[Inhibitory effects of therapeutic reagents on the PCR detection of hepatitis C virus in serum and their elimination by RNA extraction methods]

Abstract

Inhibitors of enzymatic amplification in serum may cause false-negative results for direct detection of hepatitis C virus (HCV) by polymerase chain reaction (PCR). This study was undertaken to demonstrate inhibitory effects of the therapeutic reagents on the PCR detection of HCV and to evaluate the efficacy of their elimination by RNA extraction methods. RNA was extracted using a manual method based on organic extraction and precipitation of RNA (SepaGene RV-R, Sanko Junyaku) or an automated system based on specific capture of HCV-RNA with probes and magnetic bead/fluid (B/F) separation (Roche Molecular Systems). When RNA was extracted by SepaGene RV-R from serum containing 10(5) copies per ml of HCV and amplified for HCV-RNA in the presence of hemoglobin, bilirubin, or heparin by Amplicor HCV (Roche Molecular Systems), results tended to be negative. In addition to these, two therapeutic reagents, ATP and dextran sulfate sodium were also found to have inhibitory effects. When ATP at concentrations up to 5 mM was added to the sera and RNA was extracted with SepaGene RV-R, there were no inhibitory effect on the detection of either HCV-RNA or the internal control. In contrast, when dextran sulfate sodium up to 1 mM was added to the sera, there was a dose dependent inhibitory effect on detection of both HCV-RNA and the internal control. When HCV-RNA was extracted by the automated system, the inhibitory effect of dextran sulfate sodium was successfully eliminated. In conclusion, dextran sulfate sodium and ATP were newly identified as inhibitors that may be present in serum, and the efficacy of eliminating these substances varied among RNA extraction methods.