RPA-毛细管电泳检测人腺病毒55型的方法建立及初步应用
Establishment and preliminary application of RPA-capillary electrophoresis for detection of human adenovirus 55
袁若栋 1董阳超 2武福星 1段甜 3薛潘 3张剑 2袁明城 2薛智丰 3张海军 2张欠欠 3雷迎峰 2高小朋3
作者信息
- 1. 延安大学生命科学学院,陕西延安 716000;空军军医大学基础医学院病原生物学与微生物学教研室,陕西西安 710032
- 2. 空军军医大学基础医学院病原生物学与微生物学教研室,陕西西安 710032
- 3. 延安大学生命科学学院,陕西延安 716000
- 折叠
摘要
目的 建立重组酶聚合酶扩增(RPA)技术联合毛细管电泳快速检测人腺病毒55型(HAdV55)的方法及初步应用.方法 构建HAdV55-Hexon质粒作为扩增模板,针对HAdV55保守区Hexon基因序列设计RPA特异性引物,对引物筛选和优化,并建立RPA扩增及联合毛细管电泳检测体系,进行了特异性、灵敏度、可靠性评价;对18份临床样本进行RPA-毛细管电泳检测.结果 构建了含有HAdV55-Hexon全长基因的质粒,获得一对扩增效率最高的RPA引物;HAdV55的RPA-毛细管电泳检测体系最低检出限为2.9 × 103 copies/mL,整个过程30 min即可完成,冠状病毒(HCoV-229E、HCoV-OC43、HCoV-HKU1)、甲型乙型流感病毒、副流感病毒、呼吸道合胞病毒标准品提取物用该体系检测时均为阴性,且检测常见的人腺病毒类型(HAdV3、HAdV4、HAdV7、HAdV16、HAdV21)时也均为阴性,可认为对HAdV55有较强的特异性检测;建立的RPA-毛细管电泳检测能够有效地检测出临床患者咽拭子中的HAdV55.结论 RPA联合毛细管电泳检测HAdV55方法的建立为其临床诊断提供了良好的基础.
Abstract
Objective To establish a method for rapid detection of human adenovirus type 55(HAdV55)by recombinase polymerase amplification(RPA)combined with capillary electrophoresis and its preliminary application.Methods The HAdV55-Hexon plasmid was constructed as the amplification template,and RPA specific primers were designed for Hexon gene sequence of HAdV55 conserved region.The primers were screened and optimized,and the RPA amplification and combined capillary electrophoresis detection system was established to evaluate the specificity,sensitivity and reliability.Eighteen clinical samples were examined by RPA-capillary electrophoresis.Results A plasmid containing the full-length HAdV55-Hexon gene was constructed,and a pair of RPA primers with the highest amplification efficiency was obtained.The RPA-capillary electrophoresis detection system of HAdV55 had a minimum detection limit of 2.9 x 103 copies/mL,and the whole process could be completed in 30 min.The standard extracts of coronavirus(HCoV-229E,HCoV-OC43,and HCoV-HKU1),influenza A and B viruses,parainfluenza virus and respiratory syncytial virus were all negative when tested with this system.In addition,common human adenovirus types(HAdV3,HAdV4,HAdV7,HAdV16,and HAdV21)were also negative,which suggested that it had strong specificity for HAdV55.The established RPA-capillary electrophoresis assay could effectively detect HAdV55 in throat swabs of clinical patients.Conclusion The establishment of RPA combined with capillary electrophoresis for the detection of HAdV55 provides a good basis for its clinical diagnosis.
关键词
人腺病毒55型/重组酶聚合酶扩增/毛细管电泳Key words
human adenovirus type 55/recombinase polymerase amplification/capillary electrophoresis引用本文复制引用
基金项目
陕西省重点研发计划(2021ZDLSF01-05)
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据