2.4∣Fluorescence detection
To further validate the visual detection accuracy, the signals of the
VDAP were quantified using a
fluorescence detection system. Briefly, 2 μL of the VDAP reaction
products were added to the plate and then the fluorescence intensity was
determined on a fluorescence microplate reader synergy H1 (Biotek
Instruments, Winooski, Vermont, USA) using excitation at 470 nm and
emission detection at 510 nm. In each replicate, fluorescence
intensities of VDAP reactions with various detection concentrations were
normalized against the one
containing the highest target concentration.
2.5∣ Real-time
RT-PCR
RNA was extracted from 300 μL nasopharyngeal swab samples in a
biological safety cabinet using the Bio-germ nucleic acid extraction and
purification Kits. Real-time RT-PCR was conducted for comparison with
the VDAP. Real-time RT-PCR assays
of N and ORF1ab genes of SARS-CoV-2 were used in accordance with the
respective manufacturers’ instructions (2019-nCoV detection kits from
fluorescence PCR, Shanghai Bio-germ medical science and Technology Co.,
Ltd, China). Reactions were
conducted in 25 μL volumes in an ABI 7500 real-time PCR system using 5
μL of nucleic acid. Usually, the
reaction cycle parameters were set as reverse transcription at 50°C for
10 min, denaturation at 95°C for 5 min, then followed by 45 two-step
cycles of 10 s at 95°C and 40 s at 55°C.
2.6∣Statistical Analysis
Statistic significance were calculated by GraphPad Prism (Vision8.0.2)
and all the data were shown as mean ± s.d. All illustrations were drawn
by GraphPad Prism. The bar chart and line chart were drawn with column
and group respectively. The heat map was initially drawn by Graphpad
Prism and adjusted by Adobe Illustrator. Two-tailed Mann-whitey U test
was used to compare two groups with a P value < 0.05 as
a threshold for significance. Three technical replicates were performed
to improve the statistics. Pearson correlation coefficient was used to
analyze the correlation.
3 ∣RESULTS
3.1∣Development
and optimization of VDAP
In this work, we first optimized the RPA assay system. Three front
primers and three reverse primers were designed according to the RPA
primer design principle, 9 pairs of primers were detected and then
selected one pair of primers with the highest amplification efficiency.
We first employed both RPA and VDAP reactions for 30 min at 37 ° C. High
and low concentrations of template (1.0×106 copies/μL,
1.0×104copies/μL) were employed and the
fluorescence intensities were measured to reflect the amplification
efficiency of each primer combination. The results of the RPA primer
combination assay for ORF1ab gene and N gene were shown in Figure 2A and
Figure 2B, respectively. The abscissa and the ordinate represent the
forward primers and the reverse primers, respectively. In each box, the
left side represents the result under the high concentration template
and the right side represents the result under the low concentration
template. The fluorescence intensity of primer combination (F3R1) was
significantly higher than the cutoff value (Figure 2A), and therefore,
this primer pair was used in subsequent experiments. We performed an RPA
primer screening test for the N gene using the same reaction conditions.
The amplification efficiency of primer combination (F1R2) was optimal at
both high and low template concentrations and exceeded the cutoff value
of 4.0 (Figure 2B). Therefore, F1R2 was used in subsequent experiments.
Next, to achieve better reaction sensitivity and efficiency, we studied
the effect of different reaction conditions on the results. Since the
RPA manufacturers had verified that the RPA used in the reaction system
had the best activity at 37°C, we focused on VADP by timing of RPA
amplification. The fluorescence intensity was examined after
RPA reaction at 37℃ for 0 min, 5
min, 10 min, 15 min, 20 min, 25 min and 30 min, respectively. It was
easy to find that the obvious fluorescence can also be seen by naked eye
(Figure 2C) and the normalized fluorescence intensity has exceeded the
cutoff value of 4.0 at 5 minutes
(Figure 2E). However, the fluorescence intensity remained essentially
unchanged after 15 min, which meant the reaction entered a plateau
period. Therefore, we chose 15 min as the optimal time for subsequent
RPA reactions.
Next, the reaction time of the VDAP was optimized.
The VDAP reactions were performed
using the same amplicon samples at 37 ° C for 0 min, 5 min, 10 min, 15
min, 20 min, 25 min, and 30min. The visual detection results and the
time-dependent curves of the VDAP reaction were shown in Figure 2D and
Figure 2F, respectively. The fluorescence intensity increased with time
significantly from 0 to 15 minutes, but it did not change with time
after 15 minutes (Figure 2F). Therefore, we chose 15 min as the reaction
time for subsequent VDAP.
Finally, the optimal temperature of the VDAP was explored at different
temperatures (from 27 to 52 °C) for 15 min. As shown in figure 2G, the
fluorescence intensity at 37°C was the highest at 15 minutes. Therefore,
37°C was used as the optimal reaction temperature for subsequent
studies.
3.2∣Limit of detection
and specificity of VDAP
After the VDAP reaction system was established and optimized, we used
standard substances to determine the limit of detection (LOD) and
specificity of the system. To identify the LOD of the VDAP assay,
SARS-CoV-2 standard substances were serially diluted 10-fold from 1.0 ×
107 to 1.0 × 101copies/μL.
All examinations were performed
in triplicate. We first quantified the fluorescence of the reaction
products, after which we set the
normalized fluorescence cutoff
for VDAP detection at 4.0. As shown in figure 3,
the normalized fluorescence was
above 4.0 and obvious fluorescence could be seen by naked eye. The
results showed that the LODs for ORF1ab gene and N gene were 70
copies/μL (Figure 3A, C)) and 500
copies/μL (Figure 3B, D), respectively.
Next, to demonstrate the specificity of the VDAP assay, we selected
four
standards as targets for the
specificity assay, including
SARS-CoV, parainfluenza virus,
influenza A virus and influenza B virus. The results were detected by
naked eye and the fluorescence detection system. As shown in figure 3E.
the detection results of the four standard samples in VDAP systems were
all negative, the normalized fluorescence was below the cutoff value and
no fluorescence was observed by naked eye. Therefore, the VDAP assay was
highly specific due to the fact that the CRISPR-based methods relied on
specific sequences for recognition, with no observed cross-reactivity
with other respiratory virus and suitable for the examination of
clinical specimens.