一种同时检测丙型与庚型肝炎病毒的逆转录-巢式聚合酶链反应方法

目的庚型肝炎病毒（ＨＧＶ）与丙型肝炎病毒（ＨＣＶ）同属黄病毒科，且传播途径相似，重叠感染率高，本研究旨在建立一种同时检测ＨＧＶ与ＨＣＶ感染的方法。方法根据ＨＣＶ与ＨＧＶ的基因序列分别选取５’－ＵＴＲ（ＨＣＶ）与ＮＳ３（ＨＧＶ）的两套引物，在同一管内进行逆转录－巢式聚合酶链反应，并初步应用于１５３例标本。结果该方法能同时检出ＨＧＶ与ＨＣＶ感染，扩增片段大小与设计相符。结论该方法简便特异，适用于临床检测     

输血后丙型肝炎与抗丙型肝炎病毒（HCV）阳性献血？…

采用丙型肝炎病毒（ＨＣＶ）５’－非编码区（５’－ＮＣＲ）和Ｃ区具有型特异性的引物建立的逆转录－聚合酶链反应方法检测了２５例丙型肝炎（ＨＣ）和２６例抗－ＨＣＶ阳性献血员的ＨＣＶ ＲＮＡ及基因型。结果２５例ＨＣ患者ＨＣＶ ＲＮＡ阳性率为１００．０％，２６例抗－ＨＣＶ阳性献血员ＨＣＶ ＲＮＡ阳性率为８４．５％，（２２／２６）。２５例ＨＣ中Ｉ、Ⅱ和混合型（Ｉ＋Ⅱ、Ⅱ＋Ⅲ、Ｉ＋Ⅲ、Ｉ＋Ⅱ＋Ⅲ）分别占８．０％     

逆转录—巢式聚合酶链反应检测庚型肝炎病毒RNA方法…

庚型肝炎病毒基因组为单链，正肌ＲＮＡ，全长９３９２ｂｐ。根据５‘－非编码区基因序列设计合成两对引物。随机选取酶联抗－ＨＧＶ阳性病人血清３份，阴性病人血清６份，应用逆转录－巢式聚合酶链反应进行检测。结果２份抗－ＨＧＶ阳性血清可见较强的特异扩增带，１份抗－ＨＧＶ阳性血清可见较弱的特异扩增带，其余６份抗－ＨＧＶ阴性血清均无特异性扩增带。     

丙型肝炎病毒抗原检测方法的建立

目的以特异性单克隆抗体为基础建立丙型肝炎病毒(HCV)抗原检测的酶联免疫吸附(ELISA)法,探索从血浆或血清中检测HCV抗原的可能性.方法利用我们制备的抗-HCV核心及NS3区单克隆抗体(McAbs),进行多种交叉组合模式的分析,确立实验室模式,并测定348份义务献血员血样,确定此方法的Cutofff值,并分析146份抗-HCV阳性及225份抗-HCV阴性血浆,阳性结果用套式PCR试剂盒确证.结果构建了以抗-HCV核心区单抗C39及NS3区单抗C7-6为包被抗体,以C39及NS3区C7-57为标记抗体的夹心ELISA检测模型,以C7为抗原,其检出灵敏度为5ng/ml,其Cutoff值为阴性对照均值±0.25.146份抗-HCV阳性样本中,11份为抗原反应阳性,225份抗-HCV阴性样本中,16份为抗原阳性,这些阳性样本经PCR检测后,23份为HCVRNA阳性.结论用单抗构建的HCV抗原检测方法分别从抗-HCV阳性及阴性样本中检测出HCV抗原反应阳性样本,并经PCR确证,表明直接从血浆样本中检测HCV抗原是可能的,这将对于HCV和基础研究及控制HCV的传播有重要意义.     

