engleski

Quantification of hepatitis C virus with homologous RNA standards

In the absence of an efficient culture system for hepatitis C virus (HCV), or viral antigen assays, direct detection of HCV is based on nucleic acid amplification and hybridization methods. The measurement of HCV RNA levels in viremic persons would increase our understanding of the transmission and pathogenesis of HCV infection and may also facilitate the clinical staging and treatment of patients with chronic hepatitis C. We developed a quantitative competitive RT-PCR assay for measurement the levels of HCV RNA in biological fluids samples that uses a synthetic part of HCV RNA genome as internal control. Developed assay consists of the co-extraction, co-amplification and the co-analysis of the sequences of interest together with known amounts of internal standards. Amplification is done using fluorescent dye-labelled primers followed by the quantitation of the PCR products by laser-induced fluorescence and GeneScan analysis software system. 5'-untranslated region (5'UTR) of HCV genome was amplified using specific primers. After sequencing of PCR product we determined Tth111I on site 167 and NheI on site 232 like unique restriction sites that were used to construct internal HCV RNA standards. By insertion of 66 bp and deletion of 61 bp, we constructed two internal HCV RNA standards HCVins+ and HCVdel-, respectively. Aliquotes internal HCV RNA standards were stabile within one month of preservation on - 80*C. Despite of that, we calibrated the internal standards in each assay. Quantification of both HCV RNA internal standards in samples of NIBSC HCV RNA working reagent was highly reproducible. Using described method, high HCV RNA levels were found in samples of patient’ s sera with acute HCV infection (105 - 106 geq/ml), in contrast to low HCV RNA levels found in samples of positive plasma pools and spent culture fluids of human leukocytes (102 - 103 geq/ml) derived from healthy donors. Quantification by both internal HCV RNA standards gave results in the same range. Constructed internal HCV RNA standards and used quantification method are specific and sensitive for determination HCV RNA level in different biological fluid samples which contains high or low copy number HCV RNA.

quantification; HCV RNA; internal standard

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