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Folia Medica

The Journal of Medical University-Plovdiv

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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 Abe1 / Corinne Klett1 / Eberhard Wieland1 / Sascha Gille1 / Olfert Landt1

Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart Katharinenhospital, Stuttgart, Germany1

TIB MOLBIOL GmbH, Berlin, Germany2

This content is open access.

Citation Information: Folia Medica. Volume 52, Issue 3, Pages 21–30, ISSN (Online) 1314-2143, ISSN (Print) 0204-8043, DOI: 10.2478/v10153-010-0003-4, October 2010

Publication History

Published Online:
2010-10-22

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

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