Microchip and capillary electrophoresis for quantitative analysis of hepatitis C virus based on RT-competitive PCR

Kung Chia Young, Hsiang Mei Lien, Chun Che Lin, Ting Tsung Chang, Gwo Bin Lee, Shu Hui Chen

Research output: Contribution to journalArticlepeer-review

17 Citations (Scopus)

Abstract

A method to quantitatively perform reverse transcription-competitive PCR (RT-cPCR) of hepatitis C virus followed by both microchip and capillary electrophoretic separation and detection was described. In this method, HCV wild-type (WT) RNA extracted from serum was coretrotranscribed and coamplified with a constant amount of recombinant internal standard (IS) RNA which had the same primer binding region as the target RNA and was constructed by removing a centrally located 25-bp segment from the target template. A linear calibration curve was constructed by adding IS RNA at a constant concentration of 8000 copies μl-1 into a series of RNA target standards ranging from 400 to 106 copies μl-1. The amplified IS and target DNA were detected by both capillary and microchip electrophoresis via laser-induced fluorescence (LIF) using Cy5-labelled primer as the fluorescence probe. The method was further demonstrated for the quantitation of clinical patients with low, medium, and high viral titer and the results were found to be comparable to those determined by the commercial bDNA assay.

Original languageEnglish
Pages (from-to)323-330
Number of pages8
JournalTalanta
Volume56
Issue number2
DOIs
Publication statusPublished - 2002 Feb 11

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

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