Production of Encapsidated RNA Particles as a Working Standard in Detecting Foodborne Viruses in Oysters

Abstract

Monitoring foodborne viruses via nucleic acid amplification tests rely on stable RNA standards to obtain reliable testing. This study aimed to produce RNA-based standard reagents for hepatitis virus (HAV) or norovirus detections which relies on viral-like particle (VLP) technology. Using a plasmid packaging system, plasmids containing DNA encoding Qβcoat protein (CP) monomer and the VP1 gene of viruses were co-transformed into E. coli host cells. In cell lysates, expressed CP was characterized by western blot and the whole icosahedral formation of VLPs was proved by electron microscope analysis. Encapsidated RNAs were measured and assessed as a standard by a two-step reverse transcription recombinase polymerase amplification (RT-RPA). Our results showed that CP has a distinguished protein band with a molecular weight of 14.5 kDa but a few variabilities of particle size were visualized. When adjusting the pH of the lysate to lower than 6, a more intense protein band and substantial particles with homogenous particle size were observed. These VLPs were found to enclose HAV and norovirus RNA contents to 1.2×107 copies/ng and 1.9×107 copies/ng, respectively. When analyzed by RT-RPA, linear regression analysis confirmed the alternative application of RNAs enclosed in VLPs to naked RNA synthesized from in vitro transcription. Using the E. coli expression system to produce Qβ VLPs allows cost-effective production and, therefore, can be implemented in laboratories with basic equipment. These encapsidated RNAs may become an ideal “standard” for detecting foodborne viruses via a molecular test in food and clinical samples. Keywords: Molecular testing, Nanoparticles, Nucleic amplification, RNA standards, Viral-like particles