期刊
JOURNAL OF VIROLOGICAL METHODS
卷 102, 期 1-2, 页码 119-128出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/S0166-0934(02)00007-1
关键词
HCV; real-time RT-PCR; molecular beacon; TaqMan
资金
- NIAAA NIH HHS [AA13547] Funding Source: Medline
- NIDA NIH HHS [DA112815] Funding Source: Medline
- NIMH NIH HHS [MH49981] Funding Source: Medline
A newly developed real-time RT-polymerase chain reaction assay for quantitation of hepatitis C virus (HCV) RNA in human plasma and serum was applied. A pair of primers and a probe (molecular beacon) were designed that are specific for the recognition of a highly conservative 5'-non-coding region (5'-NCR) in HCV genome. HCV real-time RT-PCR assay had a sensitivity of 1000 RNA copies per reaction, with a dynamic range of detection between 101 and 101 RNA copies. The coefficient variation of threshold cycle (Ct) values in intra- and inter-runs were less than 1.37 and 4.66%, respectively. The real-time RT-PCR assay on the HCV sero-positive samples yielded reproducible data, with less than 2.09% of the inter-assay variation. In order to determine its potential for clinical diagnosis, real-time RT-PCR was used to examine the HCV RNA levels in plasma from sero-positive and negative subjects.. showing that the assay is highly sensitive and has specificity of 100%. It was demonstrated that the real-time RT-PCR was able to amplify HCV RNA in reference sera with seven genotypes (1A, 1B, 2B, 3A, 4, 5A and 6A) that include six major HCV genotypes circulated in the world. Since HCV is a major pathogen of post-transfusion and community-transmitted non-A, non-B hepatitis, this assay has a broad application for basic and clinical investigations. (C) 2002 Elsevier Science B.V. All rights reserved.
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