Construction and application of MS2 bacteriophage virus-like particles for SARS-CoV-2 detection using a single-plasmid system.

Research on the compatibility of MS2 bacteriophage virus-like particles (VLPs), which are used for the detection of a range of RNA viruses, with commercially available detection kits is limited. Here, we aimed to construct a positive control for RNA virus nucleic acid detection using MS2 bacteriophage VLPs and evaluate its compatibility with commercial nucleic acid detection kits to support the standardization of molecular diagnostic quality control systems. We generated recombinant plasmids expressing MS2 bacteriophage maturation enzyme, capsid proteins, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target genes (ORF1ab, N, and E) using a single-plasmid dual-expression system (pACYCDuet-1). The plasmids were expressed in Escherichia coli BL21, and then MS2 VLPs were purified. The VLPs were characterized, and their performance as a positive control was validated for homogeneity and stability; their detection compatibility with five commercial SARS-CoV-2 nucleic acid detection kits was also examined. We successfully generated MS2 VLPs carrying SARS-CoV-2 target genes, with a uniform particle size of 23-28 nm and target gene copy number of 1.99 × 1010 copies/μL. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the expression of MS2 proteins, whereas functional validation revealed excellent nuclease resistance, batch-batch homogeneity, and stability at 4 °C for ≥20 days. Importantly, the positive control exhibited consistent detection performance across all five commercial kits, confirming its practicality for routine laboratory workflows. The established positive control is readily applicable for standardizing quality control in molecular diagnostics.