丙型肝炎病毒基因酶切分型及其在干扰素治疗中的…

为了解广州地区丙型肝炎病毒感染的基因型及其与对干扰素应签效果的关系，应用逆转录－聚合酶链反应对１３３例丙型肝炎病人血清进行ＨＣＶ５’端非编码区基因片段扩增，对１１６例阳性ＰＣＲ产物进行酶切分型。并了国产基因工程干扰素α１对其中５１例慢性丙型肝炎的疗效。     

多重引物聚合酶链反应扩增丙型肝炎病毒基因及基 …

利用聚合酶链反应（ＰＣＲ）技术对丙型肝炎病毒（ＨＣＶ）的５’－非编码区（５’－ＮＣＲ）、Ｃ及ＮＳ４基因区的３对引物分别及同时扩增，检测８０例抗－ＨＣＶ阳性患者的血清ＨＣＶ ＲＮＡ，并进行了ＨＣＶ基因分型研究。各不同引物所介导的ＰＣＲ检出ＨＣＶ ＲＮＡ的结果为：５’－ＮＣＲ基因区６０％（４８／８０），Ｃ基因区３７％（３０／８０），ＮＳ４基因区３０％（２４／８０）。以上３对引物同时扩增仅４２％（３４／     

山西省不同人群丙型肝炎病毒的基因分型研究

目的探索丙型肝炎病毒(HCV)基因型在山西省不同人群中的分布规律及流行的优势型。方法用RT-PCR和型特异性引物逆转录巢式PCR法,对山西省271例抗HCV阳性的丙型肝炎病人、原发性肝细胞癌患者、非肝癌癌症患者、性关系混乱者和性病患者、职业献血员、吸毒者及公共场所从业人员进行了HCVRNA的检测和基因分型。结果271份抗HCV阳性标本中,HCVRNA检出率为45.45%～89.66%,平均67.52%。以丙型肝炎病人、献血员和吸毒者的HCVRNA检出率较高(76.9%～89.7%),χ2=30.44,P<0.01。在133份HCVRNA阳性血清中,仅检出了108例1b型、2a型和此两种基因型的混合感染者。未检出1a型、2b型和3a型。其中1b型占80.00%(88例),2a型占11.81%(13例),混合型占6.36%(7例)。在肝癌患者和献血员中,仅检出1b型的感染;非肝癌的其他癌症患者中,未发现混合感染。各基因型在各人群中的分布比例也有差别,丙型肝炎患者、非肝癌的其他癌症患者、吸毒者和从业人员的各基因型构成比较接近,均以1b型为主。而性病患者和性关系混乱者中1b型和2a型感染者比例相等。结论山西省HCV的基因以1b型占优势。     

逆转录─巢式PCR在一管中同时检测丙型与庚型肝炎病毒

逆转录┐巢式ＰＣＲ在一管中同时检测丙型与庚型肝炎病毒谭文杰夏宁邵丛郁庚型肝炎病毒（ＨＧＶ）与丙型肝炎病毒（ＨＣＶ）同属黄病毒科，且传播途径相似，重叠感染率高。本室根据对ＨＧＶ与ＨＣＶ基因序列的分析，分别选取５′ＵＴＲ（ＨＣＶ）与ＮＳ３（ＨＧＶ）区的两...     

二重逆转录—聚合酶链反应及微孔板反向杂交法检？…

目的 为适应临床上需同时检测庚型肝炎病毒（ＨＧＶ）与丙型肝炎病毒（ＨＣＶ）感染情况，建立了二重逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）及微孔板反向杂交法。方法 根据ＨＣＶ与ＨＧＶ基因与ＨＧＶ特异探针微孔板反向杂交检测ＣＰＲ产物。结果 ＰＣＲ产物经测序，ＨＣＶ与Ｔａｋａｍｉｚａｗａ等及Ｃｈｏｏ等报道的核苷酸同源性分别为９３．１％￣９４．１％与９２．５％￣９３．７％，ＨＧＶ与Ｓｉｍｏｎｓ等、Ｌｉｎｎｅｎ等     

聚合酶链反应鉴定丙型肝炎病毒母婴传播的状态及其…

采用多种方法,动态检测了１１例丙型肝炎病毒感的孕妇所生的婴儿血抗－ＨＣＶ和ＨＣＶＲＮＡ。发现用合成肽酶联免疫吸附试验检测婴儿抗－ＨＣＶ阳性率显著低于第二低重组抗原ＥＬＩＳＡ;用２ｎｄＥＬＩＳＡ检测,６例婴儿脐血和静脉血抗－ＨＣＶ阳性,５例持续１－５月阴转,１例阳性持续１３个月。     

用逆转录—套式聚合酶链反应检测我国不同临床型肝？…

目的 为了研究庚型肝炎病毒（ＨＧＶ）在我国的感染状况。方法 概括已发表的ＨＧＶ的５‘端非编码区（５’－ＵＴＲ区）及螺旋酶区（ＮＳ３区）两段高度保守的基因序列分别设计两套引物，用逆转录－套式聚合酶链式反应检测ＨＧＶＲＮＡ。结果 从北京、秦皇岛、河南等地采集各种肝病患者及职业献血员血清３５４份，ＨＧＶＲＮＡ阳性９７份，阳性率为２２．３％。其中已确定的临床型肝炎／肝病患者２５４例，ＨＧＶＲＮＡ阳性者为５     

Single-step nested polymerase chain reaction for detection of different genotypes of hepatitis C virus

Hepatitis C virus (HCV) exhibits considerable sequence variability and circulates in the blood at extremely low levels. Current methods for detecting HCV RNA are based mostly on nested polymerase chain reaction (PCR), in which part of the first amplification product is reamplified in the second tube by an internal primer pair. A novel nested PCR method was developed in which the two successive amplification processes are carried out in the same tube with a single step of physical manipulation. Careful selection of highly conserved sequences of the 5′ noncoding region as primers enabled successful detection of all three major genotypes circulating in France, including the one with variation in this region. Retaining high sensitivity of the conventional nested PCR, the novel method reduced greatly the risk of carry-over contaminations. It was also cost- and time-saving. The one-step nested PCR method is especially suitable for routine diagnosis of HCV infection in clinical laboratories. © 1995 Wiley-Liss, Inc.     

聚合酶链反应鉴定丙型肝炎病毒母婴传播的状态及其意义

采用多种方法，动态检测了１１例丙型肝炎病毒（ＨＣＶ）感染的孕妇所生的婴儿血抗－ＨＣＶ和ＨＣＶＲＮＡ。发现用合成肽酶联免疫吸附试验（ｓｐＥＬＩＳＡ）检测婴儿抗－ＨＣＶ阳性率（２３．５２％）显著低于第二代重组抗原ＥＬＩＳＡ（２ｎｄＥＬＩＳＡ）（４１．１８％）（Ｐ＜０．０５）；用２ｎｄＥＬＩＳＡ检测，６例婴儿脐血和静脉血抗－ＨＣＶ阳性，５例持续１～５月阴转，１例阳性持续１３个月。经重组免疫印迹试验（ＲＩＢＡ）鉴定，４例阳性，２例可疑阳性。用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）检测ＨＣＶＲＮＡ，５例阳性，３例于生后１～６个月自然阴转，２例持续阳性分别达９个月和１３个月。提示检测抗－ＨＣＶ判断ＨＣＶ母婴传播的状态受到婴儿抗－ＨＣＶ产生水平低下、母体抗－ＨＣＶ的被动输入和不同检测方法的影响，用ＲＴ－ＰＣＲ检测ＨＣＶＲＮＡ是判断母婴传播更可靠的指标。     

Detection of hepatitis C virus sequences in sera with controversial serology by nested polymerase chain reaction.

The specificity of first-generation enzyme-linked immunosorbent assays (ELIAs) for antibody detection in individuals with hepatitis C virus (HCV) infection has been questioned in some pathological situations. We observed a surprisingly high prevalence of anti-HCV antibodies in alcoholic patients, and thus, false-positive reactions in anti-HCV tests were strongly suspected. The introduction of new epitopes, particularly a core protein, C22 (second-generation tests), seems to increase the sensitivity of anti-HCV detection. In order to study the specificity of the second-generation tests, 60 serum samples from alcoholic patients found to be positive by the first-generation anti-HCV ELISA (Ortho) were reexamined by a second-generation anti-HCV enzyme immunoassay (Abbott) and a recombinant immunoblot assay (RIBA II; Chiron). Fifteen serum samples gave contradictory results when they were tested by the two assays. We performed nested polymerase chain reactions (PCRs) to confirm that the discrepancies that we observed could be due to the presence of low levels of anti-HCV antibodies, which were detected by a more sensitive test, or to unspecific positive reactions. Nested PCR revealed the presence of HCV RNA sequences in all anti-HCV-positive sera or sera that were weakly positive by ELISA. Anti-HCV positive by RIBA II was always correlated with the presence of viral RNA in serum, but HCV RNA was detected in RIBA II-negative sera. These results indicate that the specificity of the second-generation tests is an important improvement but that an HCV infection can still persist without detectable antibodies. PCR remains the reference assay to clear up controversial serology results and to detect HCV infection in patients with no anti-HCV-detectable immune response.