Determination of hepatitis C virus genotypes in the United States by cleavase fragment length polymorphism analysis
Determination of hepatitis C virus genotypes in the United States by cleavase fragment length polymorphism analysis
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通过切割酶片段长度多态性分析确定美国丙型肝炎病毒基因型
DOI：
10.1128/jcm.35.12.3156-3162.1997
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发表时间：
1997
影响因子：
9.4
通讯作者：
Arruda
中科院分区：
文献类型：
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作者：
D. Marshall;L. Heisler;V. Lyamichev;C. Murvine;D. M. Olive;G. Ehrlich;B. Neri;Monika;De;Arruda
我们描述了一种新的DNA扫描方法的应用，该方法被称为裂解酶片段长度多态性（CFLP; Third Wave Technologies，Inc.，麦迪逊，威斯康星州），用于丙型肝炎病毒（HCV）基因型的测定。CFLP分析导致产生允许区分不同DNA序列的结构指纹。我们分析了逆转录-PCR产生的251-bp的cDNA产物的高度保守的5 '-非编码区的HCV。我们通过DNA测序确定了87个样本的基因型，发现了代表98%的美国常见类型的分离株，即，类型1a、1b、2a/c、2b、3a和4。这些样本的盲法CFLP分析与DNA测序结果100%一致，因此密切相关的基因型产生具有强烈家族相似性的模式，而更趋异的序列产生具有明显差异的模式。在每种情况下，总的模式是指示基因型分组，而更精细的变化表明亚基因型差异。我们还通过分析属于单一亚型的三种不同分离株，评估了CFLP分析在HCV基因分型中的重现性。这三个分离株产生了不可区分的CFLP模式，作为一个单一的分离株重复分析。这项研究证明了这种技术对HCV基因分型的适用性，并表明它可能提供一种低成本，高通量的替代DNA测序或其他更昂贵或繁琐的基因分型方法。
We describe the application of a new DNA-scanning method, which has been termed Cleavase Fragment Length Polymorphism (CFLP; Third Wave Technologies, Inc., Madison, Wis.), for the determination of the genotype of hepatitis C virus (HCV). CFLP analysis results in the generation of structural fingerprints that allow discrimination of different DNA sequences. We analyzed 251-bp cDNA products generated by reverse transcription-PCR of the well-conserved 5'-noncoding region of HCV. We determined the genotypes of 87 samples by DNA sequencing and found isolates representing 98% of the types typically encountered in the United States, i.e., types 1a, 1b, 2a/c, 2b, 3a, and 4. Blinded CFLP analysis of these samples was 100% concordant with DNA sequencing results, such that closely related genotypes yielded patterns with strong familial resemblance whereas more divergent sequences yielded patterns with pronounced dissimilarities. In each case, the aggregate pattern was indicative of genotypic grouping, while finer changes suggested subgenotypic differences. We also assessed the reproducibility of CFLP analysis in HCV genotyping by analyzing three distinct isolates belonging to a single subtype. These three isolates yielded indistinguishable CFLP patterns, as did replicate analysis of a single isolate. This study demonstrates the suitability of this technology for HCV genotyping and suggests that it may provide a low-cost, high-throughput alternative to DNA sequencing or other, more costly or cumbersome genotyping approaches.
了解丙型肝炎的最新进展。
影响因子：
--
作者：
Douam,Florian;Ding,Qiang;Ploss,Alexander
通讯作者：
Ploss,Alexander