从MT2、HeLa细胞DNA及人和黑猩猩PBMC DNA中扩增GBV-C核苷酸

目的　检测MT2和HeLa细胞DNA、6例人和 1例黑猩猩PBMCDNA中是否存在与GBV C同源的核苷酸序列。方法　直接以上述DNA为模板、采用GBV C 5′ NCR和NS3区引物的直接套式PCR(dPCR)、核苷酸序列分析、引物介导原位扩增 (PRINS)NDA序列特异荧光标记技术。结果　从MT2和HeLa细胞DNA、4例人PBMCDNA标本中获得 5′ NCR引物扩增片段 ,从MT2和HeLa细胞DNA、5例人和黑猩猩PBMCDNA标本中获得NS3区引物扩增片段。这些扩增产物的核苷酸序列与GBV C同源性分别为 74 2 9%～ 77 14%和 73 80 %～ 79 15 %。PRINS检测结果显示 ,dPCR阳性的PBMC及其染色体上有荧光着色。结论　MT2和HeLa细胞DNA、人和黑猩猩PBMCDNA中存在与GBV C 5′ NCR和 或NS3区同源性较高的核苷酸序列 ,这些序列可能位于dPCR阳性的PBMC染色体上。

Study on amplification of the nucleotide sequences homologous to GBV-C from the DNAs of MT2 and HeLa cells and the PBMC DNAs of human and chimpanzee

Objective　To detect the nucleotide sequences homologous to GBV-C existed in the DNAs from MT2 and HeLa cells and the PBMC DNAs from 6 human samples and 1 chimpanzee sample. Methods　Direct nested-PCR (dPCR) respectively amplified with GBV-C 5′-NCR and NS3 primers by using the DNAs as templates, nucleotide sequence analysis, primed in situ (PRINS) sequence-specific labeling with fluorescein. ResultsDNA fragments amplified with GBV-C 5′-NCR primers were obtained from the DNAs of MT2 and HeLa cells and the PBMC DNAs in 4 of 6 human samples. DNA fragments amplified with GBV-C NS3 primers were obtained from MT2 and HeLa cells and the PBMC DNAs in 5 of 6 cases of human cases and chimpanzee. The homology of the nucleotide sequences of these amplification products compared with GBV-C genomic sequence were 74.29%-77.14% and 73.80%-79.15% respectively. The fluorescence staining by PRINS was shown in PBMCs and their chromosomes with positive PCR results. Conclusion　The nucleotide sequences with higher homology to GBV-C 5′-NCR and/or NS3 region existed in the DNAs from MT2 and HeLa cells and in the PBMC DNAs of human and chimpanzee. The sequences of the PBMCs with positive PCR results were expected to locate in their chromosomes.