Preparation of nuclease-resistant RNA standard for hepatitis C virus quantification.

Abstract

Positive control (or standard) is an indispensable ingredient in molecular biology assays widely used for the quantification of nucleic acid. The commonly used standards are plasmid DNA, cDNA, or naked RNA, which are unstable and easily degraded by nucleases in the surrounding environment; this might affect the accuracy of quantitative results. In this study, the authors designed and created a positive control for the hepatitis C virus (HCV) quantification based on armored RNA technology. The 5’UTR non-encoding sequence of HCV was cloned into the BH20 plasmid. Armored RNA HCV (AR-HCV) was induced for expression in the E. coli BL21 (DE3) by the addition of an IPTG inducer. AR-HCV was collected by sucrose density gradient ultracentrifugation followed by gel filtration chromatography using Superdex 75 column. Created AR-HCV was determined the concentration and examined the formation of pseudo viral particles by transmission electron microscopy (TEM). Stability assessment of AR-HCV to DNase and RNase treatment simultaneously has demonstrated its ability to resist these nucleases. Moreover, AR-HCV is stable over time and storage conditions. Strikingly, AR-HCV can be directly added to the specimen, allowing better and more accurate control of the whole quantitative procedure of HCV.