丙型肝炎病毒基因分型的反向杂交检测法评估

Evaluation of a reverse hybridization assay for genotyping of hepatitis C virus.

作者信息

Zeuzem S, Ruster B, Lee J H, Stripf T, Roth W K

机构信息

Medical Department II, University Hospital, Frankfurt, Germany.

出版信息

J Hepatol. 1995 Dec;23(6):654-61. doi: 10.1016/0168-8278(95)80030-1.

DOI:10.1016/0168-8278(95)80030-1

PMID:8750163

Abstract

BACKGROUND/AIMS: Several strains of the hepatitis C virus exist; distinct genotypes and subtypes can be identified by sequence comparison of the viral genomes. Recent evidence that the genotype/subtype of hepatitis C virus may influence the clinical course of chronic hepatitis C and the response to interferon-alpha therapy for this disease suggests that methods to identify the genotype may become clinically useful. In the present study we evaluated a recently introduced reverse hybridization assay.

METHODS

HCV-RNA was isolated from serum samples from 61 consecutive patients attending our out-patient clinic and subsequently sequenced in the 5'-noncoding and the nonstructural-5 region by the dideoxynucleotide chain termination method. HCV-genotyping was performed by phylogenetic analysis of nonstructural-5 sequences. The amplification product for the reverse hybridization assay was obtained by "nested" polymerase chain reaction using biotinylated primers corresponding to the 5'-noncoding region. The assay is based on hybridization of the resulting polymerase chain reaction product with oligonucleotide probes immobilized as parallel lines on membrane strips.

RESULTS

According to the phylogenetic analysis of the nonstructural-5 region the prevalence of hepatitis C virus subtypes was as follows: 1a 18%, 1b 51%, 2a 3%, 2b 3%, 2c 7% and 3a 18%. The reverse hybridization assay correctly identified each hepatitis C virus genotype (1, 2, and 3). However, differentiation of hepatitis C virus subtypes was insufficient. 1/11 HCV-1a isolates was incorrectly classified by the reverse hybridization assay as HCV-1b and vice versa 3/31 HCV-1b isolates as HCV-1a. Classification of hepatitis C virus subtypes 2a, 2b and 3a was correct, but 4/4 HCV-2c isolates were misinterpreted by the assay as HCV-2a.

CONCLUSIONS

The reverse hybridization assay can differentiate between hepatitis C virus genotypes 1, 2, and 3, but is not completely reliable for hepatitis C virus subtyping.

摘要

背景/目的：丙型肝炎病毒存在多种毒株；通过对病毒基因组进行序列比较可识别出不同的基因型和亚型。近期有证据表明，丙型肝炎病毒的基因型/亚型可能会影响慢性丙型肝炎的临床病程以及对此疾病的α干扰素治疗反应，这表明识别基因型的方法可能会在临床上发挥作用。在本研究中，我们评估了一种最近推出的反向杂交检测法。

方法

从连续61例到我们门诊就诊的患者的血清样本中分离出丙型肝炎病毒RNA，随后通过双脱氧核苷酸链终止法对5'非编码区和非结构5区进行测序。通过对非结构5序列进行系统发育分析来进行丙型肝炎病毒基因分型。反向杂交检测法的扩增产物通过使用与5'非编码区对应的生物素化引物进行“巢式”聚合酶链反应获得。该检测法基于所得聚合酶链反应产物与固定在膜条上呈平行线排列的寡核苷酸探针的杂交。

结果

根据非结构5区的系统发育分析，丙型肝炎病毒亚型的流行情况如下：1a型18%，1b型51%，2a型3%，2b型3%，2c型7%，3a型18%。反向杂交检测法正确识别了每种丙型肝炎病毒基因型（1、2和3）。然而，丙型肝炎病毒亚型的区分不够充分。1/11例丙型肝炎病毒1a型分离株被反向杂交检测法错误分类为丙型肝炎病毒1b型，反之，3/31例丙型肝炎病毒1b型分离株被错误分类为丙型肝炎病毒1a型。丙型肝炎病毒2a、2b和3a亚型的分类正确，但4/4例丙型肝炎病毒2c型分离株被该检测法误判为丙型肝炎病毒2a型。