The application of a new fluorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant o...The application of a new fluorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.展开更多
目的：采用二聚体蝎型探针技术建立一种高敏感性和特异性的检测梅毒螺旋体（T P ）的荧光定量聚合酶链反应（PCR）方法。方法根据T P特异性外膜蛋白Gpd的基因序列设计引物和荧光探针，采用基因工程技术构建可用于T P定量的标准品，优化...目的：采用二聚体蝎型探针技术建立一种高敏感性和特异性的检测梅毒螺旋体（T P ）的荧光定量聚合酶链反应（PCR）方法。方法根据T P特异性外膜蛋白Gpd的基因序列设计引物和荧光探针，采用基因工程技术构建可用于T P定量的标准品，优化荧光定量PCR的反应体系和反应条件，建立检测T P的二聚体蝎型探针定量PCR。采用本研究建立方法和商品化荧光定量PCR试剂盒检测40例临床标本，对比分析检测结果的统计学差异。结果成功构建了T P重组质粒标准品和T P的二聚体蝎型探针定量PCR ，该方法线性范围为101～108 copy／mL ，灵敏度为10 co py／m L ，特异性和敏感性均为100．0％；二聚体蝎型探针定量PC R对疑似梅毒病例阳性检出率显著高于目前商品化Taqman荧光定量PCR试剂盒（82．5％ vs 62．5％，P＜0．05）。结论成功建立了二聚体蝎型探针荧光定量PCR快速检测T P的方法，为临床上T P的早期诊断和防控奠定了基础。展开更多
基金the National Natural Science Foundation of China (No. 20075012) and the Outstanding Scholar Program of Nankai University.
文摘The application of a new fluorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.
文摘目的：采用二聚体蝎型探针技术建立一种高敏感性和特异性的检测梅毒螺旋体（T P ）的荧光定量聚合酶链反应（PCR）方法。方法根据T P特异性外膜蛋白Gpd的基因序列设计引物和荧光探针，采用基因工程技术构建可用于T P定量的标准品，优化荧光定量PCR的反应体系和反应条件，建立检测T P的二聚体蝎型探针定量PCR。采用本研究建立方法和商品化荧光定量PCR试剂盒检测40例临床标本，对比分析检测结果的统计学差异。结果成功构建了T P重组质粒标准品和T P的二聚体蝎型探针定量PCR ，该方法线性范围为101～108 copy／mL ，灵敏度为10 co py／m L ，特异性和敏感性均为100．0％；二聚体蝎型探针定量PC R对疑似梅毒病例阳性检出率显著高于目前商品化Taqman荧光定量PCR试剂盒（82．5％ vs 62．5％，P＜0．05）。结论成功建立了二聚体蝎型探针荧光定量PCR快速检测T P的方法，为临床上T P的早期诊断和防控奠定了基础。
