Development of Real-Time PCR Assays for the Quantitative Detection of CD81 Receptor Gene of Hepatitis C Virus in Tupaia Belangeri

Abstract

—A real-time PCR assay based on the TaqMan chemistry was developed for reliable and quantitative detection of CD81 in Tupaia belangeri. The assay is performed with the ABI 7300 system using TaqMan probe and primers amplifying 118bp CD81 fragment of Tupaia belangeri. The standard curve for quantitation of target gene showed linearity over an at least 5-log DNA concentration range, represents10 3 to10 7 copies per reaction, with a correlation coefficient of 0.9996 (the slopes value-3.39 VS-3.32). Moreover, this protocol enabled detection of as little as 10 copies of CD81 cDNA in Tupaia belangeri liver tissues. The overall % coefficient of variation (%CV) for this assay was lower 5% with statistical significance due to in intra-assay 1.08% and inter-assay 0.16%, which indicated its high reproducibility. The new assay greatly improves current detection methods for CD81 evaluation, and this is the first report on the standardization and evaluation of a Tupaia belangeri CD81 RNA quantitation assay from China. As TaqMan real-time PCR enable the rapid and easy processing of a large number of samples, and can be used as a tool for monitoring progression of CD81 expression.