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目的建立脑心肌炎病毒( EMCV) TaqMan real-time PCR检测方法。方法根据GenBank中公布的EMCV 3D基因保守区段设计并合成1对引物和1条TaqMan 探针,建立EMCV TaqMan real-time PCR检测方法,且对体系进行优化;对该法进行灵敏性、特异性验证;采用建立的方法对98份猪血清样本进行检测,并与ELISA结果进行比较。结果建立的EMCV TaqMan real-time PCR检测方法线性关系较好,以质粒标准品构建的标准曲线相关系数R2为0.995;灵敏性比普通PCR高100倍,且仅能特异性检出 EMCV;对猪血清样本的检测与 ELISA 法检测结果符合率为98.0%。结论已建立了EMCV TaqMan real-time PCR检测方法,该法灵敏性高、特异性好,可用于EMCV的检测及定量分析。 相似文献
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登革病毒1~4型(dengue virus type 1-4,DV1-4)、流行性乙型脑炎病毒(Japanese encephalitis virus,JEV)及黄热病病毒(yellow fever virus,YFV)均为黄病毒属的重要传染病病原,其流行季节及临床症状都极为相似,且暴发日益频繁,发展特异、敏感的快速诊断及鉴别诊断方法,对这类疾病的预防及控制具有重要意义。本研究在对其基因组序列系统分析的基础上,建立了快速检测及鉴别诊断上述病毒的微孔杂交(PCR-ELISA)方法。 相似文献
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目的探讨乙型脑炎病毒反向遗传学系统在临床中的应用价值。方法对在我院自2011年12月至2013年1月接受治疗的80例乙型脑炎病毒患者资料进行回顾性分析,医护人员根据患者住院时间将其分为实验组和对照组,每组有患儿40名。对照组患者采用传统方法进行治疗,实验组患者在传统方法基础上利用分子生物学技术构建获得乙型脑炎病毒复制子,比较两组患者的诊断效果及其价值。结果实验组患者总有效率为95%,优于对照组患者;两组患者神经功能状况指数、患者意识评分等指标治疗前没有差异性,患者治疗后,两组患者指标明显改善,实验组患者改善效果更好FEVl(87.2±10.5VS70.1±8.2)。结论临床上医护人员对乙型脑炎病毒在常规诊断和治疗基础上利用反向遗传学技术临床上效果较好,患者确诊率较高,能够为治疗乙型脑炎病毒提供依据,值得推广使用。 相似文献
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目的建立实时定量PCR检测血浆病毒载量的方法,对马传染性贫血病毒(equine infectious anemia virus,EIAV)强毒株攻毒马和疫苗免疫攻毒马血浆中病毒载量进行了跟踪检测,探讨病毒载量和临床疾病状态的相关性。方法以EIAV强毒株LN40序列为标准,在gag保守区设计1对引物和Taqman探针,用于实时定量PCR扩增,用含扩增目的基因的体外转录RNA作标准品,获得标准曲线,对扩增样品进行准确定量。强毒株LN40直接攻毒,或者疫苗株DLV免疫马6个月后用强毒株LN40进行攻毒,跟踪检测攻毒马及免疫攻毒马血浆中EIAV载量情况。结果反应在10^1~10^9copies/ml之间具有良好的线性关系,反应的检出下限为10copies/ml。强毒攻毒马出现发热并最终死亡,发热期间马血浆中EIAV载量与体温呈正相关,载量最高达10^7copies/ml。免疫攻毒马未出现发热,其血浆EIAV载量的总体水平低于强毒攻毒马,攻毒后3个月低至10copies/ml以下。结论成功建立了实时定量PCR检测血浆EIAV载量的方法,并证实了用实时荧光定量PER检测EIAV病毒载量的方法来监测动物感染状态具有可行性,为EIAV致病机制研究和弱毒疫苗免疫保护机制的研究提供良好的技术平台。 相似文献
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目的:建立快速荧光灶试验(fluorescence focus assay,FFA)滴定乙型脑炎病毒(Japanese encephalitis virus, JEV)的方法,验证该法替代病毒蚀斑法测定乙型脑炎减毒活疫苗(简称乙脑减毒活疫苗)滴度的可行性并将该法作初步应用。方法:利用原核表达方式构建JEV非结构蛋白1(... 相似文献
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目的 建立特异、敏感、快速检测肝螺杆菌的TaqMan MGB探针实时荧光定量PCR方法.方法 针对肝螺杆菌flaB 基因的保守区设计特异性引物和探针,建立肝螺杆菌TaqMan MGB探针实时荧光定最PCR方检测方法,验证方法的特异性、敏感性和稳定性.对2008-2011年期间采集的1081份临床样本中的肝螺杆菌进行检测,同时进行分离培养和常规PCR检测.结果 建立的TaqMan MGB探针实时荧光定量PCR方法对肝螺杆菌的检测具有高度的特异性,对幽门螺杆菌、空肠弯曲菌、泰泽氏菌、侵肺巴斯德氏菌、大肠埃希菌、铜绿假单胞菌均无交叉反应,检测的灵敏度达8.3拷贝.标准曲线显示各浓度范围内具有良好的线性关系,相关系数为0.999,斜率为-3.227,TaqManMGB探针实时荧光定量PCR效率为100%.对1081份临床样本进行检测,TaqMan MGB探针实时荧光定量PCR和常规PCR均能检出86份肝螺杆菌阳性样本,而细菌分离培养则仅检出4份阳性.结果显示,建立的TaqMan MGB探针实时荧光定量PCR方法比细菌分离培养方法更敏感,能够直接从临床样本中检出肝螺杆菌DNA,检测时间仅为2h.结论 研究建立的TaqMan MGB探针实时荧光定量PCR方法具有可靠、特异、敏感的特点,适用于肝螺杆菌的快速检测. 相似文献
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目的建立TaqMan探针实时荧光定量RT-PCR方法,测定登革热病毒(DV)及DV病毒的RNA拷贝数。方法利用TaqMan探针,建立实时荧光定量RT-PCR方法,通过对登革热病毒RNA定量外标准品的定量分析,优化反应体系,检测TaqMan探针实时荧光定量RT-PCR方法的灵敏度、特异性和重复性。结果该方法检测灵敏度可达1×103copies/mL,特异性及重复性良好,对同一样品进行5次重复检测,其循环阈值的平均标准偏差为0.792。结论TaqMan探针实时荧光定量RT-PCR法特异性、敏感性高,稳定性好,可用于定量测定登革热病毒及DVRNA载量。 相似文献
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HAI YAN ZHANG WEN LI MA XIAO MING ZHANG WEN LING ZHENG Institute of Molecular Biology Southern Medical University Guangzhou P.R. China Center of Disease Control of Guangzhou Military District Guangzhou P.R. China Liu Hua Qiao Hospital Guangzhou P. R. China 《中华微生物学和免疫学杂志(英文版)》2006,4(2):96-99
