Abstract

Tularemia is a natural focal zoonotic infection that can cause epidemic manifestations of an emergency nature. The purpose of the study is to develop a method for detecting DNA strains of the tularemia pathogen Francisella tularensis by loop mediated isothermal amplification (LAMP). Primers for the selected targets were calculated using the on-line Primer Explorer 5 program and tested for the specificity using the BLAST program. The primers were synthesized by Synthol, Moscow. Isolation of DNA from vaccine and virulent strains of epidemically significant subspecies of the tularemia microbe, as well as pathogens of other infectious diseases, was performed using the commercial DNA-sorb-B kit (InterLabService, Russia). The amplification reaction was carried out at a temperature of 63°C for 60 min (without loop primers) or 30 min (with loop primers) with preliminary heating at a temperature of 92°C for 2 min on a Tertsik amplifier (DNA-Technology, Russia) in the presence of the thermostable SD polymerase. Sequences of the genes encoding acid phosphatase A (acpA), outer membrane protein (fopA), and a region of the iglC gene of the pathogenicity island were chosen as DNA targets for the detection of the tularemia pathogen. Of the two sets of outer, inner, and loop original primers synthesized for each selected marker gene, the sets acpFt101, fopFt132, and iglCFt1 reproducibly and specifically detected DNA of 100–1000 F. tularensis microbial cells. The opportunity to reduce the analysis time by half appeared due to the introduction of loop primers and visual detection of amplification products stained with the SYTO 82 intercalating dye without subsequent electrophoresis and visualization of the gel after staining with ethidium bromide. An easy-to-use test that does not require sophisticated equipment is proposed for clinical and field diagnostics of the tularemia pathogen.

中文翻译：

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通过环介导等温扩增检测兔热菌病原体 DNA

摘要

兔热病是一种自然局灶性人畜共患感染，可引起紧急情况的流行病表现。本研究的目的是开发一种通过环介导等温扩增 (LAMP)检测土拉热病病原体土拉弗朗西斯菌DNA 菌株的方法。使用在线 Primer Explorer 5 程序计算所选靶标的引物，并使用 BLAST 程序测试特异性。引物由莫斯科 Synthol 合成。使用商业 DNA-sorb-B 试剂盒（InterLabService，俄罗斯）从疫苗和兔热病微生物流行显着亚种的毒力菌株以及其他传染病的病原体中分离 DNA。扩增反应在Tertsik放大器(DNA-Technology)上在63°C的温度下进行60分钟(无环引物)或30分钟(有环引物)，并在92°C的温度下预加热2分钟。 ，俄罗斯）在热稳定性 SD 聚合酶的存在下。选择编码酸性磷酸酶 A ( acpA )、外膜蛋白 ( fopA )的基因序列和致病岛iglC基因的区域作为检测兔热病病原体的 DNA 靶标。在为每个选定的标记基因合成的两组外部、内部和环原始引物中，acpFt101、fopFt132 和 iglCFt1 组可重复且特异性地检测 100-1000 个土拉菌微生物细胞的 DNA。由于环引物的引入以及用 SYTO 82 嵌入染料染色的扩增产物的目视检测，无需随后的电泳以及在用溴化乙锭染色后对凝胶进行可视化，出现了将分析时间缩短一半的机会。提出了一种不需要复杂设备的易于使用的测试，用于兔热病病原体的临床和现场诊断。