Archives of Pharmacal Research

The Pharmaceutical Society of Korea (대한약학회)
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Rapid Detection of Serum HCV RNA by Combining Reverse Transcription and PCR without RNA Extraction

  • Published : 1996.12.01
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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 $95^{\circ}C$ 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 and Taq DNA polymerase. The detection of HCV RNA from $10^{-3}{\mu}l$. 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.

Keywords

References

  1. N. Engl. J. Med. v.325 Hepatitis C virus infection in post-transfusion hepatitis: an analysis with first- and second-generation assays Aach,R.D.;Stevens,C.E.;Hollinger,F.B.;Mosley,J.W.;Peterson,D.A.;Taylor,P.E.;Johnson.R.G.;Barbosa,L.H.;Nemo,G.J.
  2. N. Engl. J. Med. v.321 Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis Alter,H.J.;Purcell,R.H.;Shih,J.W.;Melpolder,J.C.;Houghton,M.;Choo,Q.L.;Kuo,G.
  3. Anal. Biochem. v.162 Single-step method of RNA isolation by acid guanidium-thiocyanate-phenol-chloroform extraction Chomczynski,P.;Sacchi,N.
  4. Science v.244 Isolation of a cDNA derived from a blood-borne non-A, non-B hepatitis genome Choo,Q.L.;Kuo,G.;Weiner,A.J.;Overby,L.R.;Bradley,D.W.;Houghton,M.
  5. Lancet v.335 Detection of hepatitis C viral sequences in blood donations by 'nested' polymerase chain reaction and prediction of infectivity Garson,J.A.;Tedder,R.S.;Briggs,M.
  6. Lancet v.336 Demonstration of viraemia patterns in haemophiliacs treated with hepatitis C virus contaminated factor VIII concentrates Garson,J.A.;Tuke,P.W.;Makris,M.;Briggs,M.;Machin,S.J.;Preston,F.E.;Tedder,R.S.
  7. Lancet v.335 Differentiation between specific and non-specific hepatitis C antibodies in chronic liver disease Gary,J.J.;Wreghitt,T.G.;Friend,P.J.;Wight,D.G.D.;Soundaresan,V.;Calne,R.Y.
  8. J. Clin. Microbiol. v.31 Detection of hepatitis C virus RNA: comparison of one-stage polymerase chain reaction(PCR) with nested-set PCR Gretch,D.R.;Wilson,J.J.;Carithers,R.L.Jr.;Rosa,C.D.;Han,J.H.;Corey,L.
  9. J. Clin. Invest. v.89 Direct detection of circulating hepatitis C virus RNA using probes form 5' untranslated region Hu,K.Q.;Yu,C.H.;Vierling,J.M.
  10. J. Clin. Microbiol. v.30 Detection of hepatitis C virus sequence in sera with controversial serology by nested polymerase chain reaction Lazizi,Y.;Elfassi,E.;Pillot,J.
  11. Jpn. J. Exp. Med. v.60 Detection of hepatitis C virus by a two-stage polymerase chain reaction with two paris of primers deduced from 5'-noncoding region Okamoto,H.;Okada,S.;Sugiyama,Y.;Tanaka,T.;Sugai,Y.;Akahan,Y.;Machida,A.;Mishiro,S.;Yoshizawa,H.;Miyakawa,Y.;Mayumi,M.
  12. J. Hepatology. v.17 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 Sakamoto,N.;Sato,C.;Haritani,H.;Maekawa,S.;Kurosaki,M.;Enomoto,N.;Hoshino,Y.;Tazawa,J.I.;Nishimura,M.;Marumo,F.
  13. Lab. Invest. v.65 Detection of hepatitis C virus sequences in liver tissue by the polymerase chain reaction 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.;Gerber,M.A.
  14. Lancet v.335 Detection of hepatitis C viral sequences in non-A, non-B hepatitis Weiner,A.J.;Kuo,G.;Bradley,D.W.;Bonini,F.;Saracco,G.;Lee,C.;Rosenblatt,J.;Choo,Q.L.;Houghton,M.
  15. J. Clin. Microbiol. v.31 Detection of hepatitis C virus by combined reverse transcription-polymerase chain reaction assay Young,K.K.Y.;Resnick,R.M.;Myers,T.W.