With the emergence of the COVID-19 outbreak, many methods have been developed for the diagnosis of SARS-CoV-2 in the past year. These are mainly divided into nucleic acid-based amplification tests and serological tests. The nucleic acid-based amplification methods, such as rRT-PCR, are based on the amplification of viral RNA . On the other hand, serological tests are based on detecting either the proteins of the COVID-19 virus or human antibodies generated in response to infection such as the immunoglobulin type M (IgM) or immunoglobulin type G (IgG). The most important disadvantage of serological tests is their limited sensitivity to early detection . In general, the development of specific antibodies against the virus begins after the first week, and IgM and IgG production occurs mostly in the second week . Therefore, the sensitivity of serological tests is limited in the acute stage of infection. Another drawback is the possibility of similar antibody responses to the viruses in the same or close families and the possibility of cross-reactivity. This possibility is a serious concern as most human coronaviruses are antigenically close related to each other. Therefore, the WHO and CDC do not recommend the use of point-of-care immunodiagnostic tests for clinical decision-making. Instead, serological tests can be used for research or clinical support purposes [21–23].
Since rRT-PCR method is considered the gold standard in the diagnosis of SARS-CoV-2, WHO and CDC recommend it as the diagnostic test for asymptomatic and mildly symptomatic patients [21, 24]. However, rRT-PCR methods also have some drawbacks such as possible false-negative or false-positive results, the cost, etc . In order to eliminate or minimize those drawbacks, multiplex rRT-PCR methods have been developed that target more than one gene at the same time. By doing this, it is aimed to improve rRT-PCR efficiency and sensitivity. Until now, many studies have been conducted to find the method that can detect the SARS-CoV-2 RNA with the highest sensitivity. For this, different combinations of targeted viral genes were tested in multiplex. According to the WHO recommendations, four viral genes (RdRp, E, N and S genes) can be used in multiplex rRT-PCR reactions in different combinations . Along with these genes, primer and probe sequences of the human internal / positive control RNase P (RP) gene have been published and their use has been recommended by US CDC . The studies to improve these protocols are still ongoing. Designing primers with the highest sensitivity towards the target gene, eliminating their cross reactivity, minimizing possible false negative and positive results, optimizing rRT-PCR conditions are examples of what can be improved by those studies. It is worth mention here that recently, Dekker et al.  demonstrated a faulty design in RP primer sets defined by the CDC, which is another example of the importance of those improvement studies.
In this study, a multiplex rRT-PCR method has been developed that simultaneously targets the viral N2 and RdRP genes and the human RP gene. The primer and probe sets were designed to obtain the best PCR efficiency and target specificity. The rRT-PCR efficiencies of 10-fold dilutions series of the standards were > 99 for both N2 and RdRP genes, which matches the criteria for an efficient RT–qPCR assay . Besides, the current protocol allows the diagnosis of SARS-CoV-2 RNA with a limit of detection (LOD) value of 1.25 copies/µL or 5 copies/reaction for both N2 and RdRP genes. The LOD of the CDC’s 2019-nCoV Real-Time RT-PCR Diagnostic Panel was found to be 10 copies/µL. According to Vogels et al. , all SARS-CoV-2 primer and probe sets that has capacity to detect 500 copy/reaction can be used to diagnose SARS-CoV-2. Therefore, the current assay can be said to have a higher sensitivity than recommended and well-known assays.
The validation of the assay was tested in 28 SARS-CoV-2 positive samples. It is revealed that three samples out of 28 did not match with the results of the commercial kits. In both assays, either commercial or the current one, it is estimated that the Ct score of those negative samples was higher than > 37.01 which is out of the WHO recommendation . Therefore, in the current mCoV-2 assay, they are called as negative. Accordingly, the Ct value equals and lower than 37 is accepted as positive.
For COVID19, RT-PCR detection kits are commonly destined to amplify the genes S, E, N, RdRP, and ORF1a/b, but ORF1a/b and E were mostly applied [30, 31]. In China, Orf1ab and N genes are regularly used, while N1, N2 and N3 genes were utilized in US CDC and E, N, and RdRP genes in Europe . The importance of N1 and N2 primer-probes is for providing a less conservative but more sensitive than the RdRP-SARSr primer-probes especially in samples that have low viral titers [33, 34]. In a recent study, where swabs from confirmed cases were taken from nasopharynx and pharynx targeting ORF1ab and N genes yielded the best sensitivity when compared to positive confirmed samples . Chu et al.  have reported two assays that had the capability to achieve a large dynamic range and recommended targeting N gene for screening and the ORF1b gene to confirm the result. The abovementioned studies confirmed the capability of targeting N gene to be utilized in the detection of COVID-19 with other genes. While Li et al.  concluded that the more stable E gene is the target for the standardization of coronavirus tests, but N and RdRP genes are mostly targeted to confirm the results. In our study, it seems that targeting RdRP and N2 genes will make the test more sensitive. However, more studies are needed for fast and accurate COVID19 detection.