Abstract:Abstract: Objective: Norovirus (NoV) represents the preeminent etiological agent of foodborne viral gastroenteritis worldwide, accounting for over 50% of all foodborne disease outbreaks and constituting a substantial public health burden across diverse populations. This non-enveloped, single-stranded RNA virus belongs to the Caliciviridae family and is categorized into at least seven genogroups (GI-GVII), with GII being the predominant genogroup responsible for the vast majority of human infections. Real-time fluorescent reverse transcription PCR (RT-PCR) has been established as the gold standard method for NoV molecular detection due to its superior sensitivity, specificity, and capacity for quantitative analysis. However, numerous methodological challenges persistently compromise assay reliability, including the presence of complex RT-PCR inhibitors in clinical and environmental matrices, suboptimal RNA recovery rates during nucleic acid extraction procedures, and spontaneous degradation of RNA templates prior to amplification. These technical variables frequently culminate in false-negative results, thereby undermining diagnostic accuracy and epidemiological surveillance efforts. Consequently, the implementation of robust quality control materials throughout the entire analytical workflow-from nucleic acid extraction through reverse transcription and final amplification-represents a critical safeguard for ensuring result validity. To address the notable deficiency in biosafety standard reference materials specifically designed for monitoring NoV contamination within laboratory environments, we engineered a novel Qβ bacteriophage-based armored RNA (AR) standard reference material. This innovative construct was designed to simultaneously encapsulate detection targets for GII genogroup NoV and the giant panda (Ailuropoda melanoleuca) RPS15a gene, serving as an internal control for comprehensive biosafety monitoring. The development and rigorous characterization of this dual-target armored RNA standard, designated AR-NS15, enable more thorough and dependable surveillance of potential biological safety hazards within laboratory settings, thereby substantially elevating overall laboratory biosafety management standards. Methods: The full-length cDNA fragment was artificially synthesized through de novo gene assembly, comprising the Qβ bacteriophage maturase-encoding gene, capsid protein-encoding gene, packaging signal, and the concatenated detection targets (NoV GII-specific region and RPS15a sequence). This synthetic cassette was subcloned into the pET-28a(+) expression vector using NdeI and XhoI restriction sites to construct the recombinant plasmid pET-QN-S15. The sequence-verified construct was transformed into Escherichia coli BL21(DE3) competent cells for high-level protein expression. Bacterial cultures were grown at 37°C in LB medium containing 50 μg/mL kanamycin until reaching an optical density of 0.6 at 600 nm. Protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 6 hours at 37 °C with vigorous shaking. The clarified lysate was subjected to ultracentrifugation at 85 000 r/min for 4 hours at 4 °C to pellet the VLPs. The concentrated VLPs were further purified by size-exclusion chromatography using a Sephacryl S-500 HR gel filtration column equilibrated with phosphate-buffered saline. Purified AR-NS15 preparations were characterized by transmission electron microscopy (TEM) after negative staining with 2% uranyl acetate. For value assignment and stability assessment, absolute quantification was performed using a validated real-time fluorescent reverse transcription PCR (RT-PCR) assay with a standard curve generated from serial dilutions of linearized plasmid DNA of known concentration. Homogeneity testing was conducted, analyzing 10 randomly selected units from the batch, while stability studies were performed under accelerated conditions (4 °C and -20 °C) over defined time intervals. Results: SDS-PAGE analysis of IPTG-induced E. coli lysates revealed a prominent protein band at approximately 14.2 kDa, corresponding to the expected molecular weight of the Qβ capsid protein monomer, confirming robust expression of the recombinant construct. Subsequent purification steps effectively eliminated host-derived contaminating proteins and residual nucleic acids, as evidenced by the absence of extraneous bands on Coomassie blue-stained gels and undetectable plasmid DNA in agarose gel electrophoresis. Transmission electron microscopy demonstrated that the purified AR-NS15 formed uniform, non-aggregated spherical virus-like particles with structural integrity, measuring approximately 25 nm in diameter and resembling native Qβ phage morphology. The quantitative characterization indicated that AR-NS15 contained 9.59 × 101? copies/μL of encapsidated RNA targets, providing an abundant and consistent source of reference material. Homogeneity evaluation confirmed excellent batch uniformity. Stability studies demonstrated that AR-NS15 remained stable when stored at 4 °C for up to 15 days without significant degradation, while storage at -20 °C preserved full viability for at least 60 days, with minimal loss of detectable RNA targets. These findings collectively validate the suitability of AR-NS15 as a long-term reference material under standard laboratory storage conditions. Conclusion: This study successfully developed a homogeneous, and stable armored RNA standard reference material designated AR-NS15, which simultaneously encapsulates detection targets for GII norovirus and the giant panda RPS15a gene. The Qβ VLP-based delivery system ensures RNase resistance and biological safety, making it an ideal surrogate for native viral particles in molecular diagnostics. With its precisely assigned copy number, exceptional batch-to-batch consistency, and demonstrated thermal stability, AR-NS15 provides comprehensive quality control for nucleic acid extraction, reverse transcription, and amplification processes. This dual-target reference material enables laboratories to monitor both analytical variables and extrinsic contamination risks, thereby establishing a robust quality assurance framework that significantly improves the accuracy and reliability of NoV molecular detection while enhancing overall laboratory biosafety protocols.
