Abstract
Background: Currently, rapid and widespread testing is essential to
promote early intervention and curb the ongoing COVID-19 pandemic. The
reverse transcription-polymerase chain reaction (RT-PCR) for detecting
novel coronavirus (SARS-CoV-2) is restricted to professional
laboratories and well-trained personnel, thus, limiting its widespread
use in in resource-limited conditions. Methods: To overcome these
challenges, we developed a rapid and convenient assay using recombinase
polymerase amplification (RPA) and clustered regularly interspaced short
palindromic repeats (CRISPR) technology for the rapid detection
SARS-CoV-2, which was named as Visual Detection of RPA-amplified
Products (VDAP). The reaction conditions of the VDAP were optimized and
selected using pure SARS-CoV-2 RNA standards and the sensitivity and
specificity of the VDAP were further determined. Finally, the VDAP was
verified on clinical specimens. Results: The VDAP was performed at 37 °C
for 15 min, and the result was visible by the naked eye. The limits of
detection (LODs) of the VDAP for the target ORF1ab and N genes are 70
and 500 copies, respectively. No cross-reactivity was observed with the
RNA standard samples of four respiratory viruses or clinical samples of
common respiratory viral infections. These results confirmed that the
assay was highly specific. Thirty SARS-CoV-2 positive and 30 SARS-CoV-2
negative patient specimens were analyzed. We compared these results to
RT-PCR, the overall sensitivity and specificity of the VDAP compared to
RT-PCR for detection SARS-CoV-2 were 93.3% and 100.0%, respectively.
Conclusions: The VDAP is a simple, highly specific, and convenient assay
for the detection of SARS-CoV-2 in resource-limited conditions