CAJ | 학술논문

目的登革病毒核酸检测竞争性假病毒颗粒型内参照品建立与评价.方法将登革病毒核酸检测靶基因序列间引入180碱基与登革病毒无关基因序列构建内参照检测靶靶标并在其下游通过核糖体内部进入位点基因介导的荧光蛋白报告基因后引入慢病毒包装载体,在辅助质粒的帮助下转染HEK 293T细胞,收获假病毒颗粒,定量分析后加入到到待检样本和模拟样本中一起进行评价和应用.结果所构建的登革病毒病毒核酸检测竞争性假病毒颗粒型内参照品能够充当RT-PCR法检测不同型别登革病毒的内参照品,扩增片段大小和病毒目的扩增片段明显不同,易于区别,能够监测反应的整个过程.结论登革病毒核酸检测竞争性假病毒颗粒型内参照系统,容易制备,保存简单,易于改造为多种RNA病毒的核酸检测的内参照.
목적 등혁병독핵산검측경쟁성가병독과립형내삼조품건립여평개.방법 장등혁병독핵산검측파기인서렬간인입180감기여등혁병독무관기인서렬구건내삼조검측파파표병재기하유통과핵당체내부진입위점기인개도적형광단백보고기인후인입만병독포장재체,재보조질립적방조하전염HEK 293T세포,수획가병독과립,정량분석후가입도도대검양본화모의양본중일기진행평개화응용.결과 소구건적등혁병독병독핵산검측경쟁성가병독과립형내삼조품능구충당RT-PCR법검측불동형별등혁병독적내삼조품,확증편단대소화병독목적 확증편단명현불동,역우구별,능구감측반응적정개과정.결론 등혁병독핵산검측경쟁성가병독과립형내삼조계통,용역제비,보존간단,역우개조위다충RNA병독적핵산검측적내삼조.
Objective Development of pseudoviral competitive internal controls for RT-PCR laboratory detection of dengue virus. Methods The internal controls target gene were obtained by insertion of a 180bp non-related DNA fragment into RT-PCR detection target of dengue virus between the forward and reverse PCR primer binding regions. A yellow florescence protein reporter gene was induced at downstream of internal controls target gene via internal ribosome entry site gene. HEK 293T cells were transfected with plasmid containing this whole cassette and lentiviral packaging support plasmid. Pseudoviral particle was recovered from the supernatant and analyzed quantitatively and qualitatively in simulated samples at the same tube under different experimental conditions. Results The established pseudoviral competitive internal controls can be used in the RT-PCR detection of different serotype dengue virus and the whole detection process can be monitored. The obtained fragment is easy to be differentiated in agarose electrophoresis. Conclusion The pseudoviral competitive internal controls could be used for the quality control of the laboratory diagnosis process, simple to prepare, stable for storage, easy to be transformed into internal controls for other RNA virus.