SARS-CoV-2 Reference Standard Preparation

The SARS-CoV-2 Research Grate Test Material (RGTM 10169; National Institute of Standards and Technology, Gaithersburg, MD), consisting of two synthetic RNA fragments from the SARS-CoV-2 genome (including SARS-CoV-2 sequences 25949–29698 and 12409–15962 of isolate USA-WA1/2020) in a background of 5 ng/µL human Jurkat RNA, was diluted over six serial log dilutions with nuclease-free water for use as calibration standards. RNA template of SARS-CoV-2 Nucleocapsid Phosphoprotein (N protein) encapsulated in the MS2 bacteriophage construct (PerkinElmer, Waltham, MA) was prepared following manufacturer’s instructions for use as a positive control (PC). Single-use aliquots of reference RNