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

Abstract

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.