丙型肝炎病毒基因型的研究进展

丙型肝炎是一种主要经血液传播的疾病,丙型肝炎病毒慢性感染可导致肝脏慢性炎症坏死和纤维化,部分患者可能发展成为肝硬化或肝癌。HCV是单股RNA病毒易变异,目前可分为6个基因型与不同的亚型,本文就此问题的研究状况进行综述。     

基因芯片法特异性检测丙型肝炎病毒的基因分型

目的：采用基因芯片特异性检测血清中丙型肝炎病毒（HCV）并进行基因分型。方法：设计HCV基因型特异探针，将其固定在玻璃片上制成微阵列芯片。阳性组血清60份，阴性组血清15份，乙型肝炎血清5份（抗HCV阴性）。经核酸提取，多聚酶链式反应（PCR）扩增，与芯片上的探针杂交，最后分析结果并与测序分型结果比较。结果：阳性组血清全部检测到HCV-RNA，均有基因芯片分型结果。基因芯片分型结果与测序分型结果一致者56例。阴性组血清HCV-RNA全部阴性。乙型肝炎血清全部阴性。结论：基因芯片可准确对HCV感染血清做定性检测并同时检测HCV基因型，简便快捷，特异性好，并且不需荧光标记和昂贵的荧光扫描仪器，与乙型肝炎血清无交叉反应。可替代基因测序分型，适于临床大量样品的检测。     

建立DNA芯片技术检测丙型肝炎病毒基因型及其初步应用

目的 建立一种简便、快速的以DNA芯片技术为基础的丙型肝炎病毒基因分型检测方法。方法 根据丙型肝炎病毒HCV的基因序列信息设计分型探针，将采用荧光标记的寡核苷酸引物所扩增的丙型肝炎病毒基因产物与结合在芯片上的特异性探针进行快速杂交，通过扫描荧光强度值定性和定量判定结果。结果 应用本法对65例患者血清中的丙型肝炎病毒核酸进行基因分型，对部分标本检测结果与测序法进行对比，吻合率达100％，说明检测结果有很高的特异性。结论 我们所用DNA芯片技术检测HC VRNA及其基因分型，简便、快速、特异性好、灵敏度高、结果判定指标客观，可望在临床推广使用。     

四种方法检测乙型肝炎病毒基因型的比较

目的 比较四种方法检测乙型肝炎病毒基因型的差异性.方法 对36例乙型肝炎患者的血清分别采用测序技术,荧光定量PCR技术,恒温扩增技术,基因芯片技术进行乙肝病毒基因型的检测.结果 36份血清以测序技术为金标,观测荧光定量PCR技术特异性100%,敏感性83%,恒温扩增技术特异性100%,敏感性89%,基因芯片技术特异性100%,敏感度92%.结论 目前临床采用的四种方法检测乙型肝炎病毒基因型的特异性相同,敏感度以测序为金标准,基因芯片技术最灵敏,其次为恒温扩增技术,最后为荧光定量PCR技术.     

基因芯片在丙型肝炎病毒分型检测中的评价

为研究HCV基因分型芯片检测丙型肝炎患者的基因型,以测序法进行对比,并探讨了IFN治疗慢性丙肝疗效与基因型的关系。采用基因芯片的方法对20例慢性丙型肝炎患者进行分型,并通过测序验证。结果基因芯片和测序结果完全一致。20例丙肝患者中18例为1b,2例为2a,并且IFN治疗效果2a较1b型为优。HCV分型芯片检测HCV分型,特异性强、灵敏度高、结果准确,支持HCVRNA基因型在评价IFN疗效中十分重要的观点。     

丙型肝炎病毒血清型基因型与抗HCV抗体类别的关系

采用EIA法检测丙型肝炎患者血清IgG、IgM、IgA类抗HCV抗体;以丙型肝炎病毒核心区2个型特异性合成多肽对抗HCV进行血清学分型,对已确定血清型的血清进行5′非编码区逆转录套式聚合酶链反应(RT-nPCR)和限制性片段长度多态性(RFlp)分析以确定基因型.结果:23例HCV RNA阳性的丙型肝炎患者中19例抗HCV阳性(IgG、IgM、IgA三者中至少一种阳性);12例可行血清学分型,其中1型8例,2型2例,1、2混合型2例;11例未能分型.12例已知血清型的HCV中,基因型Ⅰ型9例,Ⅱ型2例,Ⅰ、Ⅱ混合型1例.两种分型方法一致率为91.7%(11/12).提示:血清学分型具有一定应用价值,联合IgA、IgM抗HCV检测可提高血清学分型的检出率.     

