Development of a versatile and stable internal control system for RT-qPCR assays

Abstract

RT-qPCR, an established method for the detection of RNA viruses, requires internal RNA controls for the correct interpretation of PCR results. Robust and versatile RT-PCR controls can be achieved for example by packaging RNA into a virus-derived protein shell. In this study a MS2-based internal control system was developed, that allows stable and universal packing of different RNAs into non-infectious, non-lytic MS2-based viral like particles (VLPs). Two competitive internal controls for a hantavirus assay and a Crimean-Congo Hemorrhagic Fever Virus (CCHFV) assay were cloned for the expression of VLPs. The expression of VLPs containing the RNA of interest could be induced with arabinose in Escherichia coli. The VLPs proved to be temperature resistant and could be frozen and thawed several times without degradation. Distinction of IC RNA from the target RNA was facilitated by a clear shift in the melting temperature or by specific hybridization signals. Furthermore, target and IC PCR amplification could be easily distinguished by their size in gel-electrophoretic analyses. Limits of detection were determined, demonstrating that the application of the IC did not reduce the sensitivity of the target RT-qPCR reactions. The system can be adapted to nearly any required sequence, resulting in a highly flexible method with broad range applications.

Keywords: Crimean-Congo Hemorrhagic Fever Virus; Field application; Hantavirus; MS2-based VLPs; RT-qPCR; Stable internal control.