A low-density oligonucleotide microarray was used for the detection of Japanese encephalitis virus (JEV) , combining with restriction display PCR labeling method. The hybridization targets were amplified from 6 plasmids containing several JEV gene fragments. Corresponding oligonucleotide probe spots were detected unambiguously. We claim that the oligonucleotide microarray technology is feasible and may have potential for clinical laboratory application. 相似文献
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逆转录-聚合酶链反应方法检测乙型脑炎病人标本 总被引:11,自引:0,他引:11
目的 逆转录-聚合酶链反应(RT-PCR)检测乙型脑炎(乙脑)病人标本方法的建立和评估。方法 建立RT-PCR法,了解该方法用于乙脑病毒检测的敏感性,特异性,并用于临床疑似乙脑病人血清及脑脊液(CSF)标本的检测,并与反向被动血凝抑制实验(RPHI)方法进行比较分析。结果 用该RT-PCR法检测高顺生株(高株)敏感性可达64PFU。共检测临床疑似乙脑病人标本38份,对CSF中乙型脑炎病毒(JEV) 相似文献
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目的 从四川省巴中市采集的蚊虫标本中分离乙型脑炎(简称乙脑)病毒(JEV),确定其基因型别,并分析相关的基因1型乙脑病毒PrM和E基因区段氨基酸序列特征.方法 对2004年采集蚊虫标本进行病毒分离,对新分离的乙脑病毒进行生物学、血清学及分子生物学鉴定.逆转录聚合酶链反应(RT-PCR)扩增新分离JEV的PrM、E区段核苷酸序列,测序后应用Clustal X软件做碱基配对分析,MEGA4软件完成病毒进化分析,GENEDOC(3.2)软件完成氨基酸位点分析,根据蜱传脑炎病毒可溶性蛋白晶体结构为模板进行乙脑病毒E蛋白三维结构模拟预测分析.结果 共采集4668只蚊虫标本,主要是骚扰阿蚊和库蚊,分离到6株病毒,经鉴定均属于基因1型的乙脑病毒.将四川省分离的6个毒株结合我国新分离的基因1型乙脑病毒与减毒活疫苗株SA14-14-2株的PrM区段和E区段氨基酸比较,发现PrM区段在PrM2、64和65位存在基因1型乙脑病毒独有的氨基酸位点差异,E区段存在14处共同的氨基酸位点差异,其中在E129、222、327和366位点为中国目前分离到的基因1型乙脑病毒所特有的位点特征.结论 从四川省巴中市首次分离到基因1型的乙脑病毒,并发现基因1型乙脑病毒与减毒活疫苗株之间PrM、E基因区段存在氨基酸差异,但现行疫苗株理论上可以保护新分离的基因1型乙脑病毒. 相似文献
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Hepatopancreatic parvovirus is an emerging disease in crustacean aquaculture. Consequently, methods of detection are needed that enable the sensitive detection and confirmation of the virus better than currently used methods such as histology and conventional polymerase chain reaction (PCR). A TaqMan based real-time PCR assay was developed for the detection of the Australian isolate of hepatopancreatic parvovirus which is only 85% similar to its nearest known relative. The TaqMan assay was developed within the capsid protein region of the genome and is optimised to detect as little as 10 copies of the targeted sequence per PCR vial. The hepatopancreatic parvovirus primers and probe were HPV140F 5′-CTA CTC CAA TGG AAA CTT CTG AGC-3′, HPV140R 5′-GTG GCG TTG GAA GGC ACT TC-3′ and HPV140probe 5′-FAM TAC CGC CGC ACC GCA GCA GC TAMRA-3′, respectively. The assay was specific for the hepatopancreatic parvovirus strain from Australian Penaeus merguiensis as it did not detect related crustacean and canine parvoviruses from Australia. In addition, the very low homology of the target sequence with published sequences from the Thai and Korean strains of hepatopancreatic parvovirus and other prawn viruses such as WSSV, suggested this assay would be specific for the Australian hepatopancreatic parvovirus isolate. Furthermore, it detected hepatopancreatic parvovirus in 22/22 wild-caught P. merguiensis clinical samples and 473/545 (87%) farmed P. merguiensis. This assay has the potential to be used for diagnostic purposes and in robotic applications, particularly for the detection and quantitation of low-grade infections. 相似文献
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Ching-Kai Chuang 《Virology》2009,394(2):286-297
