基于MS2噬菌体的西尼罗病毒装甲RNA质控品的制备

doi:10.16431/j.cnki.1671-7236.2025.06.028

摘要/Abstract

摘要： 【目的】目前国内外西尼罗病毒(West Nile virus,WNV)的核酸检测中缺乏安全、稳定的RNA标准参考样品，对检测结果的精确度和可信度造成一定的影响。本研究旨在研制一种安全稳定的WNV核酸检测质控品，为西尼罗热监测提供技术支持。【方法】将WNV核酸检测靶基因、MS2噬菌体外壳蛋白CP及成熟酶蛋白A基因序列插入到表达载体得到重组质粒pET-CPA-WN，经转化、表达、纯化后，制备WNV装甲RNA质控品，并对其进行实时荧光定量PCR和数字PCR定量、均匀性及稳定性分析，评估其作为标准物质的可能性。【结果】PCR检测、双酶切和基因测序结果均显示已成功构建重组质粒pET-CPA-WN，表达纯化后得到了大小均一、直径为23～28 nm的病毒样颗粒。核酸酶消化后实时荧光定量PCR检测结果显示，颗粒溶液中几乎无核酸残余且形成了包封靶基因的装甲RNA；其定植结果为8.80×10⁹拷贝/mL。稳定性试验结果表明，该装甲RNA可在37 ℃稳定保持20 d，随机抽取10个样本进行均匀性检验证明其均匀性良好。【结论】本研究基于MS2噬菌体制备的装甲RNA拷贝数高，均匀性和稳定性良好，可为WNV分子检测提供安全、稳定的参考样品。

关键词: 西尼罗病毒(WNV); MS2噬菌体; 装甲RNA

Abstract: 【Objective】 The absence of secure and stable RNA reference materials for the nucleic acid assays of West Nile virus (WNV) at home and abroad was compromising the precision and credibility of the testing outcomes.This study was conducted to develop a safe and stable WNV nucleic acid testing quality control product to provide technical support for the monitoring of West Nile fever.【Method】 The WNV nucleic acid detection target gene,the MS2 bacteriophage coat protein CP,and the maturation enzyme protein A gene sequences were inserted into an expression vector to obtain the recombinant plasmid pET-CPA-WN.After transformation,expression and purification,a WNV armored RNA quality control material was prepared and subjected to Real-time quantitative PCR and digital PCR quantification,homogeneity and stability to assess its potential as a reference material.【Result】 PCR detection,double enzyme digestion and gene sequencing results all confirmed the successful construction of the recombinant plasmid pET-CPA-WN.After expression and purification,uniform virus-like particles with a diameter of 23-28 nm were obtained.Following nuclease digestion and Real-time quantitative PCR detection,the particle solution showed almost no residual nucleic acid and encapsulated the target gene in the form of armored RNA.The determined value was 8.80×10⁹ copies/mL.Stability experiments demonstrated that this armored RNA could remain stable for up to 20 days at 37 ℃.Random sampling of 10 samples for homogeneity testing confirmed good uniformity.【Conclusion】 The armored RNA prepared based on MS2 bacteriophage had high copy number,good uniformity and stability,which could provide a safe and stable reference sample for WNV molecular detection.

Key words: West Nile virus (WNV); MS2 bacteriophage; armored RNA

中图分类号:

S852.65

刘丹, 高建帅, 张博源, 李慧彤, 蒋卉, 范学政, 张广智, 丁家波, 熊涛, 沈青春. 基于MS2噬菌体的西尼罗病毒装甲RNA质控品的制备[J]. 中国畜牧兽医, 2025, 52(6): 2762-2771.

LIU Dan, GAO Jianshuai, ZHANG Boyuan, LI Huitong, JIANG Hui, FAN Xuezheng, ZHANG Guangzhi, DING Jiabo, XIONG Tao, SHEN Qingchun. Preparation of West Nile Virus Armored RNA Quality Control Product Based on MS2 Bacteriophage[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2762-2771.

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