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目的建立鉴定登革病毒型别的双重实时Taqman PCR反应体系,以准确快速鉴定登革病毒型别。方法根据GenBank上已发表的登革病毒四个型别的全基因序列,进行对比分析,分别设计登革病毒的四个型别引物和探针,登革I、III型探针用FAM-TAMARA标记,登革II、IV型探针用JOE-TAMARA标记。经过条件优化后,建立检测登革病毒I/II型和III/IV型的两套双重实时荧光RT-PCR方法,扩增四型登革病毒RNA、登革病毒阴性样本和登革病毒RNA稀释样本,检测方法的特异性、重复性和检测限性。结果通过设计筛选序列和优化反应条件,建立登革病毒I、II型和登革病毒III、IV型的双重荧光PCR反应体系,通过试验证明,所建立的方法具有良好的特异性、重复性和检测限性,能准确快速地对登革病毒进行分型。结论建立了一种快速双重荧光PCR方法能同时对登革病毒进行分型和鉴定。
Objective To establish a dual real-time Taqman PCR reaction system for the identification of dengue virus types in order to accurately and rapidly identify dengue virus types. Methods According to the published sequences of the four genotypes of dengue virus in GenBank, four types of primers and probes for dengue virus were designed respectively. Dengue I and III probes were designed by FAM-TAMARA Tag, dengue II, type IV probe labeled with JOE-TAMARA. After optimized conditions, two sets of real-time fluorescence RT-PCR methods to detect dengue virus type I / II and type III / IV were established to amplify four types of dengue virus RNA, dengue virus negative samples and dengue virus RNA Sample, test method specificity, repeatability and detection limits. Results By designing the screening sequence and optimizing the reaction conditions, a dual-fluorescence PCR reaction system of dengue virus type I and type II and dengue virus type III and type IV was established. The experiment proved that the established method has good specificity, repeatability and Detection limit, accurate and rapid dengue virus typing. Conclusion A rapid double-fluorescent PCR method was established to identify and identify Dengue virus at the same time.