Due to the lack of a proofreading function and error-repairing ability of genomic RNA, accumulated mutations are known to be a force driving viral evolution in the genus Flavivirus, including the Japanese encephalitis (JE) virus. Based on sequencing data, RNA recombination was recently postulated to be another factor associated with genomic variations in these viruses. We herein provide experimental evidence to demonstrate the occurrence of RNA recombination in the JE virus using two local pure clones (T1P1-S1 and CJN-S1) respectively derived from the local strains, T1P1 and CJN. Based on results from a restriction fragment length polymorphism (RFLP) assay on the C/preM junction comprising a fragment of 868 nucleotides (nt 10-877), the recombinant progeny virus was primarily formed in BHK-21 cells that had been co-infected with the two clones used in this study. Nine of 20 recombinant forms of the JE virus had a crossover in the nt 123-323 region. Sequencing data derived from these recombinants revealed that no nucleotide deletion or insertion occurred in this region favoring crossovers, indicating that precisely, not aberrantly, homologous recombination was involved. With site-directed mutagenesis, three stem-loop secondary structures were destabilized and re-stabilized in sequence, leading to changes in the frequency of recombination. This suggests that the conformation, not the free energy, of the secondary structure is important in modulating RNA recombination of the virus. It was concluded that because RNA recombination generates genetic diversity in the JE virus, this must be considered particularly in studies of viral evolution, epidemiology, and possible vaccine safety. 相似文献
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中国基因3型乙型脑炎病毒E基因分子特征 总被引:1,自引:0,他引:1
目的 以减毒活疫苗(SA14-14-2株)为对照,分析我国分离的基因3型乙脑病毒E基因区段核苷酸及氨基酸序列分子特征.方法 从GenBank中获取相应乙脑病毒株E基因区段核苷酸序列,通过Clustal X(1.81)、DNAStar、GENEDOC(3.2)等生物学软件进行核苷酸和氨基酸位点差异分析.以蜱传脑炎病毒可溶性蛋白晶体结构为模板进行乙脑病毒E蛋白氨基酸位点分析.结果 我国不同地域、不同宿主分离的基因3型乙脑病毒与SA14-14-2株核苷酸同源性分别在96%和95%以上,氨基酸同源性在95%和94%以上.在同一地域、同一宿主类型分离的毒株之间核苷酸和氨基酸同源性非常高.在E基因区段存在10处共同的氨基酸位点差异,在结构域Ⅰ(E160)、结构域Ⅱ(E123和E227)和两个未在结构域中的氨基酸位点(E441和E487)等5个位点在部分基因3型乙脑病毒中存在差异.结论 我国分离的基因3型乙脑病毒与减毒活疫苗株(SA14-14-2株)E基因区段同源性高,存在5处基因3型乙脑病毒特异的氨基酸位点差异,但现行减毒活疫苗株理论上可以保护我国分离的基因3型乙脑病毒野毒株. 相似文献
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V. Ravi Anita S. Desai Poornima K. Shenoy P. Satishchandra A. Chandramuki M. Gourie-Devi 《Journal of medical virology》1993,40(4):326-329
Immunological and virological evidence for persistence of Japanese encephalitis virus (JEV) in the human nervous system is described in 16/323 (5%) laboratory-confirmed cases of Japanese encephalitis. In 9/16 patients, JEV specific IgM antibodies were detected in the CSF even at 50–180 days after the onset of symptoms. Similarly, in 7/16 patients, apart from IgM antibodies, viral antigen was also present in the CSF beyond the third week of illness and in one patient it could be detected even at 117 days. Infectious virus could be isolated from the CSF beyond the third week of illness in 3/16 patients. In one patient, JEV was isolated from the CSF on three consecutive occasions at 90, 110, and 117 days after onset of clinical symptoms. These findings suggest that JEV persists in the nervous system of a small proportion of patients. © 1993 Wiley-Liss, Inc. 相似文献