Marshall D J, Heisler L M, Lyamichev V, Murvine C, Olive D M, Ehrlich G D, Neri B P, de Arruda M
Third Wave Technologies, Inc., Madison, Wisconsin 53711, USA.
J Clin Microbiol. 1997 Dec;35(12):3156-62. doi: 10.1128/jcm.35.12.3156-3162.1997.
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.
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分析（CFLP；Third Wave Technologies公司，威斯康星州麦迪逊市），用于确定丙型肝炎病毒（HCV）的基因型。CFLP分析可产生结构指纹图谱，从而能够区分不同的DNA序列。我们分析了通过逆转录PCR从HCV高度保守的5'-非编码区产生的251bp cDNA产物。我们通过DNA测序确定了87个样本的基因型，发现这些分离株代表了在美国通常遇到的98%的类型，即1a、1b、2a/c、2b、3a和4型。对这些样本进行的盲法CFLP分析与DNA测序结果100%一致，因此密切相关的基因型产生的图谱具有很强的家族相似性，而差异较大的序列产生的图谱则有明显差异。在每种情况下，总体图谱都表明了基因型分组，而更细微的变化则表明了亚型差异。我们还通过分析属于单一亚型的三个不同分离株评估了CFLP分析在HCV基因分型中的可重复性。这三个分离株产生了无法区分的CFLP图谱，对单个分离株的重复分析也是如此。这项研究证明了该技术适用于HCV基因分型，并表明它可能为DNA测序或其他更昂贵或繁琐的基因分型方法提供一种低成本、高通量的替代方法。
