# Development of a Protein-Free Nucleic Acid Lateral Flow Assay for

## TL;DR

Researchers developed a protein-free, point-of-care test for trichomoniasis that is specific, cost-effective, and suitable for use in resource-limited settings.

## Contribution

The novel use of aminated DNA probes in a nucleic acid lateral flow device eliminates the need for proteins in detection, enabling a simpler and more stable diagnostic test.

## Key findings

- The RPA-NALF device detects 1.3 × 10³ cells/mL visually or 282 cells/mL with a portable reader.
- The test is positive for all culture-positive clinical samples and for qPCR samples with Cq ≤ 23.
- The device has a shelf life of at least 6.6 months at 22 °C and is specific in the presence of other microbial DNA.

## Abstract

In line with the global research priorities on sexually
transmitted
infections (STIs), we developed a molecular point-of-care test (POCT)
for the parasite that causes the STI trichomoniasis. Trichomoniasis remains the most
common curable nonviral STI. We report on the use of specific single-stranded-tailed DNA primers in
combination with recombinase polymerase amplification (RPA) in a protein-free
nucleic acid lateral flow (NALF) device for use at the point of care.
The use of aminated DNA probes eliminates the need for proteins (such
as streptavidin or hapten-specific antibodies) for detection of the
DNA amplicons, simplifying the manufacturing process and improving
reproducibility and cost-effectiveness. The estimated shelf life of
the NALF devices is at least 6.6 months at 22 °C, and the devices
exhibited high reproducibility. The RPA-NALF requires three simple
operator steps with minimal instrumentation and takes approximately
30 min from sample preparation to interpretation of the result. It
is specific to and can
detect 1.3 × 103 cells/mL with visual readout or 282
cells/mL with the aid of a portable LFA reader. Analysis of the two
sets of clinical genomic DNA extracts showed that RPA-NALF is positive
for all samples positive by culture assay and for samples with Cq
≤ 23 with the S-DiaMGTV qPCR. Finally, RPA-NALF is positive
on biobanked vaginal swabs with Cq ≤ 25 with the Allplex qPCR assays. These results demonstrate
that RPA-NALF can specifically detect a moderate load of even in the presence of other microbial
DNA and cells and other components of clinical samples.