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KMID : 0545120050150030595
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Journal of Microbiology and Biotechnology
2005 Volume.15 No. 3 p.595 ~ p.602
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Validation of One-Step Real-Time RT-PCR Assay in Combination with Automated RNA Extraction for Rapid Detection and Quantitation of Hepatitis C Virus RNA for Routine Testing in Clinical Specimens
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Kim BG
Jeong HS/Baek SY/Shin JH/Kim JO/Min KI/Ryu SR/Min BS/Kim DK/Jeong YS/Park SN
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Abstract
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A one-step real-time quantitative RT-PCR assay in combination with automated RNA extraction was evaluated for routine testing of HCV RNA in the laboratory. Specific primers and probes were developed to detect 302 bp on 5’- UTR of HCV RNA. The assay was able to quantitate a dynamic linear range of 107-101 HCV RNA copies/reaction (R2=0.997). The synthetic HCV RNA standard of 1.84±0.1 (mean±SD) copies developed in this study corresponded to 1 international unit (IU) of WHO International Standard for HCV RNA (96/790 I). The detection limit of the assay was 3 RNA copies/reaction (81 IU/ml) in plasma samples. The assay was comparable to the Amplicor HCV Monitor (Monitor) assay with correlation coefficient r=0.985, but was more sensitive than the Monitor assay. The assay could be completed within 3 h from RNA extraction to detection and data analysis for up to 32 samples. It allowed rapid RNA extraction, detection, and quantitation of HCV RNA in plasma samples. The method provided sufficient sensitivity and reproducibility and proved to be fast and labor-saving, so that it was suitable for high throughput HCV RNA test.
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