分子信标-TaqMan探针法实时荧光定量PCR新型探针及引物

发布时间：2018-03-21 20:41

本文选题：实时荧光定量PCR　切入点：TaqMan探针　出处：《生物技术通报》2016年11期 　论文类型：期刊论文

【摘要】：在TaqMan探针法实时荧光定量PCR中,排除由引物探针聚合延伸引起的假阳性问题以及由引物二聚体(Primer Dimer,PD)引起的假阴性问题。首先对不同探针进行引物探针聚合实验;然后通过双重PCR的干扰实验选择合适的内标基因,并使用内标质粒检测验证中部同序引物排除PD干扰的实用性;最后比较探针法不同体系检测HBV基因的灵敏度。结果表明,引物与探针聚合实验中,含有反义碱基的HBVP4在重复实验中未出现假阳性;竞争性双重PCR和非竞争性双重PCR两模板开始出现干扰作用的浓度差分别为20倍和100倍;对于内标基因,使用中部同序引物对以及普通引物对分别可以检测到10-9和10-8稀释度;3种HBV基因检测体系,使用普通引物对可以检测到Ct33左右,加入内标系统和使用中部同序引物对均可检测到Ct35左右。在TaqMan-分子信标结构调整的基础上引入反义碱基可以在排除由引物和探针聚合延伸引起的假阳性问题;在探针法中,使用中部同序引物对和加入内标系统均可降低PD的干扰程度,提升检测的灵敏度。
[Abstract]:In real-time fluorescent quantitative PCR with TaqMan probe method, the false positive problem caused by the extension of primer probe polymerization and the false negative problem caused by primer dimer dimer dimer were eliminated. First, primer probe polymerization experiments were carried out for different probes. Then the suitable internal standard gene was selected by double PCR interference experiment, and the practicability of eliminating PD interference was verified by using internal standard plasmid detection. Finally, the sensitivity of probe method for detecting HBV gene in different systems was compared. In primer and probe polymerization, HBVP4 containing antisense bases did not appear false positive in repeated experiments, the concentration difference of interference between competitive double PCR and non-competitive double PCR began to be 20 times and 100 times respectively. Using middle sequence primer pairs and common primer pairs, 10-9 and 10-8 dilution of HBV genes could be detected, and Ct33 could be detected by common primer pairs. Ct35 can be detected by adding internal standard system and using middle sequence primer pairs. Introducing antisense bases on the basis of structural adjustment of TaqMan- molecular beacons can eliminate false positive problems caused by the extension of primer and probe polymerization. Using the central sequence primer pair and adding the internal standard system can reduce the interference of PD and improve the sensitivity of detection.
【作者单位】：中国人民解放军总医院临检科;温州医科大学检验医学院生命科学学院;北京泰格瑞分子检验有限公司;
【基金】：国家重大科学仪器设备开发专项(2012YQ03026107)
【分类号】：R440

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