**7. International status**

RT-qPCR assays were already known, but development can lead to better results. Thus, one study was done by targeting two monoplex assays recommended by WHO that was targeted on the envelope gene (E-Sarbeco) and RNA-Dependant RNA Polymerase coding genes (RdRp-IP4). This was combined and test named as 'Duo SARS-CoV-2 RT-qPCR assay'. As already lots of duo assays are commercially available, but this study was done to combine in-house assays. Can be used to reduce the chances of false negative results as it is dual assay [60]. As dual target assays are commercially available, developed specifically to

target multiple sites in the viral genome, but mutation rates in virus found to be moderate which can cause problem with respect to results. Some of the tests were done by using 'cobas SARS-CoV-2 E gene qRT-PCR' but some failures were seen due to change in nucleotide at position 26340 of the SARS-CoV-2 gene. So the study was done to identify single nucleotide polymorphism in E gene of the virus. This includes the importance of study regarding mutations in the virus and thus to prevent false negative results [61]. The protocol provided by WHO mention the three assay that is to be performed: First line screening assay - E gene assay, Confirmatory assay - RdRp gene assay, and Additional confirmatory assay - N gene assay [24]. 'Multiplex Real Time PCR' was also use to find its efficacy for SARS-CoV-2 detection. Found to be rapid and accurate method for viral detection [62]. 'Aus Diagnostics respiratory MT-PCR assay' was also found to be reliable and sensitive [63]. Two 'Single-tube nested (STN) real-time RT-PCR assays' specifically targets RdRp/Hel and N genes, found to be 100% specific for detection of SARS-CoV-2 [64]. Even high cost assays were given preference during a pandemic situation like TaqMan-based real-time RT-qPCR which was not even accessible to lots of laboratories. So it was performed by using 2 methods that are SYBR green RT-qPCR and conventional PCR basically standardisation was done of this 2 methods which is cost effective methods and found to give equal results as that of TaqMan-based real-time RT-qPCR [65].

COVID-19 diagnosis now done by one step RT-qPCR by using primers and probes which were developed at different institutes like China CDC, Charite (Germany), The University of Hong Kong, National Institute of Infectious Diseases in Japan (Japan NIID), National Institute of Health in Thailand (Thailand NIH) and US CDC which was announced by WHO [2, 24]. One study performed the analysis of primer-probe sets specifically targeting the N region and RdRp/Orf1 of SARS-CoV-2 by N-assay and RdRp/Orf1 Assays. Primer probe set for N-assay was N (China CDC), HKU-N (HKU), NIID\_2019-nCOV\_N (Japan NIID), WH-NIC N (Thailand NIH), and 2019-nCoV\_N1, -N2, and -N3 (US CDC) and for RdRp/Orf1 Assay RdRp\_SARSr (Charite), HKU-ORF1b-nsp14 (HKU), and ORF1ab (China CDC) primer probe set was studied. Results says that NIID\_2019-nCOV\_N" from the Japan NIID and "ORF1ab" from China CDC gave a good performance for RT-qPCR analysis without any cross-reactivity and nonspecific amplifications. This can be used for further diagnosis [66]. Sensitivity of PCR also depends on primer concentration, degeneration and multi target detection. Initial concentration of primer was 300-900 nM but as its concentration rises sensitivity also improves [67]. One study found that concentration upto 400 nM rise the sensitivity [68]. Degenerate primers plays an important role with respect to diversity of SARS-CoV-2. While screening assays with a single target area are more prone to sequence differences than dual or triple-target assays for multi-target identification [67]. Other than this primer length, melting temperature, GC content and annealing temperature also affects the sensitivity of PCR assay [67]. One study attempted to deduce the specific patterns among SARS-CoV-2 isolates and accordingly primers were design targeted to nsp2 gene and further use for diagnosis of probe free real-time RT-PCR. Sensitive and rapid SARS-CoV-2-specific real-time RT-PCR assay COVID-19-nsp2 has therefore been developed [69]. In one research, the efficiency of three novel real-time reverse transcription-PCR (RT-PCR) assays targeting RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S) and nucleocapsid (N) SARS-CoV-2 genes was developed and compared. RNA polymerase (RdRp)/(Hel) assay found to be effective with no cross reactivity with other viruses among samples [27]. Some of the RT-PCR kits manufactured at International level along with sample requirement has been shown in **Table 5**.


#### *Development of RT-PCR Based Diagnosis of SARS-CoV-2 DOI: http://dx.doi.org/10.5772/intechopen.96823*


**Table 5.**

*RT-PCR kits developed by International companies and sample requirement.*
