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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 for the detection of Zaire ebola virus (ZEBOV). METHODS: Three ZEBOV genome-wide reference sequences newly isolated in West Africa in 2014 and ZEBOV strain (NC_002549) isolated from Mayinga in 1976 were selected as targets. Based on the Insignia system algorithm and the Nt library in GenBank, the ZEBOV-specific nucleic acid tag sequence . Design primers, TaqMan probe and optimize the reaction conditions, with 10-fold serial dilutions of the positive plasmid as a standard curve, to do the accuracy and reproducibility experiments. The specificity of the 8 subtypes of Ebola virus, Marburg virus, West Nile virus, Oriental equine encephalitis virus, Western equine encephalitis virus and other 48 kinds of virus and control group were compared Detection. Results The specific primers (KJ660348, 11331-11514) were designed to be specific primer sequences. The correlation coefficient of the standard curve was> 0.99, and the sensitivity was 5 × 10 ~ 1 copies. The accuracy of the reproducibility experimental results intra-assay coefficient of variation (CV) was 0.88% ~ 2.50%, inter-batch CV was 1.75% ~ 3.31%. 8 kinds of viruses and 48 cases of clinical complex samples were negative control. Conclusion The ZEBOV nucleic acid tag (KJ660348, 11331-11514) can be used to detect ZEBOV specifically in the non-coding region based on this method, and its sensitivity and repeatability are good.