IIIIIIII Technical Tips Direct PCR Amplification of HCV RNA from Human Serum Antonella Ravaggi, Daniele Primi, and Elisabetta Cariani Consorzio per le Biotecnologie, Consiglio Nazionale delle Ricerche (CNR), Institute of Chemistry, School of Medicine, University of Brescia, 25123 Brescia, Italy Hepatitis C virus (HCV) is a positive- stranded RNA virus responsible for most cases of parenterally transmitted non-A, non-B hepatitis (NANBH). 11,21 The polymerase chain reaction (PCR) is, at present, the only method allow- ing the detection of HCV RNA in biological specimens, thus providing the direct appraisal of infectivity. (3) Therefore, serum HCV RNA determina- tion ideally represents the method of choice for the prevention of HCV transmission. The routine clinical use of PCR-based technology, however, is seriously hampered by technical dif- ficulties. In the case of HCV infection, one of the main obstacles is the neces- sity of extracting viral RNA before amplification, a time-consuming step that requires RNase-free conditions that are difficult to obtain in diag- nostic laboratories. Here we describe a simple and rapid method for reverse transcription (RT)- PCR amplification of HCV RNA from human serum that does not require the RNA extraction step. After denatura- tion (30 sec at 92~ 3 ~l of serum was directly added to the RT mix (50 mM Tris HC1, pH 8.2, 70 mM KC1, 10 mM MgCl2, 4 mM DTF, 12 units of human placental ribonuclease inhibitor (HP- RI), 0.4% Nonidet P-40, 50 pmoles of a specific antisense primer (A1), 250 ~M dNTPs, 6 units of AMV-RT, in a final volume of 25 B1). RT was carried out at 42~ for 60 min, and the cDNA was added directly, after denaturation (5 min at 100~ to the PCR mix. Am- plification was performed following the nested primers protocol, (4/ as pre- viously described, is) with two sets of primers located in the 5 ' untranslated region (UTR) of the viral genome. The first round of PCR (35 cycles) was carried out using primers A1 (5'- GATGCACGGTCTACGAGACCTC-3 ' ) and $1 (5 '-AACTACTGTCTTCACGCA- GAA-3'), generating a PCR product of 289 bp. For the second round (25 cycles), we used primers A2 (5' GCGACCCAACACTACTCGGCT-3 ' ) and $2 (5 ' -ATGGCGTFAGTATGA- GTG-3'), generating a PCR product of 187 bp. PCR cycles were as follows: denaturation at 94~ for 1 min, an- nealing of primers at 45~ for 1 min, and elongation at 72~ for 2 min. This method was used to screen serial serum samples collected from a patient with post-transfusion NANBH. In this patient, positivity for the anti- HCV ELISA assay (Ortho Diagnostics), detecting antibodies against the non- structural viral antigen C-100, was first observed 13 weeks after transfusion. By contrast, the detection of the viral nucleic acid was possible as soon as 3 weeks after transfusion (Fig. 1, lane b), confirming the superior sensitivity of PCR for early diagnosis of acute NANBH. (3,41 Further prelevments, col- lected 5-56 weeks after transfusion, confirmed persistent positivity for HCV RNA. Comparison of direct amplification from serum (Fig. 1, part 1) with the standard procedure (RNA extraction (B), RT-PCR) (Fig. 1, part 2), performed on equivalent amounts of template, show- ed a perfectly comparable sensitivity. By both methods, we obtained amplifi- cation products easily visible on agarose gel from all samples. The very low circulating HCV titers present in most of patients represent a major problem for the determination of the viral nucleic acids in serum samples. The present method for direct HCV RNA determination was successfully performed using up to 12 [xl and down to 1 ~l of a 1:104 dilution of a positive control serum. This indicates that the high sensitivity of this PCR protocol al- lows the detection of circulating HCV particles in a wide range of concentra- tion. An important drawback for the clinical use of HCV RNA amplification is the extreme variability of the viral genome, especially in the regions en- coding the envelope viral proteins. The observation of conserved mutations among American and Japanese isolates suggested the existence of HCV sub- types segregated in different geog- raphic areas. (3) The choice of oligo- nucleotide primers located in highly conserved genomic regions, such as the 5' UTR, (3) is therefore critical for a clinically useful PCR protocol. By com- parative nucleotide sequence analysis of variable regions, we recently detect- ed the common distribution in our ge- ographic area of HCV genomes belong- ing to the two main subtypes (HCV-US and HCV-J) (unpublished results). Despite the presence of extensive se- quence divergence in the envelope genes, HCV genomes belonging to 1:291-292 9 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/92 $3.00 PCR Methods and Applications 291 Cold Spring Harbor Laboratory Press on April 23, 2020 - Published by genome.cshlp.org Downloaded from