Nowadays, considerable efforts are focused on advancing DNA detection methods, which are extremely important in clinical diagnostics, pathogen determination, gene therapy, and forensic analysis. A one-pot sensitive microplate-based chemiluminescent assay coupled with catalytic hairpin assembly (CHA) amplification for detection of a 35-mer DNA oligonucleotide was developed. To improve the assay sensitivity, a triple amplification strategy based on the application of CHA (1), streptavidin-polyperoxidase conjugate (Stp-polyHRP) (2), and an enhanced chemiluminescent reaction (3) was used. The one-pot format of the assay, where all steps of the DNA determination are performed in the same well without transfer of samples from one test tube to another, increased its precision. The proposed assay detected the target DNA in the fM range and distinguished the target DNA from related DNAs, demonstrating its high sensitivity and high selectivity. Moreover, the assay was applied successfully for the quantitative determination of the target in spiked samples of human plasma. A microplate format of the assay was convenient for the analysis of a large number of samples. This study provides a prospective tool for DNA detection. Graphical abstract.

中文翻译：

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一锅基于微孔板的化学发光测定法与催化发夹装配扩增相结合，用于DNA检测。

如今，大量的精力都集中在推进DNA检测方法上，这在临床诊断，病原体确定，基因治疗和法医分析中非常重要。开发了一种基于一锅敏感的基于微孔板的化学发光测定法，该方法与催化发夹装配（CHA）扩增相结合，用于检测35-mer DNA寡核苷酸。为了提高测定灵敏度，使用了基于CHA（1），链霉亲和素-聚过氧化物酶结合物（Stp-polyHRP）（2）和增强的化学发光反应（3）的三重扩增策略。单罐形式的测定（DNA测定的所有步骤均在同一孔中进行而无需将样品从一个试管转移到另一个试管中）提高了其准确性。拟议的检测方法检测到了fM范围内的目标DNA，并将目标DNA与相关DNA进行了区分，证明了其高灵敏度和高选择性。而且，该测定法成功地用于定量测定人血浆中加标样品中的靶标。测定的微孔板形式便于分析大量样品。这项研究为DNA检测提供了一种前瞻性工具。图形概要。