Algorithmic approach to high-throughput molecular screening for alpha interferon-resistant genotypes in hepatitis C patients.
Algorithmic approach to high-throughput molecular screening for alpha interferon-resistant genotypes in hepatitis C patients.
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丙型肝炎患者α干扰素耐药基因型高通量分子筛查的算法方法。
DOI：
10.1128/jcm.36.7.1895-1901.1998
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发表时间：
1998
影响因子：
9.4
通讯作者：
Barathur,RR
中科院分区：
文献类型：
--
作者：
Sreevatsan,S;Bookout,JB;Ringpis,FM;Pottathil,MR;Marshall,DJ;DeArruda,M;Murvine,C;Fors,L;Pottathil,RM;Barathur,RR
本研究旨在分析利用裂解酶片段长度多态性（CFLP）分析作为DNA测序的替代方法，在高容量分子病理学实验室环境中高通量筛选丙型肝炎病毒（HCV）基因型的可行性和有效性。利用HCV基因组5 '非翻译区244 bp扩增子，对61份HCV逆转录- pcr （RT-PCR）临床样本进行了基因分型。CFLP方法确定的基因型频率分别为1a型44.3%、1b型26.2%、2b型13.1%和3a型5%。核苷酸序列分析结果与CFLP分析结果在主基因型水平上的一致性为100%，5个样本在亚型指定上存在可解决的差异。CFLP分析得出的HCV基因型频率也与国家估计一致(N. N. Zein等人，Ann。实习生。医学杂志。125:634-639,1996)。在不同的研究实验室中，对42个样品进行平行重新分析，100%的样品重现了CFLP指纹图谱。同样，19个样品在不同孵育温度-时间条件下的主要亚型名称也可100%重现。用线探针法、CFLP分析法和自动DNA测序法进行HCV基因分型的成本比较分析表明，CFLP分析法每个扩增子的平均成本最低，为20美元（直接成本）。基于这些发现，我们建议CFLP分析是一种稳健、敏感、特异性和经济的方法，可用于大规模筛查HCV感染患者的α干扰素耐药HCV基因型。该论文描述了一种算法，该算法将基于rt - pcr的HCV检测样品定性筛选作为反射测试，并且还解决了模棱两可的基因型。
This study was designed to analyze the feasibility and validity of using Cleavase Fragment Length Polymorphism (CFLP) analysis as an alternative to DNA sequencing for high-throughput screening of hepatitis C virus (HCV) genotypes in a high-volume molecular pathology laboratory setting. By using a 244-bp amplicon from the 5′ untranslated region of the HCV genome, 61 clinical samples received for HCV reverse transcription-PCR (RT-PCR) were genotyped by this method. The genotype frequencies assigned by the CFLP method were 44.3% for type 1a, 26.2% for 1b, 13.1% for type 2b, and 5% type 3a. The results obtained by nucleotide sequence analysis provided 100% concordance with those obtained by CFLP analysis at the major genotype level, with resolvable differences as to subtype designations for five samples. CFLP analysis-derived HCV genotype frequencies also concurred with the national estimates (N. N. Zein et al., Ann. Intern. Med. 125:634–639, 1996). Reanalysis of 42 of these samples in parallel in a different research laboratory reproduced the CFLP fingerprints for 100% of the samples. Similarly, the major subtype designations for 19 samples subjected to different incubation temperature-time conditions were also 100% reproducible. Comparative cost analysis for genotyping of HCV by line probe assay, CFLP analysis, and automated DNA sequencing indicated that the average cost per amplicon was lowest for CFLP analysis, at $20 (direct costs). On the basis of these findings we propose that CFLP analysis is a robust, sensitive, specific, and an economical method for large-scale screening of HCV-infected patients for alpha interferon-resistant HCV genotypes. The paper describes an algorithm that uses as a reflex test the RT-PCR-based qualitative screening of samples for HCV detection and also addresses genotypes that are ambiguous.