解放军医学杂志2017，Vol.42Issue(6)：526-531,6.DOI:10.11855/j.issn.0577-7402.2017.06.09
五种出血热病毒重组酶聚合酶等温扩增方法的建立
Establishment of recombinase polymerase amplification assay for five hemorrhagic fever-related viruses
摘要
Abstract
Objective To establish a one-step recombinase polymerase amplification (RPA) method for pathogen screening and rapid detection in the field targeting for five hemorrhagic fever related viruses (Zaire ebola virus, Sudan ebola virus, Marburg virus, Lassa virus and Yellow fever virus). Methods The specific nucleic acid (NA) fragments of each virus were selected as target genes by genome sequence analysis, and the primers and probes for RPA assays were designed according to the sequence. A series of diluted template genes were used for RPA detection to determine the sensitivity. The hemorrhagic fever-related viral nucleic acids were used for RPA detection to determine the specificity. The amplification experiments were carried out at different temperature ranging from 37℃ to 42℃ to validate the reaction temperature range. Results The RPA reaction systems of the five hemorrhagic fever viruses could effectively amplify the target genes, the sensitivities were between 1.5×102 and 1.5×103 copies. No cross reactions existed with the other hemorrhagic fever-related viral genes. Meanwhile, RPA assay could effectively amplify the target genes at 37-42℃. Conclusion The isothermal RPA assays of five hemorrhagic fever viruses are established, which may amply target genes fast and react at a wide temperature range, and be potentially useful for in field pathogens detection.关键词
重组酶聚合酶检测/等温扩增/出血热病毒Key words
recombinase polymerase assay/isothermal amplification/hemorrhagic fever virus分类
医药卫生引用本文复制引用
曹雪锋,康晓平,李裕昌,张森,户义,李靖,吴晓燕,杨银辉..五种出血热病毒重组酶聚合酶等温扩增方法的建立[J].解放军医学杂志,2017,42(6):526-531,6.基金项目
国家自然科学基金(81501789) （81501789）
军队重大专项课题(AWS15J006) （AWS15J006）
国家重点实验室课题(SKLPBS1415)This work was supported by the National Natural Science Foundation of China (81501789), the Military Major Foundation (AWSJ006) and the State Key Laboratory Program (SKLPBS1415) （SKLPBS1415）
