Abstract
A simple, rapid, specific and sensitive method for the detection of serum hepatitis C virus (HCV) RNA using the reverse transcription-polymerase chain reaction (RT-PCR) technique without conventional RNA extraction was developed. HCV template RNA from serum was obtained by boiling the serum at 95°C for 2 min, cooling rapidly in ice and removing the proteins by cetrifugation. RT-PCR amplifications including the reverse transcription and first PCR amplification were performed in one vessel containing both of reverse transcriptase andTaq DNA polymerase. The detection of HCV RNA from 10−3 μl serum was possible with this method. The suitability of this method for clinical analysis was evaluated by assaying HCV RNA in 225 patient samples including anti-HCV antibody negatives (13 samples) and positives (212 samples) by enzyme-linked immunosorbent assay test (ELISA). Detections of HCV RNA with this method were in 4 of 13 anti-HCV antibody negative samples (30.8%) and 95 of 212 positive samples (44.8%). The present method can be completed in 1 hr and has a wide range of application for the clinical utilities to determine the viral RNAs.
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References Cited
Aach, R. D., Stevens, C. E., Hollinger, F. B., Mosley, J. W., Peterson, D. A., Taylor, P. E., Johnson, R. G., Barbosa, L. H. and Nemo, G. J., Hepatitis C virus infection in post-transfusion hepatitis: an analysis with first- and second-generation assays.N. Engl. J. Med., 325, 1325–1329 (1991).
Alter, H. J., Purcell, R. H., Shih, J. W., Melpolder, J. C., Houghton, M., Choo, Q.-L. and Kuo, G., Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis.N. Engl. J. Med., 321, 1494–1500 (1989).
Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidium-thiocyanatephenol-chloroform extraction.Anal. Biochem., 162, 156–159 (1992).
Choo, Q-.L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W. and Houghton, M., Isolation of a cDNA derived from a blood-borne non-A, non-B hepatitis genome.Science, 244, 359–362 (1989).
Garson, J. A., Tedder, R. S. and Briggs, M., Detection of hepatitis C viral sequences in blood donations by ‘nested’ polymerase chain reaction and prediction of infectivity.Lancet, 335, 1419–1422 (1990).
Garson, J. A., Tuke, P. W., Makris, M., Briggs, M., Machin, S.J., Preston, F. E. and Tedder, R. S., Demonstration of viraemia patterns in haemophiliacs treated with hepatitis C virus contaminated factor VIII concentrates.Lancet, 336, 1022–1025 (1990).
Gary, J. J., Wreghitt, T. G., Friend, P. J., Wight, D. G. D., Soundaresan, V. and Calne, R. Y., Differentiation between specific and non-specific hepatitis C antibodies in chronic liver disease.Lancet, 335, 609–610 (1990).
Gretch, D. R., Wilson, J. J., Carithers, R. L. Jr., Rosa, C. D., Han, J. H. and Corey, L., Detection of hepatitis C virus RNA: comparison of one-stage polymerase chain reaction (PCR) with nested-set PCR.J. Clin. Microbiol., 31, 289–291 (1993).
Hu, K-.Q., Yu, C-.H. and Vierling, J. M., Direct detection of circulating hepatitis C virus RNA using probes from the 5′ untranslated region.J. Clin. Invest., 89, 2040–2045 (1992).
Lazizi, Y., Elfassi, E. and Pillot, J., Detection of hepatitis C virus sequences in sera with controversial serology by nested polymerase chain reaction.J. Clin. Microbiol., 30, 931–934 (1992).
Okamoto, H., Okada, S., Sugiyama, Y., Tanaka, T., Sugai, Y., Akahan, Y., Machida, A., Mishiro, S., Yoshizawa, H., Miyakawa, Y. and Mayumi, M., Detection of hepatitis C virus by a two-stage polymerase chain reaction with two pairs of primers deduced from the 5′-noncoding region.Jpn. J. Exp. Med., 60, 215–222 (1990).
Sakamoto, N., Sato, C., Haritani, H., Maekawa, S., Kurosaki, M., Enomoto, N., Hoshino, Y., Tazawa, J.-I. Nishimura, M. and Marumo, F., Detection of hepatitis C viral RNA in sporadic acute non-A, non-B hepatitis by polymerase chain reaction. Its usefulness for the early diagnosis of seronegative infection.J. Hepatology, 17, 28–33 (1993).
Shieh, Y. S. C., Shim, K-.S., Lampertico, P., Balart, L. A., Jeffers, L. J., Thung, S. N., Regenstein, F., Reddy, K. R., Farr, G., Schiff, E. R. and Gerber, M. A., Detection of hepatitis C virus sequences in liver tissue by the polymerase chain reaction.Lab. Invest., 65, 408–411 (1991).
Weiner, A. J., Kuo, G., Bradley, D. W., Bonini, F., Saracco, G., Lee, C., Rosenblatt, J., Choo, Q.-L. and Houghton. M., Detection of hepatitis C viral sequences in non-A, non-B hepatitis.Lancet, 335, 1–3 (1990).
Young, K. K. Y., Resnick, R. M. and Myers, T. W., Detection of hepatitis C virus by a combined reverse transcription-polymerase chain reaction assay.J. Clin. Microbiol., 31, 882–886 (1993).
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Jang, J.S., Lee, KJ. Rapid detection of serum HCV RNA by combining reverse transcription and PCR without RNA extraction. Arch. Pharm. Res. 19, 486–489 (1996). https://doi.org/10.1007/BF02986016
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DOI: https://doi.org/10.1007/BF02986016