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Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly become a significant threat to public health. However, among the Coronaviridae family members, there are other viruses that can also cause infections in humans. Among these, severe acute respiratory syndrome (SARS-CoV) and Middle East respiratory syndrome (MERS-CoV) have posed significant threats to human health in the past. Other human pathogenic coronaviruses have been identified, and they are known to cause respiratory diseases with manifestations ranging from mild to severe. In this study, we evaluated the performance of a multiplex RT-rPCR specific to seven human pathogenic coronaviruses in mainly detecting SARS-CoV-2 directly from nasopharyngeal swabs obtained from suspected COVID-19 infected patients, while simultaneously detecting different human pathogenic coronaviruses in case these were also present. We tested 1195 clinical samples suspected of COVID-19 infection. The assay identified that 69% of the samples tested positive for SARS-CoV-2 (1195), which was confirmed using another SARS-CoV-2 RT-PCR kit available in our laboratory. None of these clinical samples were positive for SARS-CoV, MERS-CoV or HCoV. This means that during the endemic phase of COVID-19, infection with other human pathogenic coronaviruses, even the common cold coronavirus (HCoV), is very uncommon. Our study also confirmed that the multiplex RT-rPCR is a sensitive assay for detecting SARS-CoV-2 regardless of differences among the variants. This multiplex RT-rPCR is also time- and cost-saving and very easy to apply in the diagnostic laboratory due to its simple procedure and its stability in storage after preparation. These features make the assay a valuable approach in screening procedures for the rapid detection of SARS-CoV-2 and other human pathogenic coronaviruses that could affect public health.

Norovirus (NV) is considering a highly contagious virus due to the increase in the number of cases of the virus in recent years. Therefore, there is a need to develop rapid, specific and sensitive molecular detection and diagnosis methods. Using the data from the National Center of Bioinformatics Information (NCBI), the sequence of more than 1000 Norovirus genogroupII genome and 39 genome of geno group I were chosen to select a common conserve region for all genotypes within the first group and the second group suitable for the design of primers and probes. The design of the first area of the junction of Open Reading Frame one (ORF) and the open reading frame two (ORF2), and the primers and probes were tested on samples of compared to the primers and using one step reverse transcription real time PCR. The kit designed for multiplex reverse reaction possess no overlap or interaction in the components of primers and probes of the two genogroups, also it is superior on the monoplex mixture of the previous kit, Norovirus being diagnosed in 45% of samples by primers and probes designed in this study, 10% of the samples were belong to the genogroupI and 35% of the samples belong to the genogroupII in comparing with virus results that tested with monoplex previous primers and probes accounted 44% , the genogroupI represented 10% and genogroupII II represented 34% of the tested samples. The use of the multiplex kit contributes to shortening the time and the largest number of samples and reduce amount of the solutions used, more than the monoplex reaction, as it should be performed separately for each genetic group.