Justo Christine Aubrey C, Acosta Shaira Jane, Jauset-Rubio Miriam, Skouridou Vasso, Mulinganya Guy, Cools Piet, Rivera Windell L, O'Sullivan Ciara K
Interfibio Consolidated Research Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, 43007 Tarragona, Spain.
Pathogen-Host-Environment Interactions Research Laboratory, Institute of Biology, College of Science, University of the Philippines Diliman, Quezon City 1101, Philippines.
ACS Infect Dis. 2025 Jul 11;11(7):1944-1955. doi: 10.1021/acsinfecdis.5c00147. Epub 2025 Jun 5.
The globally persistent and most common sexually transmitted agent continues to be an underestimated public health threat. Infection with has been associated with reproductive complications and an increased risk of HIV acquisition. With the available diagnostic tools for trichomoniasis, there is still an unmet demand for a cost-effective, facile, rapid, and robust diagnostic tool. We report on the design and utilization of tailed primers for the molecular detection of , exploiting isothermal DNA amplification and solid-phase sandwich colorimetric assay. The use of tailed primers, in combination with enzyme-linked and surface-tethered complementary probes, is a straightforward strategy to quantitatively evaluate and maximize target-specific amplification by recombinase polymerase amplification (RPA), which does not rely on the typical nuclease-probe system. With the quantitative nature of the microplate-based enzyme-linked oligonucleotide assay (ELONA) that relies on DNA-DNA hybridization, we systematically optimized the RPA conditions for maximal and specific amplification of the target DNA fragment of the genome. The developed RPA-ELONA has a detection limit of 4.03 × 10 fg/μL (3.99 aM) synthetic dsDNA and is more sensitive than quantitative RPA and PCR-based amplification using the same tailed primers. The analysis of biobanked clinical genomic samples with the RPA-ELONA showed 95% accuracy relative to a qPCR assay using the commercial S-DiaMGTV qPCR kit. The developed RPA-ELONA exploiting the tailed primers is a sensitive molecular detection assay for and serves as a guide in further development of sandwich-based POCT for .
这种全球持续存在且最常见的性传播病原体仍然是一种被低估的公共卫生威胁。感染该病原体与生殖并发症以及感染艾滋病毒的风险增加有关。尽管有可用的滴虫病诊断工具，但对于一种经济高效、简便、快速且可靠的诊断工具仍存在未满足的需求。我们报告了用于该病原体分子检测的带尾引物的设计与应用，利用等温DNA扩增和固相夹心比色法。将带尾引物与酶联及表面连接的互补探针结合使用，是一种直接的策略，可通过重组酶聚合酶扩增（RPA）定量评估并最大化靶标特异性扩增，而RPA不依赖于典型的核酸酶 - 探针系统。借助基于微孔板的依赖DNA - DNA杂交的酶联寡核苷酸分析（ELONA）的定量特性，我们系统地优化了RPA条件，以实现对该病原体基因组靶标DNA片段的最大程度特异性扩增。所开发的RPA - ELONA对合成双链DNA的检测限为4.03×10 fg/μL（3.99 aM），并且比使用相同带尾引物的定量RPA和基于PCR的扩增更灵敏。用RPA - ELONA分析生物样本库中的临床基因组样本，相对于使用商业S - DiaMGTV qPCR试剂盒的qPCR测定，准确率为95%。所开发的利用带尾引物的RPA - ELONA是一种灵敏的该病原体分子检测方法，并为进一步开发基于夹心的该病原体即时检测提供了指导。
