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

作　　者：曹雪锋[1,2] 康晓平[2] 李裕昌[2] 张森[2] 户义[2] 李靖[2] 吴晓燕[2] 杨银辉[1,2] 

机构地区：[1]安徽医科大学研究生学院,合肥230032 [2]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《解放军医学杂志》2017年第6期526-531,共6页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(81501789);军队重大专项课题(AWS15J006);国家重点实验室课题(SKLPBS1415)~~

摘　　要：目的建立五种出血热相关病毒,即扎伊尔型埃博拉病毒、苏丹型埃博拉病毒、马尔堡病毒、拉沙病毒和黄热病毒的一步法重组酶聚合酶等温扩增(RPA)技术,为病原体的确定提供现场快速检测方法。方法通过基因组序列分析,选择每种病毒的特异核酸片段作为检测靶基因,针对靶基因序列设计RPA检测的引物和探针;取系列稀释的模板基因进行RPA检测,确定其灵敏性;取出血热相关病毒核酸为模板进行RPA检测,确定其特异性;验证不同温度反应条件(37~42℃)对检测结果的影响。结果五种出血热病毒的RPA反应体系均可有效扩增靶基因,灵敏性为1.5×102~1.5×103copies/反应,与其他出血热相关病毒的核酸无交叉反应;RPA实验在37~42℃均可实现靶基因的有效扩增。结论建立了五种出血热病毒的RPA等温扩增体系,该体系反应快速,温度范围宽,适用于现场快速检测。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 1.5×10^2 and 1.5×10^3 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 

分 类 号：R373.3[医药卫生—病原生物学]