尼帕病毒N基因核酸检测方法对假病毒阳性标准品的制备及鉴定

Preparation and identification of pseudovirus control samples for Nipah virus N gene nucleic acid detection

目的 针对致死率极高的尼帕病毒,制备安全、稳定的含有尼帕病毒N基因片段的重组假病毒颗粒阳性标准品。方法 人工合成含有尼帕病毒N基因片段的核苷酸序列并导入CD513B慢病毒载体,与包装质粒同时转染HEK293T细胞,超速离心浓缩纯化,电镜观察假病毒颗粒形态,感染实验验证假病毒功能,并进行线性、稳定性和均一性检测。结果 包装并纯化后的假病毒颗粒浓度较高,直径在100~200 nm,可见明显的G蛋白刺突,感染实验绿色荧光蛋白信号强且不同温度下储存稳定性良好,均一性检测变异系数小于1%。结论 成功构建含有尼帕病毒N基因片段的假病毒颗粒,可作为阳性对照品在尼帕病毒核酸提取和检测过程中实现全程质量控制。

Objective To prepare safe and stable control samples for Nipah virus(NiV) molecular detection by recombinant pseudovirus containing N gene fragment of NiV. Methods The nucleotide sequence containing NiV N gene fragment was synthesized and inserted into CD513b lentiviral vector, and the recombinant lentiviral vector and the lentiviruspackaging vector were co-transfected into HEK293T cells. The pseudoviral particles were concentrated and purified by ultracentrifugation, and the particle morphology was observed by transmission electron microscopy. The function of the pseudovirus was verified by infection experiment, and the linearity, stability and homogeneity were conducted by qRT-PCR.Results The packaged and purified pseudoviral particles had a high concentration, with a diameter of 100-200 nm, and obvious G protein spikes were visible. The GFP signal of infection experiment was strong and the storage stability was good at different temperatures, and the coefficient of variation of homogeneity detection was less than 1%. Conclusion The control samples of pseudovirus containing NiV N gene fragment were successfully constructed, which can be used as whole-process positive control for NiV nucleic acid extraction and detection.