三种核酸扩增方法检测临床乙脑标本的比较

目的探索三种核酸扩增方法应用于临床乙脑标本检测的敏感性,建立能够适合于临床标本的检测方法,同时比较病毒核酸检测与血清学检测的相关性。方法分别采用普通逆转录PCR、逆转录套式PCR和SyBr Green I实时荧光PCR方法对48份疑似乙脑病例的血清或脑脊液标本进行检测,并比较了三种方法的检出限。结果血清学检测表明,48份标本中有18份标本IgM抗体阳性;3种病毒核酸检测,普通逆转录PCR和SyBr Green I实时荧光PCR方法未能检出阳性标本,而逆转录套式PCR检出15份阳性标本。结论三种核酸检测方法中,逆转录套式PCR最为敏感;单独依赖血清学检测会产生相当一部分假阴性,证明了临床实验室加强乙脑病毒核酸检测对临床诊断具有重要意义。     

实时荧光定量PCR在登革热病毒快速检测中的应用

目的应用实时荧光定量PCR快速检测登革热病毒感染。方法采集疑似登革热患者血清,采用实时荧光定量PCR检测登革热病毒,同时采用ELISA检测血清登革热IgM抗体。结果患者发病第7d血清登革热IgM血清抗体A值为0.236和0.237(临界值0.250),高度疑似阳性,第13d血清IgM抗体阳性。患者发病第2d,通用型核酸检测显示血清登革热病毒核酸含量较多,经过治疗,于第7d病毒拷贝下降。发病第2d,核酸检测确定感染病毒为登革热Ⅲ型;发病第13d血清登革Ⅲ型病毒核酸检测阴性。结论实时荧光定量PCR法具有准确性好、灵敏度高等优点,可用于登革热病毒感染的快速检测。     

分子与血清学检测确诊2017年西非塞拉利昂1例猴痘病毒感染病例

目的 对发生在西非塞拉利昂南部的疑似猴痘病毒感染者进行实验室确诊。 方法 采集疑似感染病人感染后12 d和55 d的血清,利用猴痘特异性引物进行实时定量PCR检测,并对病人血清中痘苗病毒IgG、IgM和猴痘病毒IgG抗体进行ELISA检测,并结合其流行病学信息进行诊断结果 实时定量PCR检测该病人感染12 d后血清为猴痘病毒核酸阳性;ELISA检测病人感染后12 d和55 d血清,痘苗病毒IgG、IgM为阳性;猴痘病毒IgG抗体检测均为阳性,且55 d较12 d抗体滴度有4倍升高(3 200/800)。 结论 病人血清的核酸检测和血清学检测结果为阳性,结合病人的临床特征和野生动物接触史,确诊该病人为猴痘病毒感染。     

多重套式RT-PCR检测患者凝血块中登革病毒RNA

目的 建立简便、灵敏、适合于全部血清型登革病毒核酸检测的多重套式RT-PCR体系,检测临床样品中登革病毒RNA,作为实验室辅助诊断的依据。 方法 利用登革病毒标准株核酸,建立多重RT-PCR检测方法。提取患者凝血块总RNA,分别用一步法RT-PCR及多重套式RT-PCR检测。 结果 通过对检测体系进行优化,多重RT-PCR能够同时检测4种血清型登革病毒核酸。采用多重套式RT-PCR方法,从8例登革热患者凝血块中有4例检测到病毒RNA,而其它核酸检测方法近检出1例阳性。 结论 多重套式RT-PCR的方法能够从临床凝血块样品中检测到登革病毒核酸,并同时进行血清学分型,简化了登革病毒核酸检测步骤,有利于对临床样品开展病毒核酸检测。     

3种方法对发热伴血小板减少综合征检测结果比较

目的 采用实时荧光PCR法、酶联免疫吸附法及细胞培养分离病毒株的方法,对2013年临床疑似发热伴血小板减少综合征患者急性期血进行检测比较,寻求快速、准确的鉴定新布尼亚病毒感染的方法。方法 对180例临床疑似病例的急性期血液首先采用实时荧光PCR法检测,筛选出新布尼亚病毒核酸阳性的样本,同时将阳性样本采用酶联免疫吸附的方法检测抗体及采用VERO-E6细胞培养的方法分离病毒株。结果 180份样本经实时荧光PCR法检测,阳性率为42.78%(77/180 Ct值≤35曲线形态与阳性对照一致呈S型的),ELISA方法检测抗体阳性率为44.16%(34/77),将核酸检测阳性的77份样本全部感染VERO-E6细胞获得病毒株32株,阳性率为38.55%(32/77)。结论 实时荧光PCR法更适合于新布尼亚病毒感染者早期实验室快速诊断,发病在1w左右的病例不适合用ELISA的方法做确证实验。细胞培养分离病毒虽然是金标准,但耗时、费力,对早期诊断意义不大,但适于研究或疫苗研发。     

