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目的制备安全、稳定的新疆出血热病毒实时荧光RT-PCR检测阳性对照品。方法人工合成含实时荧光RT-PCR扩增片段的新疆出血热病毒核苷酸序列,连接入慢病毒载体,将重组慢病毒载体与慢病毒包装载体共转染293T细胞,培养48 h后采用实时荧光RT-PCR方法检测假病毒包装情况,随后将包装成功的假病毒颗粒加核酸保护剂制备成阳性对照品,并进行稳定性试验。结果重组慢病毒载体与慢病毒包装载体共转染293T细胞后,将收集到的细胞培养上清采用实时荧光RT-PCR进行检测,结果显示10、100、1 000、10 000倍4个倍比稀释度扩增后所得的Ct值分别为13、19、25、33,显示含高浓度的假病毒颗粒。取经1 000倍稀释后制备的阳性对照品于37℃孵箱中放置0、3、7、15、21和30 d后,提取的RNA经实时荧光RT-PCR检测,所得Ct值在25~28之间,表明阳性对照品有较高的热稳定性。结论本研究通过慢病毒包装技术制备的假病毒颗粒,是新疆出血热病毒荧光RT-PCR核酸检测过程中理想的阳性对照品。
Objective To prepare a safe and stable Xinjiang hemorrhagic fever virus real-time fluorescence RT-PCR test positive control. Methods The nucleotide sequence of Xinjiang hemorrhagic fever virus was amplified by RT-PCR and ligated into lentiviral vector. 293T cells were co-transfected with the recombinant lentiviral vector and lentivirus vector. After cultured for 48 h, Fluorescent RT-PCR method was used to detect the packaging of pseudovirus. Subsequently, the pseudotyped virus particles plus the nucleic acid protectant were packaged into positive control and stability test. Results After co-transfection of 293T cells with the recombinant lentiviral vector and lentivirus vector, the collected cell culture supernatants were detected by real-time fluorescence RT-PCR. The results showed that 10, 100, 1 000, 10 000 times 4 times Ct values obtained after dilution amplification were 13, 19, 25, 33, respectively, showing high concentrations of pseudovirions. The positive control samples prepared after 1000-fold dilution were stored at 37 ℃ for 0, 3, 7, 15, 21 and 30 days. The extracted RNA was detected by real-time fluorescence RT-PCR. The obtained Ct values ranged from 25 to 28 Between the positive control showed a higher thermal stability. Conclusions The pseudovirions prepared by lentivirus packaging technique in this study are ideal positive control samples for the detection of Xinjiang hemorrhagic fever virus by RT-PCR.