Capture and RT-PCR of hepatitis C virus RNA with safety primers

Capture and RT-PCR of hepatitis C virus RNA with safety primers The principle and practice of the polymerase chain reaction (PCR) has had a major impact on medical research. This is a powerful method but it does have its limitations, especially for clinical diagnostic work. We describe some improvements of hepatitis C virus (HCV) amplification such as simplification of specimen preparation, elimination of false negative reactions influenced by point mutations, and fluorimetric detection. The aim of the method is to make the procedure as easy and as inexpensive as possible for routine laboratories and for blood screening. After rapid chemical denaturation of the clinical specimen with guanidine thiocyanate and simultaneous hybridization of biotinylated primers to template HCV RNA, the product was fixed to streptavidin-coated magnetic beads and potential inhibitors were removed in easy washing steps. To eliminate the influence of point mutations within the primer binding sites, primer sets with different lengths at their 3′-end were developed for capture, reverse transcription, and amplification of genomic fragments by PCR. Positive results were identified by fluorescence staining. The low cost of the method allows the quantitation of templates by testing of dilution series as is common in microbiological laboratories. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Virological Methods Elsevier

Capture and RT-PCR of hepatitis C virus RNA with safety primers

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Publisher
Elsevier
Copyright
Copyright © 1996 Elsevier Ltd
ISSN
0166-0934
eISSN
1879-0984
DOI
10.1016/0166-0934(96)02003-4
Publisher site
See Article on Publisher Site

Abstract

The principle and practice of the polymerase chain reaction (PCR) has had a major impact on medical research. This is a powerful method but it does have its limitations, especially for clinical diagnostic work. We describe some improvements of hepatitis C virus (HCV) amplification such as simplification of specimen preparation, elimination of false negative reactions influenced by point mutations, and fluorimetric detection. The aim of the method is to make the procedure as easy and as inexpensive as possible for routine laboratories and for blood screening. After rapid chemical denaturation of the clinical specimen with guanidine thiocyanate and simultaneous hybridization of biotinylated primers to template HCV RNA, the product was fixed to streptavidin-coated magnetic beads and potential inhibitors were removed in easy washing steps. To eliminate the influence of point mutations within the primer binding sites, primer sets with different lengths at their 3′-end were developed for capture, reverse transcription, and amplification of genomic fragments by PCR. Positive results were identified by fluorescence staining. The low cost of the method allows the quantitation of templates by testing of dilution series as is common in microbiological laboratories.

Journal

Journal of Virological MethodsElsevier

Published: May 1, 1996

References

  • Enhanced sensitivity of a second generation ELISA for antibody to hepatitis C virus
    Bresters, D.; Cuypers, H.T.; Reesink, H.W.; Schaasberg, W.P.; van-der-Poel, C.L.; Mauser-Bunschoten, E.P.; Houghton, M.; Choo, Q.L.; Kuo, G.; Lesniewski, R.
  • Impact of specimen handling and storage on detection of hepatitis C virus RNA
    Busch, M.P.; Wilber, J.C.; Johnson, P.; Tobler, E.; Evans, C.S.
  • Quantitation of hepatitis C virus RNA in serum of asymptomatic blood donors and patients with type C chronic liver disease
    Hagiwara, H.; Hayashi, N.; Mita, E.; Naito, M.; Kasahara, A.; Fusamoto, H.; Kamada, T.
  • The polymerase chain reaction in transfusion medicine
    Jackson, J.B.
  • Patterns of serological markers in transfusion-transmitted hepatitis C virus infection using second-generation HCV assays
    Lelie, P.N.; Cuypers, H.T.; Reesink, H.W.; van-der-Poel, C.L.; Winkel, L.; Bakker, E.; van-Exel-Oehlers, P.J.; Vallari, D.; Allain, J.P.; Minims, L.
  • Risk factors in hepatitis C virus-infected blood donors
    van der Poel, C.; Cuypers, H.; Reesink, H.; Choo, Q.L.; Kuo, G.; Han, J.; Quan, S.; Polito, A.; Verstraten, J.; van-de-Wouw, J.
  • Lack of mother-to-infant transmission of hepatitis C virus in human immunodeficiency virus-seronegative women: a prospective study with hepatitis C virus RNA testing
    Roudot-Thoraval, F.; Pawlotsky, J.M.; Thiers, V.; Deforges, L.; Girollet, P.P.; Guillot, F.; Huraux, C.; Aumont, P.; Brechet, C.; Dhumeaux, D.

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