Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지

A Nucleic Acid Amplification Tests for Reliable HCV RNA Detection Method for Plasma-Derived Products

핵산증폭시험을 이용한 혈장분획물질에서 HCV RNA 검출

  • Hong, Seung-Hee (Korea Human Resource Development Institute for Health and Welfare)
  • 홍승희 (한국보건복지인력개발원)
  • Published : 2008.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

HCV is transmitted via various plasma derived products. Current methods to detect hepatitis C virus (HCV) are based on its antibody detection in the donated blood and plasma. Viral contamination can potentially escape such detection during the window period of infection, when no antibody is present or the level of antibody is too low to detect. It is trying to application of nucleic acid amplification tests (NAT) for the direct detection of HCV. The objective of this study was to develop a reliable NAT for the HCV RNA detection from plasma-derived products. The most useful primers was selected for NAT among 5 sets of primers. We have also found that QIAamp viral RNA isolation kit was the most efficient for HCV RNA isolation. The highest sensitivity and specificity was appeared in $48^{\circ}C$ annealing temperature and 30 pmol of primers. With a spiking of HCV to albumin, immunoglobulins and coagulation factors, NAT can detect up to 100 IU/ml. Meanwhile, COBAS amplicor HCV 2.0 afforded a lower sensitivity in high concentrated intramuscular immunoglobulins to below 500 IU/ml. Our results suggested that NAT appears to be a highly sensitive and specific method for HCV RNA detection in plasma-derived products.

HCV는 HIV등과 함께 수혈이나 혈장된 획물질을 통하여 감염되는 주요 바이러스이다. 주로 혈액이나 혈장에서 HCV에 대한 항체를 검출함으로서 HCV의 감염을 방지하고 있다. 그러나 바이러스에 감염되었으나 항체가 생성되기 이전이나 항체의 양이 적은 경우에는 HCV의 검출이 어렵다. 따라서 핵산중폭시험(nucleic acid amplification tests, NAT)을 이용한 HCV 유전자를 검출하려는 시도들이 진행되고 있다. 이 연구의 목적은 혈장분획물질에서 HCV RNA를 검출할 수 있는 핵산증폭시험 방법을 개발하는 것이다. 5종류의 PCR primer를선별하여 실험에 이용하였다. 혈장분획물질의 HCV RNA 추출에는 컬럼 방법을 이용하는 것이 유용한 것으로나타났다. 핵산중폭시험의 결합 온도는 $48^{\circ}C$가 가장 적절한 것으로 나타났다. 또한 2차 PCR의 경우, 1차 PCR 산물 $1{\mu}l$와 30 pmol의 primer즐 사용하였을 때 높은 민감도와 특이성을 보이는 것을 알 수 있었다. 혈장 분획물질에 HCV를 주입하여 혈장중폭시험을 수행한 결과, 100 IU/ml까지 검출 할 수 있었다. 한편 근육주사용항체(IMIG)의 경우 핵산중폭시험을 통한 검출한계는 100IU/ml로 COBAS amplicor HCV2.0의 500 IU/ml 이상의 검출한계보다 민감도가 더 높은 것으로 나타났다. 이러한 결과들로 보아 본 실험에 이용된 핵산증폭시험이 혈장분획물질에서 HCV RNA를 검출하는데 유용한 방법으로 사용될 수 있을 것으로 생각된다.