丙型肝炎病毒的病毒学检测方法

丙型肝炎病毒（Hepatitis C Virus,HCV）感染引起的丙型病毒性肝炎呈全球分布,世界人口的3％即大约1．7亿人为丙肝感染者,且发展中国家高于发达国家。1992-1995年全国病毒性肝炎血清流行病学调查显示,我国人群丙肝感染率为3．2％。HCV感染自然转阴率低,极易慢性化,治疗效果差,且与肝硬化肝癌的发生及发展密切相关。丙型病毒性肝炎不仅严重危害人民健康,而且给社会带来了巨大的经济损失。因此,丙肝感染仍是我国重要的公共卫生问题之一。     

TaqMan探针法与尿素呼气试验法检测胃镜活检标本幽门螺旋杆菌的比较

目的:通过比较幽门螺旋杆菌(Helicobacter pylori,Hp)感染的两种检测方法,探讨TaqMan探针法在诊断幽门螺旋杆菌感染中的价值.方法:抽取70例有完整病例资料的胃镜活检标本,进行Hp分型、鉴定检测,并将实验结果与临床的碳14尿素呼气试验和病理结果进行相关性分析.结果:Hp感染的两种检测方法(TaqMan探针法和呼气实验法)一致性程度尚可(Kappa=0.550),两种方法检测的阳性率都随病理炎症程度的加重而增加,而呼气实验法检测Hp的阳性率更高(P<0.05).但TaqMan探针法可以将Hp进行不同毒力类型的鉴定,且TaqMan探针法可区分Hp的阳性强度(1+,2+,3+),其与病理的炎症程度有较好的相关性(γ=0.564,P<0.05).结论:TaqMan探针法可以定性地鉴定Hp,与呼气实验法有较好的一致性.TaqMan探针法可进一步定量Hp的阳性强度,更好地反映临床的病理炎症程度.     

核酸试纸条法检测HBV基因型方法的建立

目的 建立一种快速、肉眼可观测的方法检测乙型肝炎病毒(HBV)基因型.方法 根据GenBank中已发表的明确HBV分型全序列,设计特异的引物和标记生物素探针,建立核酸试纸条法检测150例乙型患者和20例健康体检者的乙型肝炎基因型,同时采用荧光定量PCR法进行比较.结果 150例标本中B型检出率为34.00%(51/150),C型占61.33%(92/150),B/C混合型占4.00%(6/150),未分型占0.67%(1/150).与荧光定量PCR法进行比较,结果一致.结论 核酸试纸条法检测HBV基因型与荧光定量pCR法相比较灵敏度和特异性相似,但该方法更简便、快速,更适合临床开展.     

乙型肝炎病毒基因型和变异型的检测及临床意义

目前乙型肝炎的治疗仍以拉米夫定等抗病毒药物为主,而药物疗效很大程度上与HBV基因型和是否发生变异等密切相关,这使得HBV基因型和变异型的检测尤为重要.     

Genotyping of hepatitis C virus by Taqman real-time PCR.

BACKGROUND: Genotype of hepatitis C virus (HCV) is of major importance for the outcome of treatment. The response rate is considerably lower for genotype 1, the predominant genotype in western countries. OBJECTIVES: To develop and evaluate a new, simple method for genotyping of HCV based on real-time polymerase chain reaction (PCR) and Taqman probes targeting the 5' non-coding region. STUDY DESIGN: The method was compared with Innolipa on 220 serum samples representing genotypes 1-4, and was applied on a further 614 clinical samples. RESULTS: Taqman typing of the 220 samples showed genotype 1 in 69, genotype 2 in 58, genotype 3 in 57 and genotype 4 in 19, while 17 were non-reactive. There was a complete concordance with Innolipa with the exception of seven samples, which were of genotype 1 by Taqman, but genotype 4 by Innolipa. Sequencing of these samples showed a subtype 4 variant which differed at two positions compared with subtypes 4b/c/d, which are targeted by the probe. By adding a modified probe, these genotype 4 variants could also be identified. Out of 614 consecutive clinical samples, 524 could be typed by the Taqman assay; 45.2% were genotype 1, 19.3% genotype 2, 33.8% genotype 3 and 1.7%, genotype 4. CONCLUSION: The method was overall accurate and provides an attractive alternative for genotyping because processing time and costs are significantly reduced. Inclusion of probes targeting genotypes 5 and 6 is required for the method to be useful in areas where these genotypes are present.     

Genotyping of Canadian hepatitis C virus isolates by PCR.

We used PCR for hepatitis C virus (HCV) genotyping with type-specific primers from the core and NS5 genes. Type I was predominant in the general population (58% in blood donors) as well as in different risk groups, such as intravenous drug abusers (58%), blood transfusion recipients (64%), hemophiliacs (62%), and patients with HCV chronic liver disease (76%). Types II, III, and IV were less prevalent in Canada, being found in 10.92, 6.72, and 5.88% of the population, respectively. The type II core primer was not type specific and reacted with the majority of our type I HCV samples, suggesting a false-positive dual infection with two different genotypes (I and II). Digestion of these amplified type I and type II products with restriction endonuclease AccI proved to be very useful in the exclusion of false-positive dual type I and type II infections.     

