新疆出血热病毒实时荧光RT-PCR检测方法的假病毒阳性对照品制备  

Preparation of pseudoviral positive control for real-time RT-PCR detection of Xinjiang hemorrhagic fever virus

作　　者：郑夔[1] 袁帅[1] 黎东红[1] 丁国允[1] 李小波[1] 师永霞[1] 戴俊[1] 黄吉城[1] 

机构地区：[1]广东检验检疫技术中心,广东广州510700

出　　处：《华南预防医学》2016年第1期1-5,共5页South China Journal of Preventive Medicine

基　　金：国家质检总局科技计划项目(2013IK223;2015IK066)

摘　　要：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 positive control for real-time RT-PCR（ rRTPCR） detection of Xinjiang hemorrhagic fever virus. Methods The nucleic acid sequence containing a rRT-PCR amplification target of Xinjiang hemorrhagic fever virus was synthesized and inserted into a lentiviral vector,the recombinant lentiviral vector and the lentivirus-packaging vector were co-transfected into293 T cells. After 48 h of transfection,the rRT-PCR assay was used for detection of the pseudoviral particles packaging and production. Then the pseudoviral particles were collected and treated with nucleic acid protective agent to prepare a positive control,and then the determination of stability was conducted.Results After the recombinant lentiviral vector and lentivirus-packaging vector were co-transfected into293 T cells,the supernatant was harvested and detected by rRT-PCR. Ten-fold serial dilutions from 10- 1to 10- 4 of supernatant were detected,the result showed that the Ct value ranged from 13 to 33,indicating that the concentration of pseudoviral particles in the supernatant was high. The 1 000- fold diluted supernatant was used for preparing a positive control. The stability test was conducted after the positive control was placed in the incubator at 37 ℃ for 0,3,7,15,21,and 30 days. Ct value of the rRT-PCR detection was 25 to 28 approximately,demonstrating that the positive control possessed high heat stability. Conclusion The pseudoviral particles produced by using lentiviral packaging technology in this study can be used for an ideal positive control for rRT-PCR detection of Xinjiang hemorrhagic fever virus.

关 键 词：新疆出血热病毒 实时荧光RT-PCR 阳性对照品 

分 类 号：R-33[医药卫生] R34