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.