病毒学报

本研究的目的为建立一种灵敏、快速、定量检测埃博拉病毒莱斯顿亚型核酸的实时荧光定量PCR检测方法及试剂盒。首先选择埃博拉病毒莱斯顿亚型的保守基因NP基因作为检测靶目标,筛选出保守序列并设计合成一对特异性引物。然后将连有NP全长基因的重组质粒作为定量标准品,将10倍系列稀释的重组质粒进行荧光定量PCR扩增,绘制标准曲线,并进行重复性、灵敏度及特异性检测。结果显示建立的埃博拉病毒莱斯顿亚型荧光定量PCR检测方法,其灵敏度可达102拷贝/μL,不同梯度标准品间线性关系(R2)达0.997,斜率为-0.3101,扩增效率为110.145%,所有标准品均在79.94℃出现尖且窄的特异性熔解峰。利用该检测系统可以快速定量检测埃博拉病毒莱斯顿亚型核酸,灵敏度高、重复性好,可用于基础及临床实验室对埃博拉病毒莱斯顿亚型感染的快速诊断和临床效果的监测,具有实际的应用价值。

We aimed to develop a real-time polymerase chain reaction(PCR)detection method for the Reston subtype of the Ebola virus.The NP gene of the Reston subtype of the Ebola virus was selected as the detection object.Sequences of different subtypes of Ebola viruses were aligned using Clustal W software.The most unique and conserved regions of the Reston subtype of the Ebola virus were recruited as candidate sequences for specific primers.Primer Express and Primer Premier 5.0software were used to filter the optimal pair of primers for detection.Real-time PCR was carried out using optimized parameters and positive DNA prepared by serial(tenfold)dilution of a recombinant plasmid and by plotting a standard curve.In addition,the reproducibility,accuracy,and specificity of the assay were tested.Results showed that the sensitivity of detection of the Reston subtype of the Ebola virus by real-time PCR could reached102 copies/μL.The linear relationship(R2)reached 0.997,the slope of the standard curve was-0.3101,and amplification efficiency was 110.145%.A sharp and narrow melting peak appeared at 79.94°C for all standards in different dilutions.In conclusion,a fast and sensitive real-time PCR detection system for the Reston subtype of the Ebola virus was developed.This system could be used as a supplementary diagnostic and monitoring approach for basic and clinical studies on the Reston subtype of the Ebola virus.The detection system does not require expensive technology or specialist operators.