Figure 1. Development of FIND: an enhanced RT-RPA based assay for detection of SARS- CoV-2.
One sentence summary: Sensitive, specific, rapid, scalable, enhanced isothermal amplification method for detecting SARS-CoV-2 from patient samples.
AbstractRapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. We developed a molecular diagnostic test for SARS-CoV-2, FIND (Fast Isothermal Nucleic acid Detection), based on an enhanced isothermal recombinase polymerase amplification reaction. FIND has a detection limit on patient samples close to that of RT-qPCR, requires minimal instrumentation, and is highly scalable and cheap. It can be performed in high throughput, does not cross-react with other common coronaviruses, avoids bottlenecks caused by the current worldwide shortage of RNA isolation kits, and takes ~45 minutes from sample collection to results. FIND can be adapted to future novel viruses in days once sequence is available.
We selected reverse transcription- recombinase polymerase amplification (RT-RPA) as the most promising current technology.
An optimized RT-RPA reaction allows for simple detection
Our optimized RT-RPA assay’s product can be hybridized and detected with a commercial lateral flow assay (LFA) without further amplification. LFAs allow accurate read-out by eye by minimally trained personnel, and even opens up the possibility of home-based testing (22). We chose to use Milenia Biotec HybriDetect lateral flow test strips that contain a streptavidin band, an anti-Ig band, and carry gold nanoparticle-labeled anti-FAM antibodies for visualization.