实时荧光RT-PCR与细胞培养法在流感病毒检测中的应用

目的探讨实时荧光逆转录聚合酶链式反应(rRT-PCR)在流感病毒检测中的应用,并比较与常规细胞培养方法的差异,以确定两种检测方法各自更适合的应用领域。方法收集烟台市两所国家级哨点医院就诊的流感样病例(ILI)咽拭子标本,应用实时荧光RT-PCR检测流感病毒核酸,阳性标本用狗肾传代细胞(MDCK)培养、分离流感病毒,比较两种检测方法的灵敏度及相关性差异。结果在617份标本中,实时荧光RT-PCR检测阳性152份,阳性率为24.64%;MDCK细胞培养法分离流感病毒79株,阳性率为12.80%。两者的阳性率差异有统计学意义(χ2=28.38,P<0.01)。结论实时荧光RT-PCR和细胞培养方法检测流感病毒具有一致的特异性。实时荧光RT-PCR可作为一种快速有效的流感病毒核酸检测法,适用于公共卫生应急疫情的实验室快速诊断;细胞培养法作为流感病原学监测的基础,用于核实暴发疫情的实验室诊断,对病毒进行抗原性和基因特性分析。     

实时荧光定量PCR在手足口病肠道病毒快速检测中的应用

目的建立快速检测手足口病肠道病毒的实时荧光定量PCR法,并作出应用分析。方法采用卫生部《手足口病预防控制指南》(2008年版)推荐的RT-PCR法,对丽水市2008年4～5月间发生的172例临床诊断手足口病患者的324份标本进行人肠道病毒、柯萨奇病毒A组16型和肠道病毒EV71型特异性核酸的检测,同时采用实时荧光定量PCR法进行平行检测,比较两者的实验结果,分析实时荧光定量PCR方法的特异性和灵敏度。结果实时荧光定量PCR检测324份标本,肠道病毒通用核酸阳性70份,柯萨奇A16核酸阳性22份,肠道EV71病毒核酸阳性15份,与RT-PCR结果一致,符合率100%。结论实时荧光定量PCR法具有特异性强、灵敏度高等优点,是一种理想的手足口病肠道病毒的检测方法。     

输血传播病毒PCR-DHPLC检测技术的建立及初步应用

目的应用聚合酶链式反应(PCR)结合变性高效液相色谱(DHPLC)技术检测输血传播病毒。方法根据输血传播病毒的核酸序列特点设计特异性引物进行PCR,将扩增产物进行DHPLC检测分析,以验证方法的灵敏度、特异性、重复性,同时进行临床检测的初步应用。结果以乙型肝炎病毒、丙型肝炎病毒、戊型肝炎病毒进行特异性试验,无交叉反应,具有较好的特异性;重复性良好;灵敏度可达1.0×10~1拷贝/反应。分别应用实时荧光PCR法、普通PCR法及本文所建立的PCR-DHPLC法同时检测32份血清样本,发现有17份样本实时荧光PCR阳性,17份样本PCR-DHPLC阳性,15份普通PCR阳性。结论本文建立的PCR-DHPLC法具有特异、敏感、重复性好等优点,可用于输血传播病毒感染的分子流行病学调查。     

