Nolte Frederick S, Green Alicia M, Fiebelkorn Kristin R, Caliendo Angela M, Sturchio Cynthia, Grunwald Aileen, Healy Mimi
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Clin Microbiol. 2003 Apr;41(4):1558-64. doi: 10.1128/JCM.41.4.1558-1564.2003.
We compared the performance characteristics of a standardized direct sequencing method (TRUGENE HCV 5'NC; Visible Genetics Inc., Toronto, Ontario, Canada) and a reverse hybridization line probe assay (INNO-LiPA HCV II; Bayer Corp., Tarrytown, N.Y.) for genotyping of hepatitis C virus (HCV). Both methods are based on detection of sequence heterogeneity in the 5' noncoding (5'NC) region. Concordance between the genotyping methods was assessed by testing 172 samples representing the six major genotypes. Sequence analysis of the more phylogenetically informative nonstructural 5B (NS5B) region was also done with 148 (86%) samples to confirm the accuracy of and resolve discrepancies between the 5'NC genotyping results. The sensitivities of the methods were assessed by using the 5'NC amplicon from both the qualitative and quantitative AMPLICOR HCV tests (Roche Diagnostics Corp., Indianapolis, Ind.). The ability of the methods to detect mixed-genotype infections was determined with mixtures of two different genotypes at relative concentrations ranging from 1 to 50%. Both 5'NC methods were able to genotype 99.4% of the samples with type agreement for 99.5% and subtype agreement for 68.2% of the samples. No or ambiguous subtype results were found by the line probe assay for 16.5% and by the TRUGENE 5'NC test for 17.1% of the samples. Discrepancies occurred between the line probe assay and NS5B results at the type level for 1.4% of the samples and at the subtype level for 14.2% of the samples. Discrepancies also occurred between the TRUGENE 5'NC and NS5B results at the type level for 2% of the samples and at the subtype level for 8.1% of the samples. We also found two distinct strains of HCV classified as type 2 by analysis of the 5'NC region that were type 1 by analysis of the NS5B region. The sensitivities of the two 5'NC genotyping methods were comparable and dependent on the amplification test used ( approximately 10(3) IU/ml with the qualitative HCV RNA tests and approximately 10(5) IU/ml with the quantitative HCV RNA tests). Genotype mixtures were successfully identified at a relative concentration of 5% by the line probe assay and 10% by the TRUGENE 5'NC test. In conclusion, the performance characteristics of the 5'NC methods were similar and both methods produced accurate results at the genotype level but neither method should be used for subtyping.
我们比较了标准化直接测序法（TRUGENE HCV 5'NC；加拿大安大略省多伦多市Visible Genetics公司）和反向杂交线性探针检测法（INNO-LiPA HCV II；美国纽约州塔里敦市拜耳公司）对丙型肝炎病毒（HCV）进行基因分型的性能特征。两种方法均基于检测5'非编码（5'NC）区域的序列异质性。通过检测代表六种主要基因型的172份样本评估基因分型方法之间的一致性。还对148份（86%）样本的非结构5B（NS5B）区域进行了更具系统发育信息的序列分析，以确认5'NC基因分型结果的准确性并解决其中的差异。通过使用定性和定量AMPLICOR HCV检测（美国印第安纳州印第安纳波利斯市罗氏诊断公司）的5'NC扩增子评估方法的灵敏度。通过使用两种不同基因型的混合物，相对浓度范围为1%至50%，来确定方法检测混合基因型感染的能力。两种5'NC方法均能够对99.4%的样本进行基因分型，其中99.5%的样本型别一致，68.2%的样本亚型一致。线性探针检测法对16.5%的样本未得出或得出模糊的亚型结果，TRUGENE 5'NC检测法对17.1%的样本未得出或得出模糊的亚型结果。线性探针检测法与NS5B结果在型别水平上对1.4%的样本、在亚型水平上对14.2%的样本存在差异。TRUGENE 5'NC与NS5B结果在型别水平上对2%的样本、在亚型水平上对8.1%的样本存在差异。我们还发现，通过5'NC区域分析被归类为2型的两种不同HCV毒株，通过NS5B区域分析则为1型。两种5'NC基因分型方法的灵敏度相当，且取决于所使用的扩增检测（定性HCV RNA检测约为10³IU/ml，定量HCV RNA检测约为10⁵IU/ml）。线性探针检测法能够成功鉴定相对浓度为5%的基因型混合物，TRUGENE 5'NC检测法能够成功鉴定相对浓度为10%的基因型混合物。总之，5'NC方法的性能特征相似，两种方法在基因型水平上均能得出准确结果，但两种方法均不适用于亚型分析。
