Centro de Desenvolvimento Científico e Tecnológico, Fundação Estadual de Produção e Pesquisa em Saúde (CDCT/FEPPS), Porto Alegre, Brazil.
J Virol Methods. 2009 Dec;162(1-2):75-80. doi: 10.1016/j.jviromet.2009.07.017. Epub 2009 Jul 28.
This study describes a colorimetric method for detecting and genotyping hepatitis C virus (HCV) in which four different oligonucleotide probes are fixed onto microwell plates and hybridized separately with biotinylated PCR amplification products derived from clinical samples. The first probe capable of hybridizing with all seven known HCV genotypes was used for overall detection, and the remaining probes were used to recognize specifically genotypes 1-3. When combined with an improved silica-based RNA extraction method, the sensitivity of the test was 50 IU/mL. Eighty-five of the 86 samples analyzed (98.8%) yielded results in agreement with reference detection methods. The remaining sample was HCV-RNA positive in the COBAS Amplicor qualitative assay, but was negative using the reverse-hybridization method. The usefulness of the new genotyping test was confirmed by comparison with direct sequencing of PCR products: 98% of samples tested (54/55) were in agreement using the two methods (21, 7 and 27 from genotypes 1-3, respectively). The single discrepancy might have been due to a mixed HCV infection. The new method is an alternative to the use of commercially available genotyping kits and should be particularly convenient in developing countries where genotypes 1-3 represent a high proportion of HCV isolates.
本研究描述了一种用于检测丙型肝炎病毒 (HCV) 的比色法，其中四个不同的寡核苷酸探针固定在微孔板上，并分别与从临床样本中获得的生物素化 PCR 扩增产物杂交。能够与所有七种已知 HCV 基因型杂交的第一个探针用于总体检测，其余探针用于特异性识别基因型 1-3。与改进的基于硅胶的 RNA 提取方法结合使用时，该检测方法的灵敏度为 50IU/mL。分析的 86 个样本中的 85 个（98.8%）与参考检测方法的结果一致。其余样本在 COBAS Amplicor 定性检测中 HCV-RNA 为阳性，但反转杂交法为阴性。通过与 PCR 产物的直接测序进行比较，证实了新的基因分型测试的有用性：两种方法的测试样本中有 98%（54/55）一致（分别来自基因型 1-3 的 21、7 和 27 个样本）。唯一的差异可能是由于混合 HCV 感染。新方法是替代市售基因分型试剂盒的一种方法，特别是在 HCV 分离株中基因型 1-3 占很大比例的发展中国家，这种方法应该特别方便。
