扎伊尔型埃博拉病毒型特异性标签序列鉴定及其荧光定量PCR检测  

Identification of Zaire ebola virus specific signature sequence and its fluorescence quantitative PCR detection

作　　者：李泽[1] 王松建[2] 杨涛 李旭[4] 郭衍志 但芸婕[1] 徐宝艳 邓国宏[1] 

机构地区：[1]第三军医大学西南医院全军感染病研究所,感染病研究重庆市重点实验室 [2]重庆大学计算机学院 [3]解放军77128部队 [4]中国科学院计算技术研究所

出　　处：《第三军医大学学报》2016年第13期1471-1474,共4页Journal of Third Military Medical University

基　　金：全军后勤科研"十二五"计划重大项目(AWS11C001);西南医院军事医学预研专项资助课题(SWH2013JS03)~~

摘　　要：目的建立一种快速准确检测扎伊尔型埃博拉病毒（Zaire ebola virus,ZEBOV）的方法。方法以2014年西非新分离的3株ZEBOV全基因组参考序列及1976年从Mayinga分离的ZEBOV株（NC_002549）为目标,基于Insignia系统算法与GenBank中Nt库比对,求出ZEBOV特异性核酸标签序列。设计引物、TaqMan探针和优化反应条件,以10倍系列稀释的阳性质粒做标准曲线,做准确性、重复性实验。并以其余4个亚型的埃博拉病毒、马尔堡病毒、西尼罗河热病毒、东方马脑炎病毒、西方马脑炎病毒等8种病毒对照及临床48例患者复杂样本为对照做特异性检测。结果选择比对得到的特异性标签序列（KJ660348,11331-11514）设计探针引物,标准曲线相关系数〉0.99,灵敏度可达5×10^1拷贝。准确性重复性实验结果批内变异系数（CV）为0.88%～2.50%、批间CV为1.75%～3.31%。8种病毒及临床48例患者复杂样本对照均为阴性。结论 ZEBOV核酸标签（KJ660348,11331-11514）在非编码区,在此基础上建立的荧光定量PCR方法可特异性检测ZEBOV,且灵敏、重复性好。Objective To establish a rapid and accurate method to detect Zaire ebola virus （ZEBOV）. Methods According to the 3 new strains of ZEBOV genome-wide reference sequences separated from West Africa in 2014 and the Zaire ebola virus strain isolated from Mayinga （NC_002549） in 1976, specific sequence signature of ZEBOV was calculated out based on Insignia system algorithm comparison with GenBank Nt library. Primers and TaqMan probe were designed, and the reaction conditions were optimized. Ten-fold series dilution positive plasmid were used as the standard samples to do amplification plots and standard curve. Also the repeatability and accuracy of the method were tested. The rest of the 4 types of ebola virus, marburg virus, west Nile virus, eastern equine encephalitis virus, and Western equine encephalitis virus, and the 48 clinical comparison samples were employed to contrast for specificity test. Results The specific sequence （KJ660348, 11331 - 11514） design probe and primers were designed. Standard curve＇ s correlation coefficient was greater than 0.99 and the sensitivity was up to 5 × 10^1 copies. Repeatability and accuracy test＇ s results showed that the coefficient of variation （CV） of the intra assay reproducibility was 0. 88% to 2.50%, and the CV of the inter assay reproducibility was 1.75% to 3.31%. Eight strains ofviruses and 19 clinical comparison samples were all negative. Conclusion The specific sequence signature of Zaire ebola virus in the non-coding regions （KJ660348, 11331 -11514）, was verified to be specific. Our established fluorescence quantitative PCR is a rapid, sensitive and reproducible method for ZEBOV detection.

关 键 词：扎伊尔型 埃博拉病毒 荧光定量PCR 标签序列 鉴定 

分 类 号：R373.9[医药卫生—病原生物学] R394.3[医药卫生—基础医学]