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