基于DPO引物特异性检测小肠结肠炎耶尔森氏菌的PCR方法

发布时间：2018-10-10 19:04

【摘要】：目的引入一种设计简易、特异性强、退火温度范围宽的双启动寡核苷酸引物(dual-priming oligonucleotide,DPO)设计,建立基于DPO引物特异性检测小肠结肠炎耶尔森氏菌的PCR方法。方法以小肠结肠炎耶尔森氏菌16S-23S rRNA基因为靶基因,设计一对DPO引物,经过PCR反应体系优化,建立小肠结肠炎耶尔森氏菌DPO-PCR检测方法。测定了检测灵敏度,以常规PCR方法作为参照,分析DPO-PCR方法的特异性及退火温度。结果建立的小肠结肠炎耶尔森氏菌DPO-PCR检测方法的灵敏度为1.43×102 CFU/mL;与常规PCR方法相比,DPO-PCR方法在49~69℃退火温度范围内均能保持高效率扩增;特异性强,所测试17种病原菌中,仅小肠结肠炎耶尔森氏菌为阳性结果,且无非特异性扩增。结论 DPOPCR方法不需要对引物参数特别是退火温度进行优化,特异性强,为致病微生物的快速准确检测提供了新方法。
[Abstract]:Objective to establish a simple, specific and wide range of annealing temperature based double primer oligonucleotide (dual-priming oligonucleotide,DPO) design for the detection of Yersinia enterocolitica by using DPO primers. Methods A pair of DPO primers were designed using the 16S-23S rRNA gene of Yersinia enterocolitica as the target gene. The DPO-PCR detection method of Yersinia enterocolitica was established by optimizing the PCR reaction system. The detection sensitivity was determined and the specificity and annealing temperature of DPO-PCR method were analyzed using conventional PCR method as a reference. Results the sensitivity of the established DPO-PCR detection method for Yersinia enterocolitica was 1.43 脳 10 ~ 2 CFU/mL;. Compared with the conventional PCR method, the DPO-PCR method could maintain a high efficiency in the range of 49 ~ 69 鈩，

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