Microchip and capillary electrophoresis for quantitative analysis of hepatitis C virus based on RT-competitive PCR.
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 mul(-1) into a series of RNA target standards ranging from 400 to 10(6) copies mul(-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.
Young
Kung-Chia
KC
Department of Medical Technology, National Cheng Kung University Hospital, Tainan, Taiwan, ROC.
Lien
Hsiang-Mei
HM
Lin
Chun-Che
CC
Chang
Ting-Tsung
TT
Lee
Gwo-Bin
GB
Chen
Shu-Hui
SH