Validation of certified reference material
The sensitivity of the qRT-PCR kits for SARS-CoV-2 detection was evaluated using a recently developed certified reference material (CRM) that was derived from genomic RNA of the SARS-CoV-2 as the standard. The CRM has known concentrations of ORF 1ab, N and E gene, the three major targets used to detect the virus in qRT-PCR systems, although the specific amplification regions may differ among the various diagnostic laboratories.
Digital PCR was performed to validate the concentrations of the three targets in the reference material. In the dot plots, all positive reactions were obviously separated from the negative, and only a few smears appeared in the middle part, which indicated that the reaction systems were well optimized for quantification (Figure 1 A-C). The resulting RNA inputs for the three target genes quantified by digital PCR was 921 copies/μL for ORF 1ab, 1689 copies/μL for N and 1098 copies/μL for E gene, respectively, which was consistent with the reference values of concentrations with the measurement uncertainty of the CRM (Figure 1D). And the no reverse transcriptase control and no template control were negative. Thus, the reference material was applied for subsequent sensitivity analysis of the commercial qRT-PCR kits produced for SARS-CoV-2 detection.
Preliminary detection by qRT-PCR assays
In our preliminary test, the certified reference material was 10-fold serially diluted four times (S1–S4) with RNA storage solution and carrier RNA (4.5 to 4.5 x 103 copies per reaction for ORF 1ab and 8.7 to 8.7 x 103 copies per reaction for N gene). Dilutions S1–S4 were then used as the templates for qRT-PCR assays of the nine types of commercial qRT-PCR kits according to the manufacturer’s instructions.
The results revealed that all these test kits were capable to amplify the ORF 1ab and N gene fragments using samples from S1 to S3 (Figure 2, Additional file, while variations in detection rate between kits was found when testing lowest concentration of the dilutions. Among them, 5 of 9 kits were highly sensitive to detect the S4 sample in all replicates, and the assays using other 3 kits were partially positive. For the rest Kit-7, all tested replicates targeting ORF 1ab and N were negative (Table 4). This indicated that the analytical sensitivity varied markedly among these kits for SARS-CoV-2 detection. Based on the above result, three types of kits including two kits with high sensitivity level (Kit-1 and Kit-2) and one less sensitive Kit-7, were selected as the representative for further analysis. The amplification curves of assays generated by the three kits were shown in Figure 2.
Sequences alignment for target amplicons
To determine if the amplicons were matched to ORF 1ab and N sequences of SARS-CoV-2 sequence deposited in the GenBank, PCR products of the selected three kit targeting ORF 1ab and N genes were purified and sequenced. It demonstrated that for Kit-2 and Kit-7, the PCR products were quite consistent with amplicons amplified by the specific primers and probes for SARS-CoV-2 detection suggested by the Chinese Center for Disease Control and Prevention (CCDC). For Kit-1, both target fragments matched another region of the genomic sequence, without overlapping with those of other two kits. The amplification product targets for ORF 1ab were located at the 3’ end of ORF 1a and ORF 1ab, respectively (Figure 3A). Additionally, alignments of these amplicons with SARS-CoV-2 and SARS-CoV were complemented. The low divergence of sequences among primer binding domains (5’ and 3’ end of the amplification products) and the two virus (Figure 3B), suggested that these assays were difficult to discriminate the two types of coronavirus, as was the case in other reported qRT-PCR assays 9-10.
Standard curve and limit of quantification (LOQ) analysis
The Kit-1, Kit-2 and Kit-7 were further evaluated by standard curve generation based on quantification cycle (Cq) values of a new serial dilutions of the reference material. From the standard curves, the amplification efficiencies (E) of both targets using the three kits were ranged from 90% to 105% (R2 > 0.98), revealing that all these reactions had been well optimized to balance both targets detection (Figure 4). The LOQ of the the three kits for ORF 1ab gene was 23, 23 and 61 copies per reaction, respectively. While for the N gene, it was 26,26 and 78 copies per reaction, respectively. Thus, compared with other two kits, the kit-7 displayed poor linearity range for quantifying the target genes, which was in concordance with the above preliminary test results for analytical sensitivity.
Limit of detection (LOD) analysis
The reference material of the SARS-CoV-2 genomic RNA was initially diluted into the concentrations around S4 (4.5 copies per reaction for ORF 1ab and 8.7 copies per reaction for N gene) for the Kit-1 and Kit-2, as well as those between S3 and S4 for the Kit-7, which were estimated to be nearly at the detection end point. Probit analysis revealed a LOD of 3.5 copies for the ORF 1ab gene, and 5.6 copies for the N gene at 95% probability using kit-1 (Figure 5A). For kit-2, the LOD at 95% hit rate was 4.6 copies for ORF 1ab and 6.4 copies for N gene (Figure 5B). These results were roughly in line with the theoretical LOD of qRT-PCR reaction (3 molecules per reaction) when only sampling noise would contribute to replicate variation at 95% confidence according to the Poisson distribution 20-22. The discrepancy was probably caused by other factors in the real reaction conditions. While the resulting LOD for the Kit-7 was much higher, which was 14.3 copies for ORF 1ab and 20.4 for N assays (Figure 5C). Furthermore, for Kit-1 and Kit-2, the LOD values for both targets at 95% probability were less than 10 copies/reaction, demonstrating a sensitivity similar to that of the digital PCR.