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首页> 外文期刊>Journal of Clinical Microbiology >Evaluation of a New Assay in Comparison with Reverse Hybridization and Sequencing Methods for Hepatitis C Virus Genotyping Targeting Both 5′ Noncoding and Nonstructural 5b Genomic Regions
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Evaluation of a New Assay in Comparison with Reverse Hybridization and Sequencing Methods for Hepatitis C Virus Genotyping Targeting Both 5′ Noncoding and Nonstructural 5b Genomic Regions

机译：与针对5'非编码和非结构性5b基因组区域的丙型肝炎病毒基因分型的反向杂交和测序方法比较的新方法评估

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We report the evaluation of a new real-time PCR assay for hepatitis C virus (HCV) genotyping. The assay design is such that genotype 1 isolates are typed by amplification targeting the nonstructural 5b (NS5b) subgenomic region. Non-genotype 1 isolates are typed by type-specific amplicon detection in the 5′ noncoding region (5′NC) (method 1; HCV genotyping analyte-specific reagent assay). This method was compared with 5′NC reverse hybridization (method 2; InnoLiPA HCV II) and 5′NC sequencing (method 3; Trugene HCV 5′NC). Two hundred ninety-five sera were tested by method 1; 223 of them were also typed by method 2 and 89 by method 3. Sequencing and phylogenetic analysis of an NS5b fragment were used to resolve discrepant results. Suspected multiple-genotype infections were confirmed by PCR cloning and pyrosequencing. Even though a 2% rate of indeterminates was obtained with method 1, concordance at the genotype level with results with methods 2 and 3 was high. Among eight discordant results, five mixed infections were confirmed. Genotype 1 subtyping efficiencies were 100%, 77%, and 74% for methods 1, 2, and 3, respectively; there were 11/101 discordants between methods 1 and 2 (method 1 was predominantly correct) and 2/34 between methods 2 and 3. Regarding genotype 2, subtyping efficiencies were 100%, 45%, and 92% by methods 1, 2, and 3, respectively; NS5b sequencing of discordants (16/17) revealed a putative new subtype within genotype 2 and that most subtype calls were not correct. Although only sequencing-based methods provide the possibility of identifying new variants, the real-time PCR method is rapid, straightforward, and simple to interpret, thus providing a good single-step alternative to more-time-consuming assays.
机译：我们报告对丙型肝炎病毒（HCV）基因分型的新型实时PCR分析的评估。分析设计是通过靶向非结构性5b（NS5b）亚基因组区域的扩增来对基因型1分离株进行分型。通过在5'非编码区（5'NC）中的类型特异性扩增子检测对非基因型1分离株进行分型（方法1; HCV基因分型分析物特异性试剂测定）。将该方法与5'NC反向杂交（方法2; InnoLiPA HCV II）和5'NC测序（方法3; Trugene HCV 5'NC）进行了比较。用方法1检测了295份血清。还通过方法2键入了其中的223个，通过方法3键入了它们的89个。使用NS5b片段的测序和系统发育分析来解析不一致的结果。通过PCR克隆和焦磷酸测序证实可疑的多基因型感染。即使用方法1获得2％的不确定性，在基因型水平上与方法2和3的结果的一致性很高。在八项不一致的结果中，确认了五种混合感染。方法1、2和3的基因型1分型效率分别为100％，77％和74％。方法1和方法2之间存在11/101的不一致性（方法1主要是正确的），方法2和方法3之间存在2/34的不一致性。对于基因型2，方法1，2的分型效率分别为100％，45％和92％。和3分别; NS5b不和谐者的测序（16/17）显示基因型2中存在一个假定的新亚型，并且大多数亚型的称呼都不正确。尽管只有基于测序的方法才有可能鉴定出新的变异体，但实时PCR方法却快速，直接且易于解释，从而为耗时的测定提供了良好的单步选择。

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