【摘要】：目的引入一種設(shè)計(jì)簡(jiǎn)易、特異性強(qiáng)、退火溫度范圍寬的雙啟動(dòng)寡核苷酸引物(dual-priming oligonucleotide,DPO)設(shè)計(jì),建立基于DPO引物特異性檢測(cè)小腸結(jié)腸炎耶爾森氏菌的PCR方法。方法以小腸結(jié)腸炎耶爾森氏菌16S-23S rRNA基因?yàn)榘谢?設(shè)計(jì)一對(duì)DPO引物,經(jīng)過(guò)PCR反應(yīng)體系優(yōu)化,建立小腸結(jié)腸炎耶爾森氏菌DPO-PCR檢測(cè)方法。測(cè)定了檢測(cè)靈敏度,以常規(guī)PCR方法作為參照,分析DPO-PCR方法的特異性及退火溫度。結(jié)果建立的小腸結(jié)腸炎耶爾森氏菌DPO-PCR檢測(cè)方法的靈敏度為1.43×102 CFU/mL;與常規(guī)PCR方法相比,DPO-PCR方法在49~69℃退火溫度范圍內(nèi)均能保持高效率擴(kuò)增;特異性強(qiáng),所測(cè)試17種病原菌中,僅小腸結(jié)腸炎耶爾森氏菌為陽(yáng)性結(jié)果,且無(wú)非特異性擴(kuò)增。結(jié)論 DPOPCR方法不需要對(duì)引物參數(shù)特別是退火溫度進(jìn)行優(yōu)化,特異性強(qiáng),為致病微生物的快速準(zhǔn)確檢測(cè)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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