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Folia Medica

The Journal of Medical University-Plovdiv

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SCImago Journal Rank (SJR) 2015: 0.181

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Simultaneous Quantification and Genotyping of Hepatitis C Virus RNA by a Two-Step Real-Time PCR Assay on the Lightcycler Instrument

Manabu Abe
  • Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart Katharinenhospital, Stuttgart, Germany
/ Corinne Klett
  • Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart Katharinenhospital, Stuttgart, Germany
/ Eberhard Wieland
  • Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart Katharinenhospital, Stuttgart, Germany
/ Sascha Gille
  • TIB MOLBIOL GmbH, Berlin, Germany
/ Olfert Landt
  • TIB MOLBIOL GmbH, Berlin, Germany
Published Online: 2010-10-22 | DOI: https://doi.org/10.2478/v10153-010-0003-4

Simultaneous Quantification and Genotyping of Hepatitis C Virus RNA by a Two-Step Real-Time PCR Assay on the Lightcycler Instrument

Background: Clinically, both viral load and genotypes have been found to be major predictors of antiviral therapy outcome regarding chronic hepatitis C and they are, under normal circumstances, performed as separate assays.

Design and methods: In order to improve the diagnostic strategy and subsequently reduce the reagent costs we have developed and established the simultaneous quantification and genotyping of hepatitis C virus RNA by a two-step real-time PCR on the LightCycler Instrument (Roche Diagnostics).

Results: The quantification assay was calibrated against WHO Standard 96/790. The detection limit was 30 IU/ml, the dynamic range up to 500,000,000 IU/ml. Intra- and inter-assay imprecisions were 1.2% and 1.9% (n = 10), respectively. The HCV RNA values obtained by real-time PCR assay were highly correlated with those obtained by the Cobas Amplicor HCV monitor test (r = 0.992; p < 0.001).

Conclusions: The genotyping was performed by means of the melting temperature analysis. The concordance between our new genotyping method and the Trugene HCV 5'NC Kit was at the level of genotypes 100%. This rapid (3 h) and convenient assay is suitable for HCV genotyping, HCV detection and disease monitoring.

Одновременное количественное измерение и генотипизирование рибонуклеиновой кислоты виру са гепатита с посредством приме нения двушагового PCR теста в реальное время на аппарате Light Cycler

Введение: Вирусное обременение и вирусный генотип представляют собой самые важные предикторы для исхода антивирусной терапии при хроническом гепатите С. В нормальных условиях их обнаруживаютс помощью двух отдельных тестов.

Материал и методы: В целях улучшения диагнос тической стратегии и уменьшения стоимости лабораторных реагентов авторы разработали ме тод одновременного количественного измерения и генотипизирования РНК гепатитного вируса С посредством применения двушагового PCR теста в реальное время на аппарате Light Cycler (Roche Diagnostics).

Результаты: Для калибрирования количественноготеста применен стандарт ВОЗ 96/790. Верхняя граница теста 30 IU/ml, его динамический охват - до 500,000,000 IU/ml. Внутри- и межтестовые ошибки соответственно1.2% и 1.9% (п = 10). Стоимости РНК вируса гепатита С, полученные PCR тестом в реальное время сильно коррелируют со стоимостями, полученными тестом Cobas Am-plicor HCV Monitor (r = 0.992; р < 0.001). Гене тически типизация проведена с помощью анализа температуры плавления. Согласованность между новым авторским методом генного типизирования и методом Trugene HCV 5'NC - 100%.

Новый метод проводится в течение всего трех часов. Он оказывается очень подходящим для генного типизирования, обнаруживания и терапевтического мониторирование вируса гепатита С.

Keywords: HCV; real-time PCR; genotyping

  • Simmonds P. Genetic diversity and evolution of hepatitis C virus - 15 years on. J Gen Virol 2004;85:3173-88. [PubMed]

  • Simmonds P, Bukh J, Combet C, et al. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 2005;42(4):962-73. [PubMed] [Crossref]

  • Zein NN. Clinical significance of hepatitis C virus genotypes. Clin Microbiol Rev 2000;13:223-35. [Crossref] [PubMed]

  • Zeuzem S. Heterogeneous virologic response rates to interferon-based therapy in patients with chronic hepatitis C: Who responds less well? Ann Intern Med 2004;370-81. [PubMed]

  • Chung RT, Andersen J, Volberding P, et al. Alfa-2a plus ribavirin versus interferon alpha-2a plus ribavirin for chronic hepatitis C in HIV-coinfected persons. N Engl J Med 2004;351:451-9. [Crossref]

  • Germer JJ, Rys PN, Thorvilson JN, Persing DH. Determination of hepatitis C virus genotype by direct sequence analysis of products generated with the Amplicor HCV test. Clin Microbiol 1999;37(8):2625-30.

