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

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.

Preparation of His-Tagged Armored RNA particles as positive controls for Real-Time RT-PCR Detection of Zika virus

Armored RNA (AR) is a good candidate for creating nuclease-resistant RNA positive controls in the nucleic acid - based assay for RNA viruses. To simplify the production and purifcation of armored RNA, a single plasmid double – expressing His6-tag system was designed. His-tag armored RNA particles were purifed using his-tag affnity. A genomic fragment of the zika virus consisting of the encoding sequences of flavi-M, flavi-E-C protein targeted for zika virus was selected to prepare a positive control. In this study, we have successfully produced His-taged MS2- phage like particles carrying specifc genomic regions (M and E genes) to monitor the procedures of real-time Reverse transcription-PCR for Zika virus detection in one plasmid double expression. AR-ZIKA is completely resistant to DNase and RNase, stable in normal human EDTA plasma at room temperature for at least 60 and 15 days at 40C and room temperature respectively.