Relative Sensitivity of Common Target Genes For The Detection of SARS-Cov-2 In Real Time-PCR

SARS-CoV-2(COVID-19) currently is the main cause of the severe acute respiratory disease and fatal outcomes in human beings worldwide. Several genes are used as targets for the detection of SARS-CoV-2, including the RDRP, N, and E genes. The present study aimed to determine the RDRP, N, and E genes expressions of SARS-CoV- 2 in clinical samples. For this purpose, 100 SARS-CoV-2 positive samples were collected from diagnostic laboratories of Mazandaran province, Iran. After RNA extraction, the real time RT-PCR assay was performed for differential gene expressions’ analysis of N, E, and RDRP. The CT values for N, RDRP, and E targets of 100 clinical samples for identifying SARS-CoV-2 were then evaluated using qRT-PCR. This result suggests N gene as a potential target for the detection of the SARS ‐ CoV ‐ 2, since it was observed to be highly expressed in the nasopharyngeal or oropharynges of COVID-19 patients (P < 0.0001). Herein, we showed that SARS-CoV- 2 genes were differentially expressed in the host cells. Therefore, to reduce obtaining false negative results and to increase the sensitivity of the available diagnostic tests, the target genes should be carefully selected based on the most expressed genes in the cells.


Introduction
SARS-CoV-2(COVID- 19), which is currently known as the global pandemic of Coronavirus, is responsible for the severe acute respiratory disease and fatal outcomes in human beings worldwide (Korber, Fischer et al. 2020). Coronaviruses as a group of enveloped viruses with positive-sense single-stranded RNA belong to the family Coronaviridae, which are able to spread between humans and animals (Holshue, DeBolt et al. 2020).
Unlike three previous epidemics of β-coronaviruses such as Middle East respiratory syndrome-related Coronavirus (MERS-CoV), severe acute respiratory syndrome Coronavirus (SARS-CoV), and severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), which have been potentially associated with acute respiratory distress syndrome (ARDS), most Human Coronaviruses (HCoV) such as OC43, HKU1, 229E, and NL63 cause a moderate upper respiratory infection in human (Hu, Lu et al. 2014). It has been shown that the mortality rate in more than 10,000 cases with both SARS-CoV and MERS-CoV was 10% and 37%, respectively (Prete, Favoino et al. 2020). Some previous studies have suggested that the epidemic potential of the COVID-19 outbreak with the fatality rate of 3.42% is higher than that of both SARS and MERS (Lai, Wang et al. 2020) (Del Rio and Malani 2020).
Coronaviruses ORFs encode four structural proteins, including S-spike, M-membrane, E-envelope, and Nnucleocapsid. Of note, several genes are used as targets identi cation such as the (RDRP and S), (N and S), and E genes. In this regard, studies have previously shown that SARS-COV-2 N protein is produced in large quantities in infected cells, which is related to the processes of replication, translation, and transcription. Moreover, it causes cell cycle deregulation, consequently inhibiting interferon production and inducing apoptosis (Astuti 2020). In order to have the best RT-PCR performance, the components of these targets should be optimized (Tombuloglu, Sabit et al. 2021). Accordingly, reverse transcription polymerase chain reaction (RT-PCR) using uorescent dyes is considered as a gold standard method for detecting bacterial and viral nucleic acid (DNA / RNA). RT-q PCR can also be used as a rapid and accurate assay for screening SARS-CoV-2 in throat samples, nasopharyngeal swabs, and feces (Chaimayo, Kaewnaphan et al. 2020 There are some commercial RT-PCR kits such as Primer Design (Chandler's Ford, UK), Seegene (Seoul, South Korea), and CerTest Biotec (Zaragoza, Spain), with different qualities, which are available to be used for the diagnosis of SARS-CoV-2 (van Kasteren, van Der Veer et al. 2020). According to this point that molecular diagnostic method for the diagnosis of SARS-COV-2 must have good sensitivity and speci city, so the present study attempted to analyze the RDRP, N, and E genes expressions of SARS-COV-2 using qRT-PCR through speci c primer pairs in the obtained clinical samples.