Genotyping of hepatitis C virus in South Africa.

The six major hepatitis C virus genotypes were investigated by using samples from 79 seropositive and PCR-positive blood donors from three different regions of South Africa as well as 9 patients with chronic renal failure, 19 with liver disease, and 23 with hemophilia. PCR products of the genome were typed by restriction fragment length polymorphic analysis by RsaI-HaeIII and MvaI-HinfI double digestion. Type 5 occurred in 40% of this population group; type 1 occurred in 33%; and types 2, 3, and 4 were found in 13.8, 7.7, and 2.3%, respectively.     

Genotyping hepatitis C virus by heteroduplex mobility analysis using temperature gradient capillary electrophoresis

The genotype of the infecting hepatitis C virus (HCV) helps determine the patient's prognosis and the duration of treatment. Heteroduplex mobility analysis (HMA) is a rapid, inexpensive method for genotyping of HCV that does not require sequencing. We developed an HMA that uses temperature gradient capillary electrophoresis (TGCE) to differentiate HCV genotypes. A 56-bp region of the HCV 5' untranslated region (UTR) that was conserved within a genotype yet whose sequence differed between genotypes was amplified for HMA-TGCE analysis. HCV amplicons of types 1, 2a, 2b, 3a, 4, and 6a were hybridized in pairs and analyzed by TGCE. Amplicons hybridized to the same subtype yielded one homoduplex peak, while hybridization of different subtypes resulted in heteroduplexes and generated multiple TGCE peaks. Heteroduplexes contain thermodynamically unstable nucleotide mismatches that reduced their TGCE mobilities compared to those of homoduplexes. Three HCV subtypes (subtypes 1a, 3a, and 4) generated unique peak patterns when they were combined with each genotype analyzed and were chosen as the reference genotypes. A blinded study with 200 HCV-infected samples was 97% accurate compared to genotyping by 5' UTR sequence analysis. The majority of discordant results were unexpected sequence variants; however, five of nine sequence variants were correctly genotyped. The assay also detected and correctly genotyped mixed HCV infections. Compared to conventional HMA, TGCE improves the resolution, with better separation of heteroduplexes and homoduplexes. All common HCV genotypes can be detected and differentiated by this HMA-TGCE assay.     

线探针核酸杂交分析贵州地区HCV感染株基因型

目的　分析贵州地区丙型肝炎病毒（ＨＣＶ）感染株的基因型。方法　用第二代线探针核酸杂交方法,对９８ 例ＨＣＶ感染者血清进行基因分型。结果　１２ 例献血员血清,均为ＨＣＶ１ｂ 型;１５ 例血液病患者中,１４ 例（９３－３％ ）为ＨＣＶ１ｂ ,１ 例（６－７％ ）为ＨＣＶ１ｂ ＋ ２ 型。慢性丙肝患者３７ 例中,ＨＣＶ１ｂ 型３４例（９１－９％ ） ,混合感染基因型３ 例（ＨＣＶ１ａ ＋ １ｂ 、ＨＣＶ１ｂ ＋ ２ 、ＨＣＶ１ａ ．１ｂ ＋ ２ａ２ｃ 各１ 例）。静脉药瘾者３４ 例,其中ＨＣＶ１ｂ 感染１３ 例（３８－２％ ） ,ＨＣＶ６ａ１０ 例（２９－４％ ） ,ＨＣＶ３ｂ４ 例（１１－８％ ） ,ＨＣＶ３ａ１ 例（２－９ ％） ,混合感染６ 例（１７－６ ％） ,大多为ＨＣＶ２ 复合其他基因型。结论　贵州地区ＨＣＶ感染者中,以ＨＣＶ１ｂ 基因型为主,其次为ＨＣＶ６ａ ,尚存在ＨＣＶ３ａ 和ＨＣＶ３ｂ 。其中ＨＣＶ３ａ、３ｂ 、６ａ ,在本地区系首次报告,但主要存在于静脉药瘾者中。虽然有混合感染基因型的存在,但其基因基础需测序分析     

Genotyping and the quantification of hepatitis C virus by the melting curves in light cycler

