A simplified method for PCR detection of hepatitis C virus RNA from human serum.

1The Laboratory of Virology and Parasitology, The Lindsley F. Kimball Research Institute of the New York Blood Center, New York, New York 10021; 2Roche Molecular Systems, Branchburg, New Jersey 08876-1760 Hepatitis C virus (HCV) is responsible for most cases of post-transfusion non-A, non-B hepatitis. (1~ PCR is a highly sensitive test for detection of HCV RNA in biological specimens and is a potentially powerful tool in laboratory diagnosis of HCV infection. Serum HCV RNA determinat ion may eventually be required for prevention of blood-borne HCV transmission. ~2~ To control contaminat ion this would require automation of the PCR technology, a development that is presently hampered by technical difficulties. One of the main obstacles is the necessity of extracting viral RNA before amplification, a t ime-consuming step that is not readily automatable. Several alternative methods for sample processing for HCV reverse transcription (RT)PCR have been reported, ~3'4~ all tending toward a much shorter and simpler procedure. Here, we describe a simple method for RT-PCR amplification of HCV RNA from h u m a n serum that does not require any extra RNA extraction step. The method is based on the f inding that Triton X-100 can release HCV RNA from the viral envelope and that this can be efficiently reverse-transcribed from the crude lysate. The protocol is as follows: One to two microliters of serum diluted with 8-9 i~l of fetal calf serum (HyClone) is added directly to the RT mix (50 mM KCI, 10 mM Tris-HCl at pH 9.0, 4 mM MgC12, 0.1% Triton X-100, 20 units of RNasin ribonuclease inhibi tor (Promega Corp., Madison, WI), 150 ng of an HCVspecific antisense primer 20 (5'-GCT CAT GGT GCA CGG TCT A, position 13 to + 6 of the viral genome), 200 I~M dNTPs, and 7.5 units of avian myeloblastosis virus (AMV)-RT (Promega) in a final volume of 20 i~l. Fetal calf serum is used as a stabilizer to prevent coagulation during the denaturation step following cDNA synthesis. In view of the known homology between HCV and animal pestiviruses, ~s~ and the possibility of contaminat ion of fetal calf serum by bovine viral diarrhea virus (BVDV), ~6~ care should be taken in both the design of PCR primers and in the use of virusscreened fetal calf serum. BVDVscreened fetal calf serum is widely available. New lots of fetal calf serum are evaluated for their ability to yield expected titer of our positive control HCVinfected serum and for negative results with a panel of noninfected control sera. cDNA synthesis is carried out at 43~ for 60 min followed by denaturat ion for 15 min at 90~ PCR mix is added [50 mM KC1, 100 mM Tris-HC1 at pH 9.0, 2 mM MgC12, 0.1% Triton X-100, 2.5 units of Taq DNA polymerase (Promega), 200 IM 22, 5'-ACT CGC AAG CAC CCT ATC A (nucleotides 293-312)] located in the 5' nonstructural region of the viral genome, generating a PCR product of 267 bp. PCR cycles are as follows: The first five cycles (94~ for 1.5 min, 72~ for 5 rain) were followed by 35 cycles of 94~ for 1.5 min, 60~ for 2 min, and 72~ for 3 min, followed by 72~ for 7 min. Amplified products were detected by oligomer hybridizat ion (7~ with a 32p. end-labeled probe (positions 161-180; 5'-GAG TAC ACC GGA ATT GCC AG) located between the primers. The probetarget duplex was separated from unhybridized probe by NuSieve 3:1 agarose gel electrophoresis. The gel was then dried and autoradiographed. Alternatively, PCR, using a biotinylated antisense primer, was followed by detection of PCR product using an experimental enzyme-l inked hybridization capture assay kindly provided by Roche Molecular Systems (Branchburg, NJ). The present method detected HCV RNA using 1 i~l of 10s to 107 dilutions of our positive control serum that titered 10-s/50 I~l by nested PCR after a standard guanid in ium/phenol /ch loroform extraction and alcohol precipitation. (2~ This control serum was chosen as having the highest titer among 19 PCR-positive blood donors sera tested. (2~ Thus, the present method is 50-500 times more sensitive than nested PCR. This was confirmed by analysis of five different plasma specimens that had been titrated by RT-PCR after guan id in ium/pheno l / chloroform extraction. These sera had been chosen to have intermediate titer (10 -2) in nested PCR titrations. As shown in Table 1, the direct method was 10to lO0-fold more sensitive for dilution of HCV RNA in ~'s sera even when the volume of sample tested is not taken into account. In one serum the two methods were of equivalent sensitivity. The specificity of the method was demonstrated by our experience in