Johanna Kreuter, Katharina Bica-Schröder, Ádám M Pálvölgyi, Rudolf Krska, Regina Sommer, Andreas H Farnleitner, Claudia Kolm, Georg H Reischer
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引用次数: 0
摘要
基于 DNA 和 RNA 的诊断方法在准确检测和鉴定与健康相关的细菌方面发挥着关键作用,可帮助人们深入了解细菌的存在、生存能力和治疗效果。在本文中,我们介绍了一种新型 DNA 和 RNA 提取方案,旨在实现简单快速的分子诊断。该提取方法基于亲水性离子液体(IL)1-乙基-3-甲基咪唑鎓醋酸盐和硅包被磁珠。首先,我们开发了一种基于离子液体的细菌细胞裂解方案,可在室温下操作。随后,我们建立了一套磁珠纯化程序,可以高效、可重复地从IL裂解液中提取DNA和RNA。IL 不仅能裂解细胞,还能促进核酸(NAs)吸附到磁珠表面,从而无需使用混沌结合缓冲液,并能在不花费大量精力和材料的情况下纯化 NA。最后,我们将细胞裂解步骤和纯化步骤结合起来,在围病原菌培养物上评估了基于 IL 的新型提取方法,并通过 (RT)-qPCR 将其与商用 DNA 和 RNA 提取试剂盒进行了比较。与参考方法相比,基于 IL 的提取方案得到了相似或更优的结果。此外,该方法成本较低,所需材料和设备最少,提取过程快速(30 分钟)、简单且可自动化。这些特点有利于将所开发的方法用于常规和高通量检测,以及用于护理点、现场和低资源环境,从而推动分子诊断领域的发展。
A novel ionic liquid-based approach for DNA and RNA extraction simplifies sample preparation for bacterial diagnostics.
DNA- and RNA-based diagnostics play a pivotal role in accurately detecting and characterizing health-relevant bacteria, offering insights into bacterial presence, viability and treatment efficacy. Herein, we present the development of a novel extraction protocol for both DNA and RNA, designed to enable simple and rapid molecular diagnostics. The extraction method is based on the hydrophilic ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate and silica-coated magnetic beads. First, we developed an IL-based cell lysis protocol for bacteria that operates at room temperature. Subsequently, we established a magnetic bead purification procedure to efficiently and reproducibly extract DNA and RNA from the IL-lysates. The IL not only lyses the cells, but also facilitates the adsorption of nucleic acids (NAs) onto the surface of the magnetic beads, eliminating the need for a chaotropic binding buffer and allowing for purification of NAs without significant effort and materials required. Lastly, we combined the cell lysis step and the purification step and evaluated the novel IL-based extraction method on periopathogenic bacterial cultures, comparing it to commercial DNA and RNA extraction kits via (RT)-qPCR. In comparison to the reference methods, the IL-based extraction protocol yielded similar or superior results. Furthermore, costs are lower, required materials and equipment are minimal and the process is fast (30 min), simple and automatable. These characteristics favour the developed method for use in routine and high-throughput testing as well as in point-of-care, on-site and low-resource settings, thereby advancing the field of molecular diagnostics.
期刊介绍:
Analytical and Bioanalytical Chemistry’s mission is the rapid publication of excellent and high-impact research articles on fundamental and applied topics of analytical and bioanalytical measurement science. Its scope is broad, and ranges from novel measurement platforms and their characterization to multidisciplinary approaches that effectively address important scientific problems. The Editors encourage submissions presenting innovative analytical research in concept, instrumentation, methods, and/or applications, including: mass spectrometry, spectroscopy, and electroanalysis; advanced separations; analytical strategies in “-omics” and imaging, bioanalysis, and sampling; miniaturized devices, medical diagnostics, sensors; analytical characterization of nano- and biomaterials; chemometrics and advanced data analysis.