1∣INTRODUCTION
The coronavirus disease 2019
(COVID-19), caused by the severe
acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a
worldwide pandemic and rapidly spread. The disease causes a series of
non-specific symptoms such as fever, sleepiness and cough in infected
patients. However, subsequent studies have found that patients with
SARS-CoV-2 exhibit high heterogeneity in their symptoms. Mild cases can
be asymptomatic, while severe cases can have acute kidney injury, liver
dysfunction, acute respiratory distress syndrome and other systemic
organ dysfunction and eventually lead to death [1-2]. As of April
2022, SARS-CoV-2 has been spreading worldwide for 29 months, infecting
508 million people cumulatively. This pandemic poses an unprecedented
challenge to the global health system and the epidemiological
statistics. In order to control further spread of the disease, early or
asymptomatic infections must be screened and isolated in a timely
manner. Rapid, highly sensitive and inexpensive laboratory tests play an
important role in screening the infected patients. Currently, there are
two main SARS-CoV-2 detection methods applied in clinical practice. The
first is immunological diagnostic techniques represented by
immunochromatography assay (ICA) [3,4] and chemiluminescence
immunoassay (CLIA) [5]. The
second is molecular diagnostic technology represented by reverse
transcription-polymerase chain reaction (RT-PCR). The products based on
ICA are portable and fast, but its specificity and sensitivity are not
satisfactory. Compared with ICA, CLIA has improved specificity and
sensitivity and can achieve quantitative detection. However, the time
lag of antibody production in infected persons makes it difficult to
screen the positive ones in the early stage. Due to its unparalleled
analytic accuracy, RT-PCR remains the gold standard for molecular
diagnosis of the SARS-COV-2
[6]. However, RT-PCR is
usually restricted to
professional laboratories with specialized equipment and well-trained
personnel. This limits its application in some remote areas, especially
in areas with inadequate medical infrastructure, as well as in secondary
medical institutions in developed areas. Therefore, there is an urgent
need to develop a convenient
assay for the
fast and early detection of
SARS-CoV-2 in resource-limited
conditions. Compared with RT-PCR, recombinase polymerase amplification
(RPA) does not require a PCR machine, being more suitable for
point-of-care testing (POCT). It is known as a nucleic acid detection
technique that can replace PCR and has the advantages of rapid, high
sensitivity and isothermal [7, 8]. However, since there is no
thermal cycling to avoid binding between primers, it is difficult to
avoid non-specific amplification in RPA amplification. CRISPR/Cas
systems were reported in the bacterial genome for the first time and
utilized to detect nucleic acids [9-11]. Cas12 protein, also known
as Cpf1, specifically binds and cleaves single and double stranded DNA
under the mediation of guiding CRISPR RNA (crRNA). It is worth
mentioning that crRNAs recognize target sequences of amplicons, thereby
improving the specificity of amplification reactions. Here, we
develop the V isualD etection of RPA A mplified P roducts
(VDAP) to improve the sensitivity
and specificity of SARS-COV-2 detection (Figure 1). Briefly, RT-RPA
amplification was first performed on nucleic acids extracted from
nasopharyngeal swabs and then
visualized by the VDAP. In this
work, the quenched fluorescent single-stranded DNA (ssDNA) reporter was
introduced, which would be cleaved by Cas12a when target DNAs were
detected in the system, and the green fluorescence signal could be
detected under the blue light. It provides a rapid, inexpensive, stable
and convenient assay for
SARS-CoV-2 nucleic acid
detection, which has great potential for diagnosing COVID-19 and curbing
disease outbreaks.