RT-PCR amplification of RNA extracted from naso-oropharyngeal swabs collected in VTM from prospective patients was carried out in the Molecular Diagnostic Laboratory, which is an ISO 15189 and ISO 15190 accredited facility.
The naso-oropharyngeal samples were extracted using MagMAX™ Viral/Pathogen II nucleic acid isolation kit with a King Fisher Flex high-throughput automated extraction system (Thermo Fisher Scientific, Waltham, MA, USA).
For the reference assay, N1 and N2 gene targets with RNaseP as human internal control were amplified using primer probes suggested by CDC (Center for Disease Control). In brief, TaqPath™ 1-Step RT-qPCR mastermix (Thermo Fisher Scientific, Waltham, MA, USA) was used in ABI 7500 Fast DX instrument (Thermo Fisher Scientific, Waltham, MA, USA) in a 20 μL reaction mix containing 5 μL of the template. The thermocycler conditions consist of a step of reverse transcription (10 min/55 °C), an initial denaturation (1 min/95 °C), and subsequent 45 cycles of denaturation (10 s/95 °C) and annealing/elongation (30 s/55 °C) [7 (link),13 (link)]. This assay was considered as a reference RT-PCR assay comprising N1 and N2 CDC primers for COVID-19.
The naso-oropharyngeal samples were extracted using MagMAX™ Viral/Pathogen II nucleic acid isolation kit with a King Fisher Flex high-throughput automated extraction system (Thermo Fisher Scientific, Waltham, MA, USA).
For the reference assay, N1 and N2 gene targets with RNaseP as human internal control were amplified using primer probes suggested by CDC (Center for Disease Control). In brief, TaqPath™ 1-Step RT-qPCR mastermix (Thermo Fisher Scientific, Waltham, MA, USA) was used in ABI 7500 Fast DX instrument (Thermo Fisher Scientific, Waltham, MA, USA) in a 20 μL reaction mix containing 5 μL of the template. The thermocycler conditions consist of a step of reverse transcription (10 min/55 °C), an initial denaturation (1 min/95 °C), and subsequent 45 cycles of denaturation (10 s/95 °C) and annealing/elongation (30 s/55 °C) [7 (link),13 (link)]. This assay was considered as a reference RT-PCR assay comprising N1 and N2 CDC primers for COVID-19.
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