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。     

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。     

抚顺地区慢性丙型肝炎患者基因分型、传播途径的分析及个体化疗效的观察

目的研究抚顺地区慢性丙型肝炎患者的基因分型与传播途径及个体化治疗的关系。方法选择2012年7月至2015年6月抚顺市传染病医院收治的236例明确诊断为慢性丙型肝炎患者,其中男性148例,女性88例;年龄8~83岁,平均年龄40.2岁。取其血清标本,提取丙型肝炎病毒(HCV)RNA,进行HCV基因分型,调查其传染途径,符合抗病毒指征者给予聚乙二醇干扰素(PEG-IFN)-α联合抗病毒药利巴韦林(RBV)治疗,观察应答相关指标。结果 236例慢性丙型肝炎患者中206例扩增成功,200例C区和NS5B区基因分型结果一致,检出基因型1a、1b、2a、3a、3b、6a、未分型,分别为6(3.00%)例、76(38.00%)例、81(40.50%)例、8(4.00%)例、15(7.50%)例、9(4.50%)例、5(2.50%)例;各HCV传播途径均有涉及,研究发现多以血液及血制品传播途径为主,为63.0%;静脉吸毒者所占比重为11%;6a型中静脉吸毒者为66.7%。治疗效果与基因型有关,不同基因型快速病毒学应答(RVR)、早期病毒学应答(EVR)、治疗结束时病毒学应答(ETVR)不尽相同,1b型的RVR为52.63%,低于2a、3a、3b、6a型的患者(P0.05),与1a型患者的RVR差异无统计学意义(P0.05)。各基因型获得的EVR无明显差异,1a、1b、3a型有1例、5例、1例未获得EVR,其余各基因型结束治疗的患者均获得ETVR。1b型的复发率大于2a型,分别为34.21%(26/76)及14.81%(12/81),两者间差异有统计学意义(P0.05)。结论抚顺地区慢性丙型肝炎患者的基因分型包括1型、2型、3型、6型4种基因型和8种基因亚型,1b、2a两型居多,HCV各个传播途径均有涉及,1b型和2a型以血液及血制品传播为主,6a型中静脉吸毒者所占比重较多。并且不同基因型治疗效果不尽相同,因此应采取个体化的治疗方式。     

吉林省延边地区朝鲜族与汉族HCV基因型特征分析

目的分析吉林省延边地区朝鲜族与汉族丙型肝炎病毒（HCV）基因型特征。方法采用荧光定量PCR方法和探针反向杂交技术对延边地区收治的62例朝鲜族和57例汉族慢性丙型肝炎患者进行HCV病毒载量检测和HCV基因型分析,比较HCV基因型在朝鲜族与汉族之间的分布差异,并分析HCV基因型与病毒载量、2型糖尿病和肝病严重程度之间的相关性。结果朝鲜族HCV基因型显示,1b型占45．16％（28／62）、2a型占38．71％（24／62）、未分型占16．13％（（10／62）;汉族HCV基因型显示,1b型占45．61％（26／57）,2a型占40．35％（23／57）,未分型占14．14％（8／57）,朝、汉族之间各基因型分布差异无统计学意义（P〉0．05）。HCV各基因型之间HCV病毒载量差异和合并2型糖尿病分布差异之间无统计学意义（P〉0．05）,而HCV1b型在中、重度慢性丙型肝炎患者与轻度慢性丙型肝炎患者中所占的比例差异具有统计学意义（P〈0．05）。结论①吉林省延边地区HCV基因型中以1b型最多,2a型次之,还有少数其他基因型;②HCV基因型在朝、汉民族之间分布无差异;③HCV基因型与HCV—RNA载量无关;④HCV基因型在合并糖尿病者与未合并糖尿病者之间分布无差异;⑤1b型HCV感染者病情相对较重。     