Multiplex primer and probe design
The speci c RT-qPCR primers and probe for the diagnosis of the target regions of the SARS-CoV-2 were designed using the following programs: PrimerPooler, PrimerPlex, and Primer3 (Tombuloglu, Sabit et al. 2021). Moreover, 5' Fluorescein amidites (FAM)-labeled probe was designed for the SARS-CoV-2 RdRp/ N/RP, as well as Hypoxanthine Phosphoribosyltransferase (HPRT) and Yakkima yellow-labeled probe for the viral E gene, which were then synthesized (Fig.1). The sequence of each primer or probe is shown in Table 1. Thereafter, Quantitation experiments were conducted using RT-PCR instrument (StepOne TM Real-Time PCR System).

RNA Extraction from the Clinical Samples
As well, qPCR was performed as follows: 1. Reverse transcription was performed for 20 minutes at 50°C, 2. Inactivation of the reverse transcriptase was done for 10 minutes at 95°C.
3. PCR ampli cation was performed with 40 cycles for 15 seconds at 95°C and for 30 second at 58°C using StepOne TM Real-Time PCR

Statistical analysis
The obtained results were examined by determining the ampli cation curve of the target gene and the housekeeping gene. Continuous variables are indicated as means (standard deviation, SD). All the statistical analyses were performed using GraphPad Prism 8 software and p-values less than 0.001 were considered as statistically signi cant.

Results
In the present study, 100 respiratory samples were collected from nasopharyngeal (NP) and throat swabs in health-care centers of Mazandarn, Iran, from December 2020 to September 2021. Thereafter, Real-time RT-PCR, using E, RDRP, and N targets, was performed for RNA detection of SARS-CoV-2. Firstly, all the primers and probes were analyzed by simplex qRT-PCR. Prior to preparing the reactions, the qRT-PCR instrument was properly calibrated in order to achieve the best uorescent signal. The simplex reactions were then performed in triplicate for three viral E, N, and RDRP genes as well as internal control genes (HPRT and RP). The criteria for the diagnosis of positive, negative, and suspicious COVID-19 samples were as follows: (0 < CT < 37.00), (NO CT or CT ≥ 40.00), and (37.00 ≤ CT < 40.00), respectively.
The average cycle threshold (Ct) and ∆Ct value with standard deviations (SD) are shown in tables 2 and 3, and the comparative Ct performances of each assay are shown in Fig 2 and 3. In this research, HPRT and RP genes were used as internal controls. Indeed HPRT and RP had signi cantly increased expression level compared to other targets (including N, E, and RDRP) (P < 0.0001). Our ndings showed that no detectable difference exists between HPRT and RP internal controls. According to the comparison of ∆Ct values among N, E, and RDRP targets, the N gene expression level was found to be higher than that of E and RDRP genes. (P < 0.0001). As shown in Fig 4, there is no signi cant difference between E and N targets (0.611). The result of our study suggest N gene as the most sensitive target compared to E and RDRP for SARS-CoV-2 detection using RT-PCR.

Discussion
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 signi cant 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 rst 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 identi cation . As well, the E gene is the rst line screening, the RDRP gene is used as con rmatory test, and the N gene is used for a con rmatory testing, all of which are used in identifying the coronavirus. A previous study has shown that the RdRP_SARSr-P2 target 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 speci c 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 identi cation (Shirato, Nao et al. 2020) has shown that N2 primer has high speci city and sensitivity in this regard. These primers were also assessed using the following commercial kits: 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 identi cation that can be used in infection's prevention and control, and in diagnostic laboratories and medical centers.

Declarations
Ethics approval and consent to participate: Not applicable.  Tables   Table 1. The sequences and concentrations of primer and probe sets used in the PCR reactions.