Keywords

References

  1. Bukh, J., R.H. Purcell, and R.H. Miller. 1992. Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay. Proc. Natl. Acad. Sci. USA 89, 187-191
  2. Chandra, S., J.E. Cavanaugh, C.M. Lin, C. Pierre-Jerome, N. Yerram, R. Week, E. lanigan, and F. Feldamn. 1999. Virus reduction in the preparation of intravenous immune globullin: in vitro experiments. Transfusion 39, 249-257 https://doi.org/10.1046/j.1537-2995.1999.39399219280.x
  3. Christopher, J., N.K. Healiy, J.D. Sabharwal, D. Fiona, Y. Pen-Lee, A.F. Kenneth, W.G. Roger, P.S. Chapman, and H. Chapel. 1996. Outbreak of acute hepatitis C following the use of anti-hepatitis C virus-screened intravenous immunoglobulin therapy. Gastroenterology 110, 1120-1126 https://doi.org/10.1053/gast.1996.v110.pm8613001
  4. Choo, Q.L., G. Kuo, A.J. Weiner, L.R. Overby, D.W. Bradley, and M. Houghton. 1989. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244, 359-362 https://doi.org/10.1126/science.2523562
  5. Echevarria, J.M., P. Len, C.J. Domingo, J.A. Lpez, C. Elola, M. Madurga, F. Salmern, P.L. Yap, J. Daub, and P. Simmonds. 1996. Laboratory diagnosis and molecular epidemiology of an outbreak of hepatitis C virus infection among recipients of human intravenous immunoglobulin in Spain. Transfusion 36, 725-730 https://doi.org/10.1046/j.1537-2995.1996.36896374377.x
  6. Fanson, B.G., P. Osmack, and A.M. Bisceglie. 2000. A comparison between the phenol-chloroform method of RNA extraction and the QIAamp viral RNA kit in the extraction of hepatitis C and GB virus-C/hepatitis G viral RNA from serum. J. Virol. Meth. 89, 23-27 https://doi.org/10.1016/S0166-0934(00)00192-0
  7. Heermann, K.H., H. Seitz, and R. Thomssen. 1996. Capture and RT-PCR of hepatitis C virus RNA with safety primers. J. Virol. Meth. 59, 33-43 https://doi.org/10.1016/0166-0934(96)02003-4
  8. Houghton, M., K. Richman, J. Han, K. Berger, C. Lee, C. Dong, L. Overby, A. Weiner, D. Bradley, G. Kuo, and Q.L. Choo. 1991. Hepatis C virus (HCV): A relative of the pestiviruses and flaviviruses, p. 328-333. In F.B. Hollinger, S.N. Lemon, and H. Margolis (eds.), Viral Hepatitis and Liver Disease. The Williams & Wilkins Co., Baltimore, Mayland, USA
  9. Hsiang, J.L., S. Naiyi, M. Masashi, and F.B. Hollinger. 1992. Polymerase chain reaction assay for Hepatitis C virus RNA using a single tube for reverse ranscription and serial rounds of amplification with nested primer pairs. J. Med. Virol. 38, 220-225 https://doi.org/10.1002/jmv.1890380312
  10. James, L.G. and D. Elizabeth. 2000. Blood screening by nucleic acid amplification technology : Current issues, future challenges. Mol. Diagn. 5, 11-22 https://doi.org/10.2165/00066982-200005010-00007
  11. Lee, D.S., Y.C. Sung, and Y.S. Whang. 1996. Distribution of HCV genotypes among blood donors, patients with chronic liver disease, hepatocellular carcinoma, and patients on maintenance hemodialysis in Korea. J. Med. Virol. 49, 55-60 https://doi.org/10.1002/(SICI)1096-9071(199605)49:1<55::AID-JMV9>3.0.CO;2-J
  12. Petrik, J., G.J. Pearson, and J.P. Allain. 1997. High throughput PCR detection of HCV based on semiautomated multisample RNA capture. J. Virol. Meth. 64, 147-159 https://doi.org/10.1016/S0166-0934(96)02153-2
  13. Sayah, N., M. Freddie, and B. Robin. 1994. Simultaneous amplification and detection of specific Hepatitis B virus and Hepatitis C virus genomic sequences in serum samples. J. Med. Virol. 42, 212-216 https://doi.org/10.1002/jmv.1890420221
  14. Waleed, A.A., J.J. Leif, and R. Peter. 2000. Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J. Clin. Microbiol. 38, 345-350
  15. Widell, A., Y.Y. Zhang, B. Anderson-Gre, and L. Hammarstrm. 1997. At least three hepatitis C virus strains implicated in Swedish and Danish patients with intravenous immunoglobulin-associated hepatitis C. Transfusion 37, 313-320 https://doi.org/10.1046/j.1537-2995.1997.37397240215.x
  16. Wilson, I.G. 1997. Inhibition and facilitation of nucleic acid amplification. Appl. Environ. Microbiol. 63, 3741-3751