基因1型狂犬病病毒一步法荧光定量RT-PCR检测方法的建立

目的在我国狂犬病均由基因1型狂犬病病毒（rabies virus,RV）引起。本研究针对RV N基因保守序列设计并合成了一套简并引物和Taqman探针,在优化反应条件的基础上,建立了检测RV核酸的一步法荧光定量RT-PCR（qRT-PCR）检测方法。方法与结果该方法能特异检测基因1型,不能检测基因2-7型和犬瘟热病毒（CDV）、犬细小病毒（CPV）、犬腺病病毒（CAV）、水泡性口炎病毒（VSV）、日本乙型脑炎病毒（JEV）等5种非狂犬病病原体,其检测灵敏度可达到4.68个TCID50的病毒含量。用该方法对29份新鲜和5份腐败的临床犬脑组织样品进行检测,并与国际金标准确诊方法狂犬病荧光抗体染色法（FAT）和乳鼠脑内接种试验（MIT）及本实验室建立的套式RT-PCR方法进行比较。结果表明所建立的qRT-PCR与套式RT-PCR的符合率为100%,二者均检测出12份阳性的新鲜样品和5份阳性的腐败样品,而FAT只检测出12份阳性的新鲜样品和1份阳性的腐败样品,MIT只检测出12份阳性,未检测出阳性腐败样品。检测中FAT和MIT确诊的所有阳性样品在qRT-PCR检测均是阳性,而在FAT检测为阴性的4份腐败样品在qRT-PCR为阳性,说明所建立的qRT-PCR方法准确性达到了FAT和MIT的水平,而且灵敏度更高,更适合于腐败样品的检测。结论研究结果表明该qRT-PCR方法特异性好、灵敏度高、污染率低、操作简单,在我国动物狂犬病临床诊断上具有巨大的应用价值。     

抗体捕捉酶联免疫吸附法在流行性乙型脑炎早期诊断上的应用

用MacELISA测定了138份临床疑似乙脑病人的血清标本,并与HI试验进行了比较。MacELLSA能特异、敏感地检出乙脑患者发病第2天的IgM抗体,阳性率为55.56％。第4、5病日,IgM抗体阳性率高达92.86％。10例正常人和10份流行性出血热病毒抗体阳性的血清标本无一份阳性。     

浙江省猪血清中乙脑病毒的分离鉴定及抗体的测定

目的对浙江嘉兴地区采集的猪血清进行乙型脑炎病毒分离鉴定以及了解该地区猪感染乙型脑炎病毒的状况。方法在浙江省嘉兴地区采集猪血清标本,利用病毒分离方法检测猪血清是否携带乙脑病毒,利用间接免疫荧光法对猪血清进行抗体检测。应用荧光定量RT PCR方法对新分离病毒进行鉴定;设计特异性引物,利用RT PCR方法扩增新分离乙脑病毒PrM-E基因,并对新分离病毒进行基因分型和E基因的分析。结果共采集240份猪血清标本,抗体检测阳性率为61.6%。其中6月中旬50%以上的猪乙脑病毒抗体呈阳性。分离出3株病毒。经荧光定量RT PCR鉴定3株病毒均为乙脑病毒,基因分析表明3株病毒均属于基因I型乙脑病毒。对这2株乙脑病毒的E基因区段进行分析,它们之间核苷酸和氨基酸的同源性均为100%。与疫苗株SA14 14 2相比,核苷酸同源性在87.7%,氨基酸同源在97.0%以上。结论浙江省嘉兴地区猪群中存着乙脑病毒的隐性感染或曾经感染过,提示在该地区自然界中存在着乙脑病毒。     

Intense transmission of Japanese encephalitis virus to pigs in a region free of epidemic encephalitis

Epidemic Japanese encephalitis recurs annually in the northern provinces of Thailand, but in the southern provinces cases of human encephalitis are rare. We investigated transmission of Japanese encephalitis virus (JEV) to pigs in southern Thailand. Blood specimens from one hundred young pigs at abattoirs in three southern provinces were tested for JEV hemagglutination inhibiting (HAI) antibodies. Seventy-four percent were positive. Ten seronegative sentinel pigs were placed at five locations in one southern province. Seven of the ten pigs developed JEV HAI and JEV IgM ELISA antibodies within two weeks of placement. JEV was isolated from all seven seroconverting sentinel pigs from blood specimens collected 3 to 11 days after placement. Fifteen light-trap mosquito collections at the five locations all included known JEV vectors, some in large numbers. We conclude that there is intense transmission of JEV to pigs in southern Thailand despite the rare occurrence of human encephalitis in the same region.     

