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

The role of GB virus C (GBV-C)/hepatitis G virus (HGV) in hepatitis has bee n controversial. To investigate its possible pathogenicity and site(s) of r eplication, it is important to develop an accurate quantitative assay for b oth positive and negative strand GBV-C/HGV RNA. In this study, a competitiv e reverse transcription-polymerase chain reaction (RT-PCR) assay for both p ositive and negative strand GBV-C,HGV RNA quantitation was developed. In de veloping the quantitative assay, heteroduplex formation was repeatedly obse rved. A heterologous competitor RNA with GBV-C/HGV primer-binding sequences was introduced, and heteroduplex artifact was circumvented successfully. T wo-hundred thirty-seven serum specimens were screened by RT-PCR for GBV-C/H GV 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 patient s 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 detect ion in synthetic RNA were introduced. A RT control containing no primer bef ore PCR is necessary to evaluate the trace amounts of template DNA remainin g in synthesized RNA. The method will differentiate reliably between positi ve and negative strand RNAs up to a 10(4)-fold difference in titer. The pos itive 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. (C) 1999 Elsevier Science B.V, All rights reserved.