Quantstudio 7 flex real time pcr detection equipment

The C36.3 lineage was confirmed by the presence of amino acid deletion HV69-70 and substitution L452R in the Spike gene using a multiplex real-time RT-PCR assay and the VIASURE (Certest Biotec SL, Zaragoza, Spain) SARS-CoV-2 detection kit on the Applied Biosystems QuantStudio 7 Flex Real-Time PCR detection equipment and QuantStudio Real-Time PCR software v.1.3 (Thermo Fisher Scientific, Waltham, MA, USA). Additionally, Alpha variant mutations were confirmed using the SNPsig-Variplex SARS-CoV-2 SNP genotyping panel with primers and probes designed to detect 20I/501Y.V1 (B.1.1.7), 20H/501Y.V2 (B.1.351), 20J/501Y.V3 (P1), and 20C/S.452R. Briefly, the amplification profile was: 10 min at 55 °C, 2 min at 95 °C, and 45 cycles, each consisting of 10 s at 95 °C and 60 s at 60 °C. All the mentioned procedures were done according to the manufacturer’s instructions. Delta lineage mutations were identified using the Thermo Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, USA) Applied Biosystems TaqMan SARS-CoV-2 Mutation Panel. Other SNP combinations, such as 501Y, 484K, and L452R and wild-types 501N, 484E, and 452L; deletion of amino acids H69 and V70, N501Y, E484K, and K417N; K417T, P681R, P681H, L452R, and K417N; and Q677H were identified with (Primerdesign Ltd., COVID-19 genesig, Chandler’s Ford, UK) following the manufacturer’s instructions.

The study examined 1076 nasopharyngeal (NP) swab samples obtained from visa applicants, tourists, and patients in isolation throughout Egypt between 1 January 2020 and 31 July 2021. Briefly, samples were collected with flocked nasopharyngeal swabs and immersed in viral transport medium for delivery to local governorate laboratories for testing with SARS-CoV-2 PCR protocols. The chemagic 360 equipment (PerkinElmer Inc, Waltham, United States) was used to extract nucleic acid from the clinical samples. The local laboratories governorates performed the initial SARS-CoV-2 qPCR screening using the Viasure SARS-CoV-2 Real-Time PCR Detection Kit (Certest Biotec SL, Zaragoza, Spain). The Applied Biosystems QuantStudio 7 Flex Real-Time PCR Detection equipment with QuantStudio Real-Time PCR software v.1.3 (Thermo Fisher Scientific, Waltham, MA, USA) was used for RT-PCR. The samples were then sent to the central public health laboratory (CPHL) under optimal storage conditions. All accepted samples had a cycle threshold (CT) value less than 35. Positive SARS-CoV-2 samples were confirmed at CPHL using the Cobas 6800 system (Roche Holding AG, Basel, Switzerland).