A competitive reverse transcription-polymerase chain reaction assay for quantitation of GB virus C/hepatitis G virus RNA that circumvents heteroduplex artifact

Abstract

The role of GB virus C (GBV-C)/hepatitis G virus (HGV) in hepatitis has been controversial. To investigate its possible pathogenicity and site(s) of replication, it is important to develop an accurate quantitative assay for both positive and negative strand GBV-C/HGV RNA. In this study, a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay for both positive and negative strand GBV-C/HGV RNA quantitation was developed. In developing the quantitative assay, heteroduplex formation was repeatedly observed. A heterologous competitor RNA with GBV-C/HGV primer-binding sequences was introduced, and heteroduplex artifact was circumvented successfully. Two-hundred thirty-seven serum specimens were screened by RT-PCR for GBV-C/HGV RNA. Two of the 62 patients infected with chronic hepatitis C virus (HCV) were found to be positive for GBV-C/HGV RNA. None of the 50 other patients with no evidence of HCV infection and none of the 125 normal individuals were positive for GBV-C/HGV RNA. The sensitivity of RT-PCR was 3000 gE/ml (30 gE in RT-PCR). Alternate methods for residual DNA removal and its detection in synthetic RNA were introduced. A RT control containing no primer before PCR is necessary to evaluate the trace amounts of template DNA remaining in synthesized RNA. The method will differentiate reliably between positive and negative strand RNAs up to a 10 4-fold difference in titer. The positive and negative strand GBV-C/HGV RNAs were detected in one patient by RT-PCR and hybridization analysis, and the strand titer was determined by RT-PCR.