3.3∣Clinical validation of the optimized VDAP
Compared with the standard substances, there are a variety of
interfering substances in clinical samples, including human-derived
substances, contamination during sampling and other pathogens [11].
Next, the assay was further tested using
60 clinical samples.
Thirty SARS-CoV-2 positive samples and thirty negative samples verified
by RT-PCR. Among those
SARS-CoV-2 positive samples,
29 cases ORF1ab genes and 28 N
cases genes were detected by the assay (Figure 4A). For three undetected
genes, we found that two of them were from the same specimen. Among
those SARS-CoV-2 negative samples, 2 cases were diagnosed with influenza
A, 5 cases with influenza B, and 2 with respiratory syncytial virus.
Neither gene of the SARS-CoV-2 was detected by this method in those
negative samples and the results were in good agreement with RT-PCR
(Figure 4B). The results shown that the negative predictive value was
100% and the positive predictive value was 93.3% (Figure 4C). The
fluorescence intensity of above results was shown in Figure 4D. In
summary, the reaction system had high specificity, good
anti-interference ability and stability, so it was suitable for genetic
testing in clinical practice.
4∣DISCUSSION
In this study, RPA amplification and CRISPR reaction were combined to
detect the nucleic acid of
SARS-CoV-2, which had the advantages of high speed, high sensitivity,
high specificity, high stability and visualization. We first set up a
method of screening RPA primers, which was using high and low
concentration templates to detect the fluorescence intensity of
different primers and selected the primer combination with the highest
amplification efficiency after considering the results of two
experiments. In addition, agarose gel electrophoresis and Sanger
sequencing were used to verify the accuracy and purity of RPA products.
In this way, the following reaction system can be assured of high
sensitivity and reliability. Then we optimized the reaction conditions,
including RPA reaction time, VDAP reaction time and reaction
temperature. The VDAP reaction conditions were finally determined to be
isothermal at 37°C for 15 minutes.
Compared with RT-PCR, the method used in this study did not require
specific, expensive, and sophisticated equipment, and a simple water
bath or heat block is sufficient to provide a constant temperature for
reactions. In addition, the detection time was significantly shorter
than conventional RT-PCR. The greatest advantage of the method was the
visual interpretation by naked eye without any testing equipment for
positive and negative results. The results shown that the LODs of VDAP
for ORF1ab and N genes was 70 copies/μL and 500 copies/μL, respectively.
In terms of detection specificity, there was no cross reaction with
SARS-CoV, parainfluenza virus, influenza A virus and influenza B virus,
which was very helpful in distinguishing this group of clinically
similar human respiratory pathogens. The aforementioned viruses caused
clinical symptoms very similar to those after SARS-CoV-2 infection and
were sometimes difficult to identify [12-14]. The sequence of
coronavirus and SARS-CoV-2 also
have some homology, so some methods are easy to confuse them and cause
false positive such as antigen detection [10].
The detection of clinical samples tested the anti-interference ability,
specificity and robust of the reaction system. The method tested 30
SARS-CoV-2 negative samples with
a variety of common respiratory pathogens and did not fluoresce in any
samples. The detection rate of 30 positive samples was 93.7%.
Application of the new method has the potential to rapidly screen out
asymptomatic SARS-CoV-2-infected cases and thereby curb further spread
of the outbreak. RT-PCR is still the gold standard for the diagnosis
of SARS-CoV-2, but it has been
reported that the false negative reaction of RT-PCR can occur due to
individual differences and sampling sites and there is significant
difference in the detection rate between mild and severe
cases [15]. Compared with
PCR, although slightly less sensitive than PCR, the VDAP is simple,
rapid, does not require specialized laboratories and expensive
instruments, and is therefore more suitable for widespread development
in primary care settings. Reverse transcription loop-mediated isothermal
amplification (RT-LAMP) is a newly developed isothermal nucleic acid
amplification technique in recent years. Compared with the VDAP assay,
the primer design and selection were very strict [16]. Antigen
detection provides the possibility of home detection for potential
infected patients, but its sensitivity and specificity cannot meet the
diagnostic requirements [17]. Thereby, the VDAP assay provides a
rapid, inexpensive, and convenient assay for SARS-CoV-2 nucleic acid
detection and more easily developed for point of care testing
applications.