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hepatology 2010_part5

Gan - Một cuốn sách giáo khoa lâm sàng với một đặc trưng của gần như 100%, độc lập của các kiểu gen HCV (Lee 2000, Konnick 2002). Đối với cao hơn nồng độ HCV RNA trước khi pha loãng của mẫu ban đầu là cần thiết. | Hepatology - A clinical textbook with a specificity of almost 100 independent of the HCV genotype Lee 2000 Kon-nick 2002 . For higher HCV RNA concentrations pre-dilution of the original sample is required. Branched DNA Hybridization Assay Versant HCV RNA Assay 3.0 Siemens Branched DNAHybridization Assay is based on signal amplification technology. After reverse transcription of the HCV RNA the resulting single-stranded complementary DNA strands bind to immobilised capture oligonucleotides with a specific sequence from conserved regions of the HCV genome. In a second step multiple oligonucleotides bind to the free ends of the bound DNA strands and are subsequently hybridised by multiple copies of an alkaline phosphatase-labeled DNA probe. Detection is achieved by incubating the alkaline phosphatase-bound complex with a chemiluminescent substrate Sarrazin 2002 . The Versant HCV RNA assay is at present the only FDA- and CE-approved HCV RNA quantification system based on a branched DNA technique. The lower detection limit of the current version 3.0 is 615 IU ml and linear quantification is ensured between 615-8 000 000 lU ml independent of the HCV genotype Morishima 2004 Ross 2002 . The bDNA assay only requires 50pl serum for HCV RNA quantification and is currently the assay with the lowest sample input. Real-time PCR-based HCV RNA detection assays Real-time PCR technology provides optimal features for both HCV RNA detection and quantification because of its very low detection limit and broad dynamic range of linear amplification Sarrazin 2006 Figure 1 . Distinctive for real-time PCR technology is the ability of simultaneous amplification and detection of the target nucleic acid allowing direct monitoring of the PCR process Higuchi 1992 . RNA templates are first reverse-transcribed to generate complementary cDNA strands followed by a DNA polymerase-mediated cDNA amplification. 108 107 106 105 104 103 102 101 100 IU ml 50 500 10 10 10 5-10 615 TMA bDNA qual. quant .