【摘要】 双启动寡核苷酸引物(dual-priming oligonucleotide,DPO)是一种新型的引物设计方法,具有设计简易、特异性强以及退火温度范围宽的特点。以产肠毒性大肠杆菌LT基因为靶基因,设计了一对DPO引物,经过对Taq DNA聚合酶、Mg2+、dNTP反应体系因子的优化,建立了产肠毒性大肠杆菌DPO-PCR检测方法,测定了该方法的灵敏度、特异性以及退火温度不敏感性。结果显示,该方法检测灵敏度为1.24×102CFU/ml,在45℃~65℃退火温度范围内,均可实现靶基因的高效扩增。该方法特异性强,测试菌株中4株产肠毒性大肠杆菌均为阳性结果,其余菌株为阴性结果,且无非特异性扩增发生。与常规PCR方法相比,DPO-PCR方法不需要对引物参数特别是退火温度反复优化,同时DPO引物的特殊结构又增强了检测特异性,为致病性微生物的快速准确检测提供了新方法。更多还原

【Abstract】 Dual-priming oligonucleotide(DPO) is a novel primer design method with the features of simple design,strong specificity and wider annealing temperature range.In this study,a pair of DPO primers was designed based on enterotoxigenic E.coli(ETEC) LT gene and following optimization of reaction system factors that Taq DNA polymerase,Mg2 +and dNTP,a DPO-PCR detection method for ETEC was established and its sensitivity,specificity and annealing temperature insensitivity were analyzed.Results showed that detection sensitivity of the method was 1.24 × 102CFU / ml and the target gene could be efficiently amplified by the DPO primers at 45℃ ~ 65℃ of annealing temperature range.The DPO-PCR method showed high specificity,in tested strains,4 ETEC strains were positive results and other strains were negative results,and no nonspecific amplifications were produced.Compared with conventional PCR methods,the DPO-PCR method should not require to repeatedly optimize primer parameters in particular annealing temperature,at the same time,the special structure of DPO primers would enhance detection specificity,which provided a new method for fast and accurate detection of pathogens.更多还原