猪乙脑病毒IgG抗体间接免疫荧光检测方法的建立与初步应用

目的建立检测猪血清中流行性乙型脑炎病毒(JEV)IgG抗体的间接免疫荧光试验(IFA)。方法用乙脑病毒感染C6/36白蚊伊蚊细胞制备抗原片,建立检测猪血清中JEV-IgG抗体的IFA方法,并用于猪乙脑血清流行病学调查。结果成功建立特异的猪JEV-IgG的IFA检测方法,并对50份母猪、55份仔猪和65份屠宰猪的血清进行检测。乙脑血清抗体效价≥1:200的分别为88.00%,10.91%和24.62%。结论建立的IFA方法能较好的用于猪血清乙脑流行病学调查。     

Japanese encephalitis: a review of the Indian perspective

Japanese encephalitis virus (JEV) causes Japanese encephalitis, which is a leading form of viral encephalitis in Asia, with around 50,000 cases and 10,000 deaths per year in children below 15 years of age. The JEV has shown a tendency to extend to other geographic regions. Case fatality averages 30% and a high percentage of the survivors are left with permanent neuropsychiatric sequelae. Currently, there is no cure for JEV, and treatment is mainly supportive. Patients are not infectious, but should avoid further mosquito bites. A number of antiviral agents have been investigated; however, none of these have convincingly been shown to improve the outcome of JEV. In this review, the current knowledge of the epidemiology and the pathogenesis of this deadly disease have been summarized.     

A serological study of Japanese encephalitis virus infections in northern Peninsular Malaysia.

This study describes the status of viral encephalitis in Perak, Malaysia during the year 1990. In addition, 14 cases selected from Penang and Perak during the years 1989 and 1990 are presented, with data showing titers of neutralizing antibodies against Japanese encephalitis virus (JEV) and dengue 2 virus, titers of antibodies against JEV and dengue virus antigens as determined by DEIA, and a comparison of these with the presence of IgM to JEV and dengue virus. These data show that there probably is far more viral encephalitis due to JEV in Malaysia than the national figures reflect.     

Increasing trend of Japanese encephalitis cases in West Bengal,India – a threat to paediatric population

ObjectiveTo detect the Japanese encephalitis virus (JEV) as the etiologic agent from the acute encephalitis syndrome (AES) cases mainly amongst the children and young adults from vaccinated and non-vaccinated districts of West Bengal.MethodsFor the detection of JEV, a total of 828 sera were referred from vaccinated and non vaccinated districts of West Bengal during 2005-2011. Japanese encephalitis (JE) positive cases were confirmed by ELISA and RT-PCR method.ResultsOut of 828 cases, 245 samples were positive by ELISA method and 46 samples were positive by RT-PCR method. Out of 291 total positive cases, 162 (55.6%) were below 20 years of age. Initially in 2005, JE cases were highest amongst the children and young adults (0-20 years). After vaccination, although the JE cases declined gradually in the vaccinated districts, but again from 2010, JE cases from the said age group showed an increasing trend from those districts. JE cases were also reported from other endemic zones of this state, which were still non-vaccinated.ConclusionsIn West Bengal, JE cases are still predominated among children and young adults till the year 2011. Mass scale vaccination programme and investigation on the circulating strains are essentially required to find out the reasons of increasing tendency of JE cases in this state.     

A diagnostic algorithm to serologically differentiate West Nile virus from Japanese encephalitis virus infections and its validation in field surveillance of poultry and horses

The detection of West Nile virus (WNV) in areas endemic for Japanese encephalitis virus (JEV) is complicated by the extensive serological cross-reactivity between the two viruses. A testing algorithm was developed and employed for the detection of anti-WNV antibody in areas endemic for JEV. Using this differentiation algorithm, a serological survey of poultry (2004 through 2009) and horses (2007 through 2009) was performed. Among 2681 poultry sera, 125 samples were interpreted as being positive for antibodies against JEV, and 14 were suspected to be positive for antibodies against undetermined flaviviruses other than WNV and JEV. Of the 2601 horse sera tested, a total of 1914 (73.6%) were positive to the initial screening test. Of these positive sera, 132 sera (5.1%) had been collected from horses that had been imported from the United States, where WNV is endemic. These horses had WNV vaccination records, and no significant pattern of increasing titer was observed in paired sera tests. Of the remaining 1782 positive sera 1468 sera (56.4%) were also found to contain anti-JEV antibodies, and were interpreted to be JEV-specific antibodies by the differentiation algorithm developed in this study. The remaining 314 horses (12.1%) for which a fourfold difference in neutralizing antibody titer could not be demonstrated, were determined to contain an antibody against an unknown (unidentified or undetermined) flavivirus. No evidence of WNV infections were found during the period of this study.