湖北地区丙型肝炎患者HCV基因分型及HCV NS5B耐药突变位点分析

目的:检测湖北地区丙型肝炎(丙肝)患者丙肝病毒(HCV)基因分型及HCV NS5B基因耐药突变位点的分布特征。方法:收集自2011-03-2014-05在本院确诊的来自湖北地区的丙肝患者外周静脉血标本273例,采用一代测序法检测每例样本的HCV和NS5B基因序列,将测得的序列与Blast进行在线比对后,统计分析HCV基因型和各亚型检出率,以及HCV NS5B耐药突变位点在HCV各基因亚型中的分布差异。结果:273例丙肝患者共检出1、2、3、6四种基因型和1a、1b、2a、3a、3b、6a六种基因亚型,其中1b亚型检出率较高,占76.19%(208/273),其次是2a亚型,占15.02%(41/273),其它各亚型检出率均不超过4.40%,各亚型检出率差异有统计学意义(P0.05)。HCV NS5B基因以L159F突变率较高,占17.95%(42/234),与其它位点的基因突变率差异有统计学意义(P0.05)。同时各个位点的耐药突变发生在2a亚型中的比例较高,达57.26%(134/234),显著高于其它亚型的耐药突变(P0.05)。结论:分析湖北地区丙肝患者HCV基因分型及HCV NS5B耐药突变位点的分布特点,可为该地区丙肝患者的个体化治疗提供指导依据。     

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.     

HCV反向点杂交基因分型方法的建立

目的利用反向点杂交技术建立HCV基因分型新方法。方法在HCV5’端非编码区（5’NCR）设计引物和分型探针（1．2．3．6型）,采用反向点杂交分型技术对53例HCVRNA阳性（浓度均在102～107IU／mL之间）血清进行分型,并以基因序列分析作为金标准,对新方法的敏感性、特异性和重复性进行评价。结果反向点杂交基因分型新方法检出的基因型有52例：1b型32例占60．38％,2a型4例占7．55％,6a型8例占15．09％．3b型8例占15．09％;未检出的基因型有1例,用基因序列分析也未能确定型别（失败的原因应是该HCVRNA扩增产物浓度偏低2．24×10^2IU／mL）。本研究的方法敏感性为98．1％,特异性为100％,随机抽取20份标本再次检测的结果与前次检测的结果完全一致,重复性好。结论相对现有的型特异性PCR分型、基因芯片、核酸序列分析等各种方法,反向点杂交技术用于HCV基因分型是一种准确有效和简便经济的方法。     

新疆维吾尔族人群丙型肝炎病毒基因分型分布分析

目的 了解新疆维吾尔族人群感染的丙型肝炎病毒（hepatitis C virus,HCV）的基因型别,为采取相应的临床治疗措施提供科学依据.方法 从医院收集维吾尔族丙型肝炎（丙肝）患者的血清样本基本信息,通过反转录巢式PCR法扩增HCV NS5b区并进行序列测定,与HCV标准株比较分析、绘制系统发生树、确定其基因型并进行比较分析.结果 39份丙型肝炎患者的样本中,HCVRNA阳性者为20份,基因型的分布状况为：1型17例,占85.0％,2型2例,只占10.0％,3型1例,占5.0％;亚型分析发现lb亚型14例（70.0％）,la亚型3例（15.0％）,2a亚型2例（10.0％）,还发现1例3a亚型（5.0％）.结论 新疆地区维吾尔族人群中流行的HCV基因型构成较为复杂,1b亚型为主要型别.     

