Hepatitis C: description of a highly sensitive method for clinical detection of viral RNA

To improve the sensitivity of hepatitis C virus RNA (HCV RNA) detection in serum by 'nested' polymerase chain reaction (PCR), primers belonging to 5' non-coding (5'NC) regions were used to compare the classical phenol/chloroform technique by using the proteinase K and silica gel technique with guanidinium thiocyanate. The silica gel techniques was found to be more efficient and sensitive for the extraction and purification of viral RNA from serum samples. The silica gel technique also avoids contact with hazardous volatile chemicals like phenol and chloroform and provides a better protection for viral RNA. Furthermore, the RNA detection sensitivity was greatly improved by modifying the buffer for reverse transcription and PCR. Using silica gel extraction, and the modified buffer, viral RNA was detected in 699 sera from anti-HCV second generation ELISA positive patients. These sera were distributed in second generation RIBA confirmed, indeterminate and non confirmed groups with PCR positive rates of 71.4%, 45.7% and 15.8%, respectively. Two out of 227 ELISA negative patients showed the presence of HCV RNA in serum. An association between the presence of antibodies against a determined viral peptide and viremia was not detected.