Isolation and characterization of ssDNA aptamers against BipD antigen of Burkholderia pseudomallei

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS

ACS Applied Bio Materials Pub Date : 2024-08-28 DOI:10.1016/j.ab.2024.115655

Kasturi Selvam , Mohamad Ahmad Najib , Muhammad Fazli Khalid , Muhammad Hafiznur Yunus , Habibah A. Wahab , Azian Harun , Ummu Afeera Zainulabid , Khairul Mohd Fadzli Mustaffa , Ismail Aziah

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Abstract

Background

Melioidosis is difficult to diagnose due to its wide range of clinical symptoms. The culture method is time-consuming and less sensitive, emphasizing the importance of rapid and accurate diagnostic tests for melioidosis. Burkholderia invasion protein D (BipD) of Burkholderia pseudomallei is a potential diagnostic biomarker. This study aimed to isolate and characterize single-stranded DNA aptamers that specifically target BipD.

Methods

The recombinant BipD protein was produced, followed by isolation of BipD-specific aptamers using Systematic Evolution of Ligands by EXponential enrichment. The binding affinity and specificity of the selected aptamers were evaluated using Enzyme-Linked Oligonucleotide Assay.

Results

The fifth SELEX cycle showed a notable enrichment of recombinant BipD protein-specific aptamers. Sequencing analysis identified two clusters with a total of seventeen distinct aptamers. AptBipD1, AptBipD13, and AptBipD50 were chosen based on their frequency. Among them, AptBipD1 exhibited the highest binding affinity with a K_d value of 1.0 μM for the recombinant BipD protein. Furthermore, AptBipD1 showed significant specificity for B. pseudomallei compared to other tested bacteria.

Conclusion

AptBipD1 is a promising candidate for further development of reliable, affordable, and efficient point-of-care diagnostic tests for melioidosis.

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针对假丝酵母伯克霍尔德氏菌 BipD 抗原的 ssDNA 短肽的分离与表征

背景：瓜虫病的临床症状多种多样，因此很难诊断。培养法耗时且灵敏度较低，这就强调了快速、准确诊断梅里埃病的重要性。假马来伯克霍尔德氏菌的伯克霍尔德氏菌侵袭蛋白 D（BipD）是一种潜在的诊断生物标志物。本研究旨在分离和鉴定特异性靶向BipD的单链DNA适配体：方法：制备重组 BipD 蛋白，然后利用配体的系统进化（Systematic Evolution of Ligands by EXponential enrichment）分离 BipD 特异性适配体。使用酶联寡核苷酸测定法评估了所选适配体的结合亲和力和特异性：结果：第五次 SELEX 循环显示，重组 BipD 蛋白特异性适配体显著富集。测序分析发现了两个簇，共有 17 个不同的适配体。AptBipD1、AptBipD13 和 AptBipD50 是根据它们的频率选出的。其中，AptBipD1 与重组 BipD 蛋白的结合亲和力最高，Kd 值为 1.0 μM。此外，与其他受试细菌相比，AptBipD1 对假丝酵母菌具有明显的特异性：结论：AptBipD1 是进一步开发可靠、经济、高效的类鼻疽护理点诊断检测的理想候选物。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

ACS Applied Bio Materials Chemistry-Chemistry (all)

CiteScore

9.40

自引率

2.10%

发文量

464