeither of the vaccination regimens is superior to the other in enhancing intestinal immunity as measured by poliovirus shedding at 52 weeks of age and the IPV regimen provides similar intestinal immunity to the four tOPV series, although the IPV regimen strongly enhances humoral immunity. The IPV-modified regimen may be considered for vaccination programs without loss of intestinal protection.     

PER.C6 cells as a serum-free suspension cell platform for the production of high titer poliovirus: A potential low cost of goods option for world supply of inactivated poliovirus vaccine

There are two highly efficacious poliovirus vaccines: Sabin's live-attenuated oral polio vaccine (OPV) and Salk's inactivated polio vaccine (IPV). OPV can be made at low costs per dose and is easily administrated. However, the major drawback is the frequent reversion of the OPV vaccine strains to virulent poliovirus strains which can result in Vaccine Associated Paralytic Poliomyelitis (VAPP) in vaccinees. Furthermore, some OPV revertants with high transmissibility can circulate in the population as circulating Vaccine Derived Polioviruses (cVDPVs). IPV does not convey VAPP and cVDPVs but the high costs per dose and insufficient supply have rendered IPV an unfavorable option for low and middle-income countries.     

Safety and immunogenicity of inactivated poliovirus vaccine based on Sabin strains with and without aluminum hydroxide: A phase I trial in healthy adults

Background

An inactivated poliovirus vaccine (IPV) based on attenuated poliovirus strains (Sabin-1, -2 and -3) was developed for technology transfer to manufacturers in low- and middle income countries in the context of the Global Polio Eradication Initiative.

Method

Safety and immunogenicity of the Sabin-IPV was evaluated in a double-blind, randomized, controlled, phase I ‘proof-of-concept’ trial. Healthy male adults received a single intramuscular injection with Sabin-IPV, Sabin-IPV adjuvanted with aluminum hydroxide or conventional IPV. Virus-neutralizing titers against both Sabin and wild poliovirus strains were determined before and 28 days after vaccination.

Results

No vaccine-related serious adverse events were observed, and all local and systemic reactions were mild or moderate and transient. In all subjects, an increase in antibody titer for all types of poliovirus (both Sabin and wild strains) was observed 28 days after vaccination.

Conclusion

Sabin-IPV and Sabin-IPV adjuvanted with aluminum hydroxide administered as a booster dose were equally immunogenic and safe as conventional IPV. EudraCTnr: 2010-024581-22, NCT01708720.     

Effects of hydrostatic pressure on the stability and thermostability of poliovirus: A new method for vaccine preservation

Viruses are a structurally diverse group of infectious agents that differ widely in their sensitivities to high hydrostatic pressure (HHP). Studies on picornaviruses have demonstrated that these viruses are extremely resistant to HHP treatments, with poliovirus appearing to be the most resistant. Here, the three attenuated poliovirus serotypes were compared with regard to pressure and thermal resistance. We found that HHP does not inactivate any of the three serotypes studied (1–3). Rather, HHP treatment was found to stabilize poliovirus by increasing viral thermal resistance at 37 °C. Identification of new methods that stabilize poliovirus against heat inactivation would aid in the design of a more heat-stable vaccine, circumventing the problems associated with refrigeration during storage and transport of the vaccine prior to use.     

Method for the simultaneous assay of the different poliovirus types using surface plasmon resonance technology

The inactivated polio vaccine (IPV) contains viral samples that belong to serotypes 1, 2 and 3. We report here a surface plasmon resonance (SPR)-based technique that permits the simultaneous assay of the individual viral types in the IPV as well as in different bulk intermediates from the industrial vaccine production process. Monoclonal antibodies specific to each of the 3 viral types along with a negative control antibody are captured via an anti-IgG antibody on the surface of the 4 flow cells of the SPR instrument. The viral samples are then injected over these flow cells and the increase in resonance units as a result of virus binding is measured. The method was calibrated by an analysis of the European Working Standard (EWS) for poliovirus vaccines. We show that the antibodies used recognize viruses with functional affinities in the picomolar range permitting an effective capture of the antigen. In addition we demonstrate that the antibodies are highly specific to a given virus type and that the heat induced destruction of the D-antigen abolishes antibody recognition entirely. The technique was found to be reproducible and robust and its response was linear to the antigen concentration. Due to the rapidity of analysis this technique permits an almost real-time follow-up of the industrial production process and may present an alternative to the established ELISA assay for the analysis of the intermediates and the final product.