现代畜牧兽医Issue(5)：1-7,7.DOI:10.20154/j.cnki.issn1672-9692.2025.05.001
HEV/HAV两联装甲RNA质控样品的研制及应用性能评价
Development and evaluation of performance characteristics for HEV/HAV dual-armored RNA quality control samples
摘要
Abstract
The experiment aims to develop a safe and highly stable dual armor RNA positive quality control product for hepatitis E(HEV)and hepatitis A(HAV).Using armored RNA technology,a design was made to tandem the target gene sequences of HEV and HAV standard detection to synthesize the MS2-HE-AV gene fragment,which was cloned into the pET-28b vector to construct the recombinant plasmid PET-28B-MS2-HE-AV608.Prokaryotic expression induced by isopropyl thiogalactoside(IPTG)was carried out in transformed Escherichia coli Rosetta(DE3),and HE-AV bivirus-like particles(VLPs)were purified and prepared.The stability and recovery rate were verified to evaluate its practical application performance.The results showed that the recombinant vector containing the MS2 shell protein and the exogenous target gene was successfully constructed in the experiment.The target protein was expressed efficiently,and VLPs with a diameter of approximately 27 nm could be seen under the electron microscope.This VLPs can tolerate the degradation effect of universal nuclease.The calibration results of HEV and HAV detection targets in VLPs were 1.88×109 and 1.17×109 copies/μL,respectively.This VLPs can be stored for at least 21 days at 4℃,which complies with the basic regulations on the storage period of VLPs in the national standard.The actual sample test results showed that when the VLPs concentration of the artificially contaminated samples was diluted to 1 000 and 10 000 times,the recovery rates of armored RNA in the four sample matrices were all greater than 1%.Studies have shown that HE-AV dual VLPs can effectively resist the degradation effect of universal nucleases,have good thermal stability and strong applicability.They can be used as positive quality control products for nucleic acid detection of HEV and HAV to monitor the entire process of viral nucleic acid detection.关键词
戊型肝炎病毒/甲型肝炎病毒/阳性质控品/装甲RNAKey words
Hepatitis E virus(HEV)/Hepatitis A virus(HAV)/Quality control product/Amored-RNA分类
农业科技引用本文复制引用
杨书仁,朱志梅,许艺凡,李慧,王术德,高慎阳..HEV/HAV两联装甲RNA质控样品的研制及应用性能评价[J].现代畜牧兽医,2025,(5):1-7,7.基金项目
辽宁省教育厅面上项目(JYTMS20231735) （JYTMS20231735）
辽宁省科学技术计划农业重大专项(2023JH110200006) （2023JH110200006）