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。     

常州地区丙型肝炎病毒基因分型及临床分析

目的:了解常州地区丙型肝炎病毒基因型分布、病毒复制与肝功能损害的关系。方法:对150例丙型肝炎病毒抗体检测为阳性的患者,采用荧光定量聚合酶链反应技术检测血清丙型肝炎病毒核糖核酸(HCVRNA)水平,并检测丙氨酸转氨酶(ALT)、天门冬氨酸转氨酶(AST)等肝功能相关指标,HCV基因分型采用基因芯片法。结果:150例患者中,HCV RNA阳性患者130例(>1×103IU/ml),HCV RNA阴性患者20例(<1×103IU/ml)。经相关性检验,HCV RNA阳性患者血清ALT、AST水平与HCV RNA含量表明无显著的相关性(P>0.05),HCV RNA阳性患者血清ALT和AST指标明显高于HCV RNA阴性患者(P<0.05)。对其中54例HCV RNA阳性标本进行HCV基因分型,1型有46例(85.19%),非1型8例(14.81%)。1型与非1型相比较,两组的血清ALT和AST水平没有明显差异(P>0.05)。结论:常州地区丙型肝炎病毒感染以1型病毒为主;持续的丙型肝炎病毒复制与肝功能损害密切相关;病毒载量与肝功能检测对丙型肝炎的治疗有重要意义。     

Use of a commercially available line probe assay for genotyping of hepatitis C virus 5a strains

To validate a commercially available line probe assay for samples containing the infrequently found hepatitis C virus (HCV) genotype 5a, we sequenced a 511-nucleotide fragment of the NS4b region of Belgian and South African HCV genotype 5a samples. Phylogenetic analysis of the sequence data was performed. For the 77 HCV genotype 5a samples collected, there was 100% concordance between the genotype assignment by the line probe assay and the genotyping based on nucleotide sequencing, despite sequence heterogeneity in the probe binding sites of some samples.     

Comparison of two hepatitis C virus typing assays in a Tunisian population

In patients with hepatitis C, it is necessary to determine the genotype of the hepatitis C virus (HCV) to tailor treatment schedules. HCV-positive sera from 60 chronically infected patients were analyzed by two methods: serotyping and genotyping, to evaluate the suitability of serotyping method for routine determination of HCV genotype; 47 men and 13 women were included in this study, nine were renal transplant patients and five were hemodialysis patients. Anti-HCV antibodies were detected by Elisa (HCV Ab III, Innogenetics SA) and confirmed by immunoblot assay (INNO-LIA-HCV Ab III update, Innogenetics SA). Genotyping analysis was performed by a line probe assay (Inno-LiPA-HCV, Innogenetics SA) and serotypes were determined by an Elisa-based serotyping assay (Murex HCV serotyping 1-6 HCO2, Murex SA) which detect type specific antibodies against NS4-derived epitopes. Among 60 patients positive for anti-HCV antibodies and confirmed by immunoblot assay only 90% show a strong reactivity with NS4. We found the following genotype distribution : 1b (73.3%), 4 (10%), 1a (5%), 3a (5%), 4c/4d (5%) and 1 not classified (1.7%). The most prevalent serotype was type 1 (60%) followed by serotype 6 (20%), 4 (8.3%), 2 (1.7%) and 10% were no type specific antibodies. The sensitivity of the serotyping and the genotyping assays was 90% with a total concordance of 68.3%. Thirteen samples revealed discrepant results with genotype : 1b (4), 1a (3), 3a (3), 4 (2) and 4c/4d (1). This study indicates that the serotyping assay is less specific than genotyping. However, the test is rapid, relatively easy to perform and represent a reliable alternative in laboratories that lack the specific expertise to typing the HCV by molecular methods.     

Hepatitis C virus genotyping by restriction fragment length polymorphism of polymerase chain reaction products generated with a HCV detection kit

Highly conserved sequence in the 5' untranslated region(UTR) of hepatitis C virus(HCV) genome have been targeted by most nucleic acid amplification-based detection assays, such as Amplicor HCV test, a commercially available assay kit. In this study, we classified HCV genotypes by direct sequencing determination for 5' UTR of nested-PCR after Amplicor HCV test. Then, based on the results of sequence, RFLP analysis after digestion of the nested PCR fragments with Hae III or Sau 3AI to classify HCV genotype was evaluated. RFLP analysis distinguished the type 1, 2a and 2b. Only one of 29 samples was not classified by RFLP analysis due to the point mutation of Hae III recognition site. HCV genotypes commonly found in JAPAN were classified into three types, 1b, 2a, and 2b. Also, RFLP analysis requires fewer resources than serotype grouping test. Hence, the present method provides an adaptable and rapid HCV genotyping in clinical laboratory in JAPAN.     

