Isothermal DNA Amplification with Tailed Primers Coupled with Enzyme-Linked Oligonucleotide Assay for the Sensitive...

The globally persistent and most common sexually transmitted agent Trichomonas vaginalis continues to be an underestimated public health threat. Infection with T. vaginalis 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 T. vaginalis, 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 T. vaginalis genome. The developed RPA-ELONA has a detection limit of 4.03 × 10^-4 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 T. vaginalis and serves as a guide in further development of sandwich-based POCT for T. vaginalis.