Proc. Nati. Acad. Sci. USA
Vol. 89, pp. 187-191, January 1992 Medical Sciences
Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay (non-A, non-B hepatltis/5' noncoding sequence/genetic heterogeneity)
JENS BUKH, ROBERT H. PURCELL*, AND ROGER H. MILLER Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
Contributed by Robert H. Purcell, October 2, 1991
United States, and Zaire) who were anti-HCV antibodypositive by a first-generation anti-HCV test (2). The German and South African samples were selected for high titers of anti-HCV from larger anti-HCV-positive groups. There were 33 patients with chronic NANB hepatitis (Italy-12, Denmark-8, Taiwan-6, Hong Kong-5, and Sweden2), 15 patients with acute NANB hepatitis (U.S.-12, Denmark-2; and Sweden-1), 2 patients with chronic hepatitis who were positive for hepatitis B virus-encoded surface antigen (HBsAg) (Denmark, Hong Kong), 3 HBsAg-negative patients with liver cirrhosis (Denmark-2, Taiwan-i), and 17 patients with hepatocellular carcinoma (HCC; South Africa13, Taiwan-3, Hong Kong-1). The remaining 44 anti-HCVpositive individuals were found by screening high-risk populations and included 10 Peruvian health care workers, 14 individuals with a history of sexual promiscuity (Zaire-9, Dominican Republic-5), 2 dialysis patients (Hong Kong), 2 HBsAg carriers (Hong Kong, India), 1 patient with hemophilia (Hong Kong), 1 Swedish blood donor who transmitted NANB hepatitis to a recipient, 6 commercial blood donors as well as 2 blood transfusion recipients from India, and 6 German recipients of anti-Rh immunoglobulin. To reduce the risk of contamination in PCR analysis, a number of precautions were taken (31). Solutions and aliquots were prepared in a laboratory that was never used for HCV research. Extraction of RNA and transfer of samples from first to second PCR reaction tubes were performed under sterile conditions with sterile reagents. These procedures as well as gel electrophoresis of the final PCR product were performed in separate, self-contained areas that had never been exposed to recombinant HCV DNA. All solutions were pipeted with positive displacement pipets or with disposable pipets or pipet tips containing an aerosol barrier. In addition, gloves were changed after each contact. A negative control was included for every test sample to monitor for contamination as a source of false-positive results. All sera were stored at -800C after receipt in our laboratory. RNA was extracted by a modification of the hot phenol method (32). Specifically, 100 1.l of serum or plasma was mixed with 400 p.1 of solution A containing 4.2 M guanidinium isothiocyanate, 25 mM Tris-HCI (pH 8.0), 0.5% sarkosyl, 0.7% (vol/vol) 2-mercaptoethanol, and 50 ,ul of solution B containing 1.0 M Tris HCI (pH 8.0), 0.1 M EDTA, and 10% (wt/vol) NaDodSO4, followed by extraction with an equal volume of water-saturated phenol/chloroform, 1:1 (vol/vol) at 65°C for 30 min. The aqueous phase was removed and saved. The organic phase (with the interphase) was extracted with 300 j. of solution A at 65°C for 5 min. The two aqueous
ABSTRACT We compared four primer sets from conserved regions of the hepatitis C virus (HCV) genome for their ability to detect HCV RNA in a "nested" cDNA polymerase chain reaction assay on sera from 114 anti-HCV antibodypositive individuals from around the world. The different primer sets had equivalent sensitivity, detecting
Vol. 89, pp. 187-191, January 1992 Medical Sciences
Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay (non-A, non-B hepatltis/5' noncoding sequence/genetic heterogeneity)
JENS BUKH, ROBERT H. PURCELL*, AND ROGER H. MILLER Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
Contributed by Robert H. Purcell, October 2, 1991
United States, and Zaire) who were anti-HCV antibodypositive by a first-generation anti-HCV test (2). The German and South African samples were selected for high titers of anti-HCV from larger anti-HCV-positive groups. There were 33 patients with chronic NANB hepatitis (Italy-12, Denmark-8, Taiwan-6, Hong Kong-5, and Sweden2), 15 patients with acute NANB hepatitis (U.S.-12, Denmark-2; and Sweden-1), 2 patients with chronic hepatitis who were positive for hepatitis B virus-encoded surface antigen (HBsAg) (Denmark, Hong Kong), 3 HBsAg-negative patients with liver cirrhosis (Denmark-2, Taiwan-i), and 17 patients with hepatocellular carcinoma (HCC; South Africa13, Taiwan-3, Hong Kong-1). The remaining 44 anti-HCVpositive individuals were found by screening high-risk populations and included 10 Peruvian health care workers, 14 individuals with a history of sexual promiscuity (Zaire-9, Dominican Republic-5), 2 dialysis patients (Hong Kong), 2 HBsAg carriers (Hong Kong, India), 1 patient with hemophilia (Hong Kong), 1 Swedish blood donor who transmitted NANB hepatitis to a recipient, 6 commercial blood donors as well as 2 blood transfusion recipients from India, and 6 German recipients of anti-Rh immunoglobulin. To reduce the risk of contamination in PCR analysis, a number of precautions were taken (31). Solutions and aliquots were prepared in a laboratory that was never used for HCV research. Extraction of RNA and transfer of samples from first to second PCR reaction tubes were performed under sterile conditions with sterile reagents. These procedures as well as gel electrophoresis of the final PCR product were performed in separate, self-contained areas that had never been exposed to recombinant HCV DNA. All solutions were pipeted with positive displacement pipets or with disposable pipets or pipet tips containing an aerosol barrier. In addition, gloves were changed after each contact. A negative control was included for every test sample to monitor for contamination as a source of false-positive results. All sera were stored at -800C after receipt in our laboratory. RNA was extracted by a modification of the hot phenol method (32). Specifically, 100 1.l of serum or plasma was mixed with 400 p.1 of solution A containing 4.2 M guanidinium isothiocyanate, 25 mM Tris-HCI (pH 8.0), 0.5% sarkosyl, 0.7% (vol/vol) 2-mercaptoethanol, and 50 ,ul of solution B containing 1.0 M Tris HCI (pH 8.0), 0.1 M EDTA, and 10% (wt/vol) NaDodSO4, followed by extraction with an equal volume of water-saturated phenol/chloroform, 1:1 (vol/vol) at 65°C for 30 min. The aqueous phase was removed and saved. The organic phase (with the interphase) was extracted with 300 j. of solution A at 65°C for 5 min. The two aqueous
ABSTRACT We compared four primer sets from conserved regions of the hepatitis C virus (HCV) genome for their ability to detect HCV RNA in a "nested" cDNA polymerase chain reaction assay on sera from 114 anti-HCV antibodypositive individuals from around the world. The different primer sets had equivalent sensitivity, detecting