High-resolution melting combines with Bayes discriminant analysis: a novel hepatitis C virus genotyping method

Current hepatitis C virus (HCV) genotyping techniques are often highly technical, costly, or need improvements in sensitivity and specificity. These limitations indicate the need of novel methods for HCV genotyping. The present study aimed to develop a novel genotyping method combining high-resolution melting (HRM) analysis with Bayes discriminant analysis (BDA). Target gene fragment including 5′-untranslated and core region was selected. Four or five inner amplicons for every serum were amplified using nested PCR, HRM was used to determine the melting temperature of the amplicons, and HCV genotypes were then analyzed utilizing BDA. In initial genotyping (HCV genotypes were classified into 1b, 2a, 3a, 3b, and 6a), both the overall accuracy rate and the cross-validation accuracy rate were 92.6 %, external validation accuracy rate was 95.0 %. To enhance the accuracy rate of genotyping, HCV genotypes were firstly classified into 1b, 3a, 3b, and 2a–6a, followed by a supplementary genotyping for 2a–6a. Both the overall accuracy rate and the cross-validation accuracy rate reached 97.5 %, and external validation accuracy rate was 100 %. Comparing adjusted HRM genotyping with type-specific probe technique, the difference in accuracy rates was not significant. However, the limit of detection and cost were lower for HRM. Comparing with sequencing, the limit detection of HRM was the same as the former, but the cost of HRM was lower. Hence, HRM combined with BDA was a novel method that equipped with superior accuracy, high sensitivity, and lower cost and therefore could be a better technique for HCV genotyping.     

Patterns of serological markers in transfusion-transmitted hepatitis C virus infection using second-generation HCV assays.

A semiautomated dot blot assay and cDNA polymerase chain reaction (PCR) were used to study longitudinal anti-hepatitis C virus (HCV) recognition patterns in relation to presence of HCV-RNA in transfusion recipients and their infectious donors. In 9 recipients, 4 different patterns of HCV infection were observed: (A) persistent HCV carriage accompanied by chronic hepatitis in 6, (B) acute resolved hepatitis, but persistent HCV replication in one, and (C) continuous HCV replication without hepatitis in one and (D) acute resolved hepatitis with clearance of infection in one. This last self-limited infection was characterized by the disappearance of HCV-RNA as well as anti-HCV reactivity. In contrast, antibody reactivity persisted in 7 of 8 patients with chronic HCV infection who could be followed until 1990. Seven of the 9 recipients developed antibodies to all recombinant peptides in dot blot assay; one became positive for anti-C33 and anti-core and one developed anti-core only. The sequence of appearance of antibodies differed among individual patients. In 7 patients with full anti-HCV recognition patterns, the sequence of events was (mean and limits in days after transfusion): onset of hepatitis at day 50 (22-74), seroconversion of anti-C33 at day 91 (59-129), anti-core at day 133 (54-203), and anti-C100 at day 143 (59-365). The incorporation of C33 and core proteins, in addition to C100, in the second generation anti-HCV ELISA enhanced the detection rate in the HCV-infected transfusion recipients from 7/9 (78%) to 9/9 (100%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between viral genotype and viral load in patients with chronic hepatitis C

To determine the distribution of hepatitis C virus (HCV) genotypes and to relate genotype to viral load, genotyping and quantification of viral RNA were carried out in 35 patients with chronic hepatitis C. Subtype 1a was most prevalent (43%), followed by subtypes 1b (23%) and 3a (14%). Mean viral load (log HCV-RNA copies/ml) for subtypes 1b, 1a and 3a was 7.1 ± 1, 5.6 ± 1.1 and 4.1 ± 2.4, respectively. The presence of immunoglobulin M was related to the duration of hepatitis and genotype 1 to a more severe hepatic injury and a higher viral load. Differences observed in viral load for a single HCV subtype justify the need to quantify HCV-RNA prior to establishing antiviral therapy.