目的 建立一种简单,快速的重组酶聚合酶核酸扩增技术检测鼠疫耶尔森氏菌的方法。方法 基于耶尔森氏菌的特异性序列设计引物及探针;通过对不同温度的检测来优化反应温度;通过对不同菌株的DNA模板进行检测评价该方法的特异性;通过对不同稀释浓度的样本DNA模板进行检测来确定灵敏性;通过模拟样品进行应用评价。结果 基于鼠疫耶尔森菌的DNA腺嘌呤甲基化酶基因设计特异性引物和探针所建立的荧光重组酶聚合酶扩增方法,35 ℃反应20 min内可成功检出低至100个拷贝的鼠疫耶尔森菌基因,而且与其他菌无交叉反应,对模拟样品的检测准确性达到98.0%。结论 重组酶聚合酶核酸扩增可广泛应用于简陋条件下鼠疫耶尔森菌的临床检测,促进鼠疫的防控。
Abstract
Objective To establish a simple, rapid method of the recombinase polymerase amplification (RPA) technology for the detection of Yersinia pestis.Methods Primers and probes were designed basing on specific sequences of Yersinia, and the reaction temperature was optimized by detecting different temperatures. The specificity of this method was evaluated by detecting DNA templates of different strains, and the sensitivity was determined by testing sample DNA templates of different dilution concentrations. The application of this method was evaluated by simulating samples.Results Specific primers and probe were designed based on the DNA adenine methylase gene and the fluorescence RPA method was established, which can successfully detect up to 100 copies of Yersinia pestis gene in 20 minutes at 35 ℃, and no cross-reaction with other bacteria, the simulation of the sample detection accuracy of 98.0%.Conclusions RPA could be used in the clinical detection of Yersinia pestis, especially under crude conditions and the method is helpful to the prevention and control of plague.
关键词
鼠疫耶尔森菌 /
重组酶聚合酶扩增技术 /
核酸检测
Key words
Yersinia pestis /
recombinase polymerase amplification /
nucleic acid detection
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基金
国家重点研发计划(2018YFC1603705)
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