渔业科学进展2026，Vol.47Issue(5)：274-284,11.DOI:10.3969/j.issn.2095-9869.20251107001
GⅡ型诺如病毒内标型装甲RNA质控品的研制与评价
Development and Evaluation of a GⅡ Norovirus Internal Standard Armored RNA Quality Control Product
摘要
Abstract
Norovirus(NoV)is a prevalent 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(G Ⅰ-GⅦ),with GⅡ being the predominant genogroup responsible for most human infections.Reverse transcription quantitative real-time PCR(RT-qPCR)has been established as the gold standard for NoV molecular detection owing to its superior sensitivity,specificity,and capacity for quantitative analyses.However,numerous methodological challenges persistently compromise assay reliability,including complex RT-qPCR 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 result 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 to final amplification,represents a critical safeguard to ensure 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 Qβbacteriophage-based armored RNA(AR)standard reference material.This innovative construct was designed to simultaneously encapsulate detection targets for the GⅡ genogroup of 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 a more thorough and dependable surveillance of potential biological safety hazards within laboratory settings,thereby substantially elevating the overall laboratory biosafety management standards. 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 Ndel and Xhol restriction sites to construct the recombinant plasmid pET-QN-S15.The sequence-verified construct was transformed into Escherichia coli BL21(DE3)cells for high-level protein expression.Bacterial cultures were grown at 37 ℃ in LB medium containing 50 μg/mL kanamycin up to an optical density of 0.6 at 600 nm.Protein expression was induced using 1 mmol/L isopropyl β-D-l-thiogalactopyranoside(1PTG)for 3 h at 37 ℃ with vigorous shaking.The clarified lysate was subjected to ultracentrifugation at 85,000 r/min for 4 h at 4 ℃ 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 using transmission electron microscopy(TEM)after negative staining with 2％uranyl acetate.For value assignment and stability assessment,absolute quantification was performed using a validated RT-PCR assay,with a standard curve generated from serial dilutions of linearized plasmid DNA at known concentrations.Homogeneity testing was conducted,analyzing 10 randomly selected units from the batch,while stability studies were performed under accelerated conditions(4 ℃ and-20 ℃)over defined time intervals. 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 recombinant construct expression.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 by agarose gel electrophoresis.TEM 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× 1010 copies/μL of encapsidated RNA targets,providing an abundant and consistent source of reference material.The homogeneity evaluation confirmed excellent batch uniformity.Stability studies demonstrated that AR-NS15 remained stable when stored at 4 ℃ for up to 15 days without significant degradation,whereas storage at-20 ℃ preserved full viability for at least 60 days,with minimal loss of detectable RNA targets.These findings collectively validated the suitability of AR-NS15 as a long-term reference material under standard laboratory storage conditions. This study successfully developed a homogeneous and stable armored RNA standard reference material,AR-NS15,that simultaneously encapsulates detection targets for GⅡ norovirus and the giant panda RPS15a gene.This Qβ VLP-based delivery system ensured 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.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.关键词
诺如病毒/内标型装甲RNA/标准样品/实时荧光定量PCRKey words
Norovirus/Internal standard armored RNA/Reference material/Reverse transcription quantitative real-time PCR(RT-qPCR)分类
轻工纺织引用本文复制引用
曲梦,李明玉,江艳华,朱文嘉,郭莹莹,李娜,王联珠,姚琳,谭志军..GⅡ型诺如病毒内标型装甲RNA质控品的研制与评价[J].渔业科学进展,2026,47(5):274-284,11.基金项目
国家自然科学基金(32172292)、中国水产科学研究院黄海水产研究所基本科研业务费(20603022025007)、中国水产科学研究院基本科研业务费(2023TD76)和国家现代农业(贝类)产业技术体系专项资金(CARS-49)共同资助. （32172292）
