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.