3.1 Development and characterization of SENA
To prepare appropriate crRNAs for SENA detection, we firstly determined the amplicon sequences from several commercial rRT-PCR kits used in China and then designed specific crRNAs corresponding to each of the distinct amplicons (Supplementary Table 1a). Candidate crRNAs were prepared and analyzed individually in a SENA system, which comprised of, in addition to the crRNAs, Cas12a, FQ-reporter and the rRT-PCR products using templates of either the positive or negative controls. The apparently most appropriate crRNAs, i.e., the lowest fluorescence with the negative control and highest with the positive control, were chosen for the final formulation of the SENA assay reagents (data not shown). In general, four formulations of regents were prepared employing crRNAs against corresponding assay targets, i.e., Orf1ab (abbreviated as O), E or N genes, individually, and the mixture of O and N (abbreviated as mix).
]. In order to mimic the clinical sampling for the titration experimentation, the RNA standards were serially diluted in buffer prepared by mixing the nucleic acid extracts from 40 COVID-19 negative people, generating RNA templates ranging from 0.025 to 25 copies per reaction (Rx).
], and extremely low, i.e., equal to and less than 1 copy/Rx. To overcome this sampling ambiguity problem, we performed 9 replicas for groups with 1 and 0.5 RNA template copies/Rx while 6 replicas for each of the rest concentrations. In addition, although the rRT-PCR assay supplier, BioGerm (BG, Shanghai, China), who follows the Chinese CDC recommended primer sets (Supplementary Table 1a), recommends 40 cycles of PCR amplification, we set 45 cycles as routine aiming at recording maximum exact Ct values if possible. After rRT-PCR reaction, all amplicons were subjected to 3 individual SENA reactions, i.e., N-SENA, O-SENA and mix-SENA with crRNAs targeting O gene, N gene and both, respectively.
] and the rigorous experimentation [
], along with the decrease of the RNA templates to less than 3 copies/Rx, the rRT-PCR Ct values in some replicas, primarily that corresponding to the N gene, passed 38 (the cut-off for positive as recommended by the rRT-PCR kit suppliers) but were less than 40, which should be considered as entering the “grey zone”. The Ct values increased steadily when the concentration of the RNA templates further decreased, with more and more replicas showing one or both Ct values entering the “grey zone” and eventually all became “negative”, i.e., greater than 40 or even 45 (Fig. 2a, Supplementary Table 2 and Supplementary Fig. 1). Employing Ct=38 as the cut-off for “positive” detection, we estimated the LoD for O and N genes with 95% confidence interval (CI) of this set of rRT-PCR assay as 3.3≤4.0≤6.1 and 4.0≤4.1≤4.4, respectively (Supplementary Fig. 2). Most likely due to the influence of the complex combination of the targeted viral genomic fragments and the clinical sampling background, the LoD determined in this study was clearly higher than the published value of 2.0≤2.5≤3.7, which analyzed single target in a pure system [
3.2 Verification of SENA in different clinical scenarios
Specifically, samples from 139 patients of Ruijin Hospital (RJ, Shanghai, China) were assayed by rRT-PCR employing diagnostic kits of Liferiver (LR) and Beijing Genomics Institute (BGI), 137 of which had consistent readouts by all those of rRT-PCR kits, indicating two positive, 123 negative and 12 suspected that fell in the “grey zone” (Supplementary Table 3). SENA detection of these samples revealed not only the 12 suspected as negative but also identified one more positive among the original 123 negative individuals, clearly a case of false negative diagnosis (Supplementary Table 3). Besides, distinct rRT-PCR assay results, positive by BGI but negative by LR were shown for samples collected from 2 close contacts of COVID-19 patients and apparently asymptomatic (ref to Supplementary Table 3). However, the amplicons of both LR and BGI were shown as negative via SENA detection. All these ambiguous rRT-PCR amplicons (17 samples, ref to Supplementary Table 3) were finally analyzed by NGS, and the results were consistent with the SENA. Noticeably, the rRT-PCR false-negative COVID-19 patient was symptomatically mild at the point of admission with all the clinical laboratory tests negative but turned positive after 24 hours. On the other hand, although those 12 suspected patients had respiratory infection symptoms, they were finally excluded from COVID-19 according to the latest guideline for diagnosis and treatment from China National Health commission (the 6th edition). Similarly, in Shenzhen Second People’s Hospital (SZII, Shenzhen, China), 5 uncertain rRT-PCR readouts for O gene were found among 139 individuals. Three of them had Ct value of 39.47, 39.7 and 40.56, respectively but the following SENA detection gave mix-FCratio values less than 1.0 for all of them, indicating all negative. The other two individuals had Ct values of 38.87 and 39.22, while their mix-FCratio values were 1.581 and 1609, respectively, indicating positive for both. In addition, there were another three individuals with Ct values larger than 40 for O gene and 36.09, 35.88 and 37.98 for N gene, respectively; however, the following SENA detection showed mix-FCratio values were 1.39, 1.55 and 1.21, respectively, indicating all positive. All these amplicons were further confirmed by NGS analysis (Supplementary Table 3), obtaining consistent results with those of SENA. Consistently, the three SENA-negative individuals were finally excluded from SARS-CoV-2 infection after being rechecked by rRT-PCR after 24 hours (Supplementary Table 3). Based on above data, it is clear, SARS-CoV-2 infection suspects with either rRT-PCR Ct values falling in the “grey zone” or with clear patient-contact epidemiological history but negative rRT-PCR tests, are strongly recommended to perform SENA detection to minimize the possibility of misdiagnosis. On the other hand, in case an rRT-PCR-positive suspect does not demonstrate any COVID-19 clinical symptoms and/or signs, SENA detection is also strongly recommended to eliminate either false-positive diagnosis or misdiagnosis of the so-called “asymptomatic carrier” or “asymptomatic patient”.
], the incomplete clearance of the SARS-CoV-2 virus ahead of discharge might be one of the possible causes. Therefore, it could be necessary to consider more sensitive detection approaches such as SENA as a potential index of viral clearance.