Detection of the HCV genome is crucial for diagnosis of HCV infection and for monitoring the efficacy of interferon treatment for patients with HCV. We developed a convenient screening test for HCV genotypes 1 and 2 based on the melting curve analysis with SYBER green I. Serum samples were drawn from 114 patients with known chronic HCV infection confirmed to be antibody-positive by immunoblot assay. A characteristic melting profile for each genotype was obtained by monitoring the fluorescence as the temperature increases through the melting point of the PCR product. Serum samples with HCV-RNA genotype (1b, 2a and 2b) were analyzed every test as standard samples and the genotype of unknown samples was determined by the comparison with the melting point of standard samples. Serum samples with known HCV-RNA genotype (1b, 2a and 2b) and HCV-RNA-negative sample were tested using the Light cycler system. The melting curve analysis indicated that melting points are 93.08 +/- 0.56 degrees C for genotype 1b (n = 63), 91.08 +/- 0.49 degrees C for genotype 2a (n = 33), and 91.77 +/- 0.28 degrees C for genotype 2b (n = 18). The melting points for genotypes 1b, 2a, and 2b differed by approximately 1 degree C in each other. The genotype was determined for all samples using Okamoto's method and Light cycler system, and both systems produced absolutely identical results for all the samples studied. Sixty-three of 114 were genotype 1b, 33 samples were genotype 2a, and 18 were genotype 2b. This melting curve analysis is a rapid and convenient screening test for differentiation of HCV genotypes 1 and 2.     

Genotyping of hepatitis C virus isolates by a new line probe assay using sequence information from both the 5'untranslated and the core regions

The correct assessment of hepatitis C virus (HCV) genotypes and subtypes by commercial assays is of utmost importance mainly for the therapeutic management of patients suffering from HCV infections. In this study, the performance characteristics of a newly designed genotyping assay were evaluated that does not rely exclusively on sequence information derived from the 5'untranslated region but also takes into account part of the HCV core. One hundred and ten clinical specimens were tested by this new assay prior to its commercialisation. The obtained typing results were compared to those recorded by the 5'UTR-based Versant HCV Genotyping Assay, version 1, the core-related Gen-Eti K DEIA, and phylogenetic analyses of partial HCV core and NS5B sequences. The HCV genotypes and subtypes identified by the newly devised kit were completely in line with the assignments achieved by DEIA and phylogenetic analyses. In particular, all 64 HCV strains belonging to subtypes 1a or 1b were recognised correctly, and HCV 6e and 6f isolates were adequately assigned to subtypes 6c-l. Thus, the second generation of the Versant genotyping assay could overcome the drawbacks of its exclusively 5'UTR-based predecessor and will turn out to be a reliable tool for HCV typing in clinical laboratories.     

Second-generation line probe assay for hepatitis C virus genotyping.

Because of the enormous variability of hepatitis C virus (HCV), the development of reliable genotyping assays is a formidable challenge. The optimal genotyping region appears to be the 5' untranslated region (UR) because of high conservation within, but considerable variability between, genotypes. In this study, 21 probes dispersed over seven variable 5' UR areas were applied to a line probe assay (LiPA) and used to analyze 506 HCV-infected sera from different geographical regions representing a multitude of subtypes. At least 31 different reactivity patterns emerged, with 404 (80%) of 506 distributed over 11 prototype patterns, in general corresponding to subtypes 1a, 1b, 2a/2c, 2b, 3a, 5a, and 6a and several type 4 subtypes. Subtyping specificity ranged from 97% in Hong Kong to 90% in Europe but was only 11% in West Africa, while typing specificity was always 100% when samples from Vietnam were excluded. In a second evaluation, the subtype prediction by LiPA of 448 GenBank 5' UR HCV sequences was scored. Of the 58 theoretically predicted patterns, 321 sequences (72%) were covered by the 11 prototype patterns. We concluded that (i) the selected probes detected the corresponding signature motifs in the seven variable regions with 100% reliability; (ii) these motifs allowed correct type interpretation of samples collected worldwide, with the exclusion of Vietnam, Thailand, or Vietnamese patients residing in European hospitals; and (iii) subtyping specificities vary according to geographical region, with 11 prototype subtyping patterns identifying the majority of samples from Europe and the Americas. These results indicate that the LiPA is a reliable assay applicable to routine typing and subtyping of HCV specimens.     

Subtyping of hepatitis C virus isolates by a line probe assay using hybridization.

A reverse hybridization test (Inno-LiPA HCV; Innogenetics, N.V., Zwijnaarde, Belgium) was used for typing hepatitis C virus. All 38 samples, typed by PCR with primers from core and NS5 genes, were also genotyped by this test. Of the samples, 33 (87%) had the same subtypes by both assays. The correlations between PCR and Inno-LiPA for individual types were 77% for type I (1a), 90% for type II (1b), 100% for type III (2a), 100% for type IV (2b), and 100% for type V (3a). One of the type III (2a) samples also reacted with type I (1a) probes in the Inno-LiPA test.