  • Germer JJ, Majewski DW, Rosser M, et al. Evaluation of the TRUGENE HCV 5'NC genotyping kit with the new GeneLibrarian module 3.1.2 for genotyping of hepatitis C virus from clinical specimen. Clin Microbiol 2003;41(10):4855-7. [Crossref]

  • Ross RS, Viazov SO, Holtzer CD, et al. Genotyping of hepatitis C virus isolates using CLIP Sequencing. Clin Microbiol 2000;38(10):3581-4.

  • White PA, Zhai X, Carter I, Zhao Y, Rawlinson WD. Simplified hepatitis C virus genotyping by heteroduplex mobility analysis. Clin Microbiol 2000;38:477-82.

  • Margraf RL, Erali M, Liew M, Wittwer CT. Genotyping hepatitis C virus by heteroduplex mobility analysis using temperature gradient capillary electrophoresis. Clin Microbiol 2004;42(10):4545-51. [Crossref]

  • Ilina EN, Malakhova MV, Generozov EV, Nikolaev EN, Govorun VM. Matrix-assisted laser desorption ionization-time of flight (Mass Spectrometry) for hepatitis C virus genotyping. Clin Microbiol 2005;43(6):2810-5.

  • Liew M, Erali M, Page S, Hillyard D, Wittwer C. Hepatitis C genotyping by denaturing high-performance liquid chromatography. Clin Microbiol 2004;42(1):158-63. [Crossref]

  • Bullock GC, Bruns DE, Haverstick DM. Hepatitis C genotype determination by melting curve analysis with a single set of fluorescence resonance energy transfer probes. Clin Chem 2002;48:2147-54. [PubMed]

  • Haverstick DM, Bullock GC, Bruns DE. Genotyping of hepatitis C virus by melting curve analysis: Analytical characteristics and performance. Clin Chem 2004;12:2405-7. [Crossref]

  • Ratge D, Scheiblhuber B, Nitsche M, Knabbe C. High-speed detection of blood-borne Hepatitis C virus RNA by single-tube real-time fluorescence reverse transcription-PCR with the LightCycler. Clin Chem 2000;46:1987-9. [PubMed]

  • Ratge D, Schreiblhuber B, Landt O, Berg J, Knabbe C. Two-round rapid-cycle RT-PCR in single closed capillaries increases the sensitivity of HCV RNA detection and avoids amplicon carry-over. Clin Virology 2002;24:161-72. [Crossref]

  • Hofgaertner WT, Kant JA, Weck KE. Hepatitis C virus quantisation: optimization of strategies for detecting low-level viremia. Clin Microbiol 2000;38:888-91.

  • Mukaide M, Tanaka Y, Kakuda H, et al. New combination test for hepatitis C virus genotype and viral load determination using Amplocor GT HCV Monitor test v2.0. World J Gastroenterol 2005;11(4):469-75.

  • Schröter M, Zöllner B, Schäfer P, Laufs R, Feucht HH. Quantitative detection of hepatitis C virus RNA by LightCycler PCR and comparison with two different PCR assays. Clin Microbiol 2001;39:765-8. [Crossref]

  • Leb V, Stöcher M, Valentine-Thon E, et al. Fully automated, internally controlled quantification of Hepatitis B virus DNA by real-time PCR by use of the MagNA Pure LC and LightCycler Instruments. Clin Microbiol 2004;42:585-90. [Crossref]

  • Abe M, Klett C, Wieland E. Gille S, Landt O. Two-step real-time PCR quantification of all subtypes of human immunodeficiency virus type 1 by an in-house method using locked nucleic acid-based probes. Folia Medica 2008;50(3):5-13.

  • Chen Z, Weck KE. Hepatitis C virus genotyping: interrogation of the 5' untranslated region cannot accurately distinguish genotypes 1a and 1b. Clin Microbiol 2002;40(9):3127-34

  • Nolte FS, Green AM, Fiebelkorn KR, et al. Clinical evaluation of two methods for genotyping hepatitis C virus based on analysis of the 5' noncoding region. Clin Microbiol 2003;41(4):1558-64.

About the article

Published Online: 2010-10-22

Published in Print: 2010-07-01

Citation Information: Folia Medica, ISSN (Online) 1314-2143, ISSN (Print) 0204-8043, DOI: https://doi.org/10.2478/v10153-010-0003-4. Export Citation

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