Reference RNA for this work was prepared by the Viral Pathogenesis Laboratory, Microbiology and Immunology Department, University of Otago, using a sample obtained from an infected patient in Dunedin, NZ (7 (link)). Briefly, the positive clinical specimen was inoculated into VERO cells and incubated for 3–7 days at 37°C, with 5% CO2. Culture material was inactivated using the Zymo ZR Viral RNA Kit™ (catalog number: R1035) and the RNA stored at −80°C. In addition, the Institute of Environmental Science and Research (ESR) laboratory has now successfully grown over 80 SARS-CoV-2 isolates for research with three of these isolates grown in substantial amounts to serve as reference material for NZ researchers.
A synthetic E-gene reference RNA was made for this work. A genome region downstream of the ORF3a gene stop codon, through the E gene, to the M gene start codon (302 bp) was synthetically generated and cloned into a pBluescript II KS(+) vector by Genscript (Piscataway, NJ, USA). RNA template was generated after linearising the plasmid by XhoI digest and subsequent in vitro transcription, from the T7 promoter, using the Invitrogen Maxiscript in-vitro transcription kit (ThermoFisher) according to the manufacturer's instructions. RNA quantity was measured using the Qubit RNA HS Assay kit (ThermoFisher).
A synthetic E-gene reference RNA was made for this work. A genome region downstream of the ORF3a gene stop codon, through the E gene, to the M gene start codon (302 bp) was synthetically generated and cloned into a pBluescript II KS(+) vector by Genscript (Piscataway, NJ, USA). RNA template was generated after linearising the plasmid by XhoI digest and subsequent in vitro transcription, from the T7 promoter, using the Invitrogen Maxiscript in-vitro transcription kit (ThermoFisher) according to the manufacturer's instructions. RNA quantity was measured using the Qubit RNA HS Assay kit (ThermoFisher).
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