In this study, CT values for the N, RDRP, and E targets were evaluated using qRT-PCR in order to detect SARS-CoV-2 in 100 clinical samples. It was observed that N gene has less Ct values (23.73±6.99) than those of E and RDRP. Moreover, our results show a significant difference among the E, N, and RDRP groups.
The diagnosis of SARS-CoV-2 using molecular tests is known as the gold standard method for the diagnosis of COVID-19 infection. Of note, the RT-PCR is a sensitive assay for the detection of SARS-CoV-2 RNA in clinical specimens (Chaimayo, Kaewnaphan et al. 2020). The study showed that after the onset of the disease’s symptoms, the SARS-CoV-2 viral load can be immediately observed in the upper respiratory tract and the antigen can also be detected in the first phase. However, some factors such as clinical manifestations, duration of disease to laboratory test, type of clinical sample, and sample collection procedure (technique process) can be effective on interpreting the results (Zou, Ruan et al. 2020).
In general, many developed laboratory methods use various tools, reagents, and targets in order to identify SRRS-COV-2 (LeBlanc, Gubbay et al. 2020). RDRP, E, and N are three targets proposed by WHO for the SARS-COV-2 identification (Corman¹, Landt et al. 2020). As well, the E gene is the first line screening, the RDRP gene is used as confirmatory test, and the N gene is used for a confirmatory testing, all of which are used in identifying the coronavirus. A previous study has shown that the RdRP_SARSr-P2 target could be specific for the coronavirus, and other probes are suitable for the detection of other types of coronavirus, and if false positive results are obtained regarding the diagnosis of Covid-19, it may possibly indicate that patients with mild symptoms are infected with other types of corona virus (Kakhki, Kakhki et al. 2020). Besides, evidence suggests that other targets such as ORF8 and specific primers / probes, may act as additional confirmatory tests in the diagnosis of SARS-COV-2 (kamali Kakhki, Aryan et al. 2020).
Houda et al. in their study have evaluated three genes of RDRP, N, and E in 187 COVID-19 samples and found gene expression as 22% and 40% in N and N, E genes, respectively. They have also shown that 6% of patients with both E and N genes and 14% of those with N gene still remained positive after a 12-day treatment period (Benrahma, Diawara et al. 2020). In addition, a study of 114 respiratory specimens has revealed that the N Ct value was more specific for laboratory diagnosis of SARS-CoV-2 (Abbasi, Tabaraei et al. 2021) .
Another study has shown that the one-step real-time RT-PCR can detect SARS-CoV-2 RNA in clinical specimens with a low detection sensitivity (Michel, Neumann et al. 2021). Since January 2020, protocols, tests, and reagents have been developed and introduced for the detection of SARS-COV-2. These laboratory tests that use SARS-CoV-2 RNA for the detection of COVID-19, were compared with commercial kits. A previous study using RT-PCR and two primers (N1 and N2) for SARS-COV-2 identification (Shirato, Nao et al. 2020) has shown that N2 primer has high specificity and sensitivity in this regard. These primers were also assessed using the following commercial kits: LN S & W-E, LN S & W-N, and LMW & RDRP (Hoehl, Rabenau et al. 2020). The results showed that the commercial LN S & W-N kit containing N primer was able to detect the virus better than the LN S & W-E (25 copies detected) and LMW & RDRP kits. It was observed that the LN S & WE targets are strongly conserved in the E gene region on SARS-COV and SARS-COV-2, while the N2 targets are a single region of N gene on SARS-COV-2 virus, so N2 is highly sensitive and specific for the detection of SARS-CoV-2 (Corman, Landt et al. 2020).
This study showed that the differences in gene expressions are associated with the genes of SARS-COV-2. Therefore, to reduce false negative results and to increase the sensitivity, diagnostic tests should be designed based on the targets that have the most differential expression. Correspondingly, RT-PCR method using of N, E, and RDRP targets is known as a reliable and accurate method for SARS-CoV-2 identification that can be used in infection’s prevention and control, and in diagnostic laboratories and medical centers.