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。     

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／     

肝病产妇丙型肝炎病毒母婴传播的可能性调查

对１９９１～１９９３年在南京市钟阜医院肝病产科住院的１０４７例肝病产妇中１２例丙型肝炎病毒抗体和／或丙型肝炎病毒核糖核酸阳性者，追踪观察了所生１２名婴儿的丙型肝炎病毒感染情况，发现１例抗－ＨＣＶ和ＨＣＶＲＮＡ均阳性的孕妇所生的婴儿，０、１、６和１２个月时抗－ＨＣＶ和ＨＣＶＲＮＡ均阳性，且该婴儿ＨＣＶ的血清型与母亲一致。结果提示，肝病产妇丙型肝炎病毒母婴传播是可能的，但不是主要的传播途径。     

乙型及丙型肝炎病毒的母婴传播

目的：用分子生物学方法研究乙型、丙型肝炎病毒在母婴之间的垂直传播和胎儿的先天性感染。方法：选择ＨＢＶＸ基因和多聚酶基因之间的片断为扩增靶序列,设计了３只引物进行套式扩增;ＨＣＶ－ＲＮＡ５’端保守的非翻译区,设计３只引物进行一步法扩增。结果：乙型肝炎表明抗原（ＨＢｓＡｇ）携带者孕妇血清中的ＨＢＶ－ＤＮＡ的阳性率为２０．３５％,乳汗中的阳性率为１０．８８％,脐血、羊水、胎盘中的平均阳性率为１２．３９％,其中４例合并ＨＣＶ－ＲＮＡ阳性。结论：乙肝、丙肝病毒的母婴传播和胎儿的先天性感染是造成人群中肝炎病毒携带者比率居高不下的原因之一,预防肝炎病毒在宫内感染、阻断肝炎病毒垂直传播是医学界的一个世界性的难题。     

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％     

乙型肝炎基因工程疫苗阻断乙型肝炎病毒母婴传播…

用转基因细胞分泌的乙型肝炎病毒表面抗原基因工程疫苗免疫母亲ＨＢｓＡｇ阳性的新生儿５０例，观察其阻断乙型肝炎病毒母婴传播的效果，随访１２个月，在３６例母亲为ＨＢｓＡｇ和ＨＢｅＡｇ均阳性的婴儿中仅１例为ＨＢｓＡｇ阳性，其余婴儿均有保护性抗体，预防保护率为９６．２％。     

定量聚合酶链反应检测肝病患者血清中乙型肝炎病毒 …

目的 了解肝病患者血清乙型肝炎病毒（ＨＢＶ）ＤＮＡ的含量，以便观察临床抗病毒治疗效果及血清ＨＢＶ ＤＮＡ水平与病情及预后的关系。方法 应用荧光素标记的半巢式引物在扩增中能量转移的定量聚合酶 链反应（ＱＰＣＲ），对６３例肝功能异常的肝病患者血清进行ＨＢＶ ＤＮＡ含量测定，并与普通ＰＣＲ及酶联免疫吸附试验（ＥＬＩＳＡ）进行比较。结果 ＱＰＣＲ阳性率为８２。５４％，普通ＰＣＲ法为７１．４３％，ＥＬＩＳＡ     

乙型肝炎病毒母婴传播

乙肝母婴传播主要通过胎盘、乳汁、羊水及唾液传播。目前主要阻断方法为应用乙肝免疫球蛋白、乙肝疫苗等治疗。     

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     

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.     

一种同时检测丙型与庚型肝炎病毒的逆转录—巢式？…

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－巢式聚合酶锭反应，并初步应用于１５３例标本。结果 该方法能同时检出ＨＧＶ与ＨＣＶ感洒，扩增片段大小与设计相符。结论 该方法简便特异，适用     

Detection of hepatitis "C" virus in formalin-fixed liver tissue by nested polymerase chain reaction.

Interpretation of antibody to hepatitis C virus (HCV) in patients with liver disease is difficult due to false-positive reactivity in some conditions. To evaluate the feasibility of HCV in archival material, HCV was sought in formalin-fixed, paraffin-embedded liver biopsy specimens. Nested polymerase chain reaction was used to detect hepatitis C virus in formalin-fixed, paraffin-embedded liver biopsy specimens after total RNA was extracted from tissue by proteinase K digestion and phenol/chloroform purification. The relative efficiency of amplification of HCV RNA from formalin-fixed material was estimated semiquantitatively by serial dilution of cDNA synthesised from RNA extracted from fresh and formalin-fixed sections from the same liver. Although HCV RNA could be detected in formalin-fixed liver tissue by nested PCR in 5/5 cases in which HCV was detected in serum, amplification was approximately 5-fold less efficient than when HCV was amplified from fresh tissue. Nevertheless, nested PCR of HCV from formalin-fixed liver tissue represents a useful technique in addressing some important questions related to the pathogenesis of liver disease.     

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.     

Detection of feline immunodeficiency virus by a nested polymerase chain reaction.

A specific and sensitive polymerase chain reaction (PCR) procedure for the detection of feline immunodeficiency virus (FIV) in peripheral blood mononuclear cells (PBMC) was developed. PBMC from both blood samples and cultures were digested by proteinase K in a lysis buffer, and after heat inactivation of the proteinase, the resultant material was used in a two step amplification protocol using nested sets of primers. Two independent amplifications, from the gag and pol genes respectively, were performed in each tube. The PCR was positive for six of 14 samples from FIV seropositive adult cats, while all 36 samples from seronegative cats were negative. In comparison with an antigen-capturing ELISA procedure, the PCR detected FIV infection in PBMC cultures on average two days earlier.     

Hepatitis C virus antibodies and hepatitis C virus RNA in Chinese blood donors determined by ELISA, recombinant immunoblot assay and polymerase chain reaction.

Antibodies to hepatitis C virus (anti-HCV) were determined in Chinese blood donors from the city of Wuhan by a second generation ELISA screening test and a confirmatory recombinant immunoblot assay (RIBA II). Two materials of 281 and 222 sera were sampled under similar conditions in 1989 and 1990, respectively. The first collection of sera was sent to Sweden in lyophilized form, the second directly as fresh unfrozen sera. A high proportion (7.1%) of the lyophilized sera reacted positively in the anti-HCV screening assay, but only seven (2.5%) were positive by the RIBA confirmatory test. In four of these sera HCV-RNA could be detected by polymerase chain reaction (PCR) analysis. In the second material of fresh sera six reacted positively in the screening anti-HCV ELISA, but only one was RIBA positive and four were RIBA indeterminate. None of these sera was positive for HCV-RNA. Thus, the overall prevalence of anti-HCV among the 503 Chinese blood donors, as identified by RIBA, was 1.6%, and of HCV-RNA by PCR 0.8%. The confirmed antibody prevalence is higher than reported from the Western world. Caution about using data from the screening ELISA only, especially if sera have been handled in an unorthodox way, is emphasized.