Mojarad Alireza Ebrahimi, Gargaria Seyed Latif Mousavi
Department of Biology, Shahed University, Tehran, Iran.
Iran J Microbiol. 2020 Aug;12(4):263-272. doi: 10.18502/ijm.v12i4.3928.
In recent years, the prevalence of diseases caused by spp. is increasing in the world, and among them species, is the most important associated with pandemic and epidemic cholera outbreaks. Therefore, the development of a reliable method for early and accurate detection of for management of diseases is a real need. Aptamers with the ability to detect targets with high specificity and accuracy can be one of the candidates used for the whole cell and thereby detection.
In this research high-affinity DNA aptamers against with two major serotypes of Inaba (ATCC 39315) and Ogawa (clinical sample) were selected from DNA aptamer library through 12 rounds of Systematic Evolution of Ligands by Exponential (SELEX) enrichment procedure using live cells as a target which monitored with flow cytometry.
The binding efficiency and dissociation constant of the isolated aptamers V.ch47 and V.ch27 were 56.4%, 53.3% and 15.404 ± 4.776 pM, 20.186 ± 3.655 pM, respectively. A sandwich Enzyme-linked aptamer sorbent assay (ELASA) was developed with the biotinylated V.ch47 aptamer and our previously developed nanobody anti-Lipopolysaccharides (LPS). We optimized this system with O1 and analyzed their cross reactivity with close physiological bacteria. The threshold of detection was obtained 10 CFU/ml in the sandwich ELASA process.
Our results showed that the sandwich ELASA is sensitive enough for the rapid detection of from other bacteria.
近年来，由 spp. 引起的疾病在全球的患病率呈上升趋势，其中 种是与霍乱大流行和疫情爆发相关的最重要的 。因此，开发一种可靠的早期准确检测 的方法以管理疾病是实际所需。具有高特异性和准确性检测靶标的适体可成为用于全细胞检测从而进行 检测的候选物之一。
在本研究中，通过12轮指数富集配体系统进化（SELEX）富集程序，以活细胞为靶标，利用流式细胞术监测，从DNA适体文库中筛选出针对稻叶型（ATCC 39315）和小川型（临床样本）两种主要血清型的高亲和力DNA适体。
分离得到的适体V.ch47和V.ch27的结合效率和解离常数分别为56.4%、53.3%和15.404±4.776 pM、20.186±3.655 pM。利用生物素化的V.ch47适体和我们先前开发的抗脂多糖（LPS）纳米抗体，开发了一种夹心酶联适体吸附测定法（ELASA）。我们用O1对该系统进行了优化，并分析了它们与密切相关的生理细菌的交叉反应性。在夹心ELASA过程中，检测阈值为10 CFU/ml。
我们的结果表明，夹心ELASA对从其他细菌中快速检测 足够灵敏。
