中国畜牧兽医2025，Vol.52Issue(6)：2762-2771,10.DOI:10.16431/j.cnki.1671-7236.2025.06.028
基于MS2噬菌体的西尼罗病毒装甲RNA质控品的制备
Preparation of West Nile Virus Armored RNA Quality Control Product Based on MS2 Bacteriophage
摘要
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.关键词
西尼罗病毒(WNV)/MS2噬菌体/装甲RNAKey words
West Nile virus(WNV)/MS2 bacteriophage/armored RNA分类
农业科技引用本文复制引用
刘丹,沈青春,高建帅,张博源,李慧彤,蒋卉,范学政,张广智,丁家波,熊涛..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):2762-2771,10.基金项目
重大外来动物疫病阻断与防控技术研发项目(2022YFD1800500) （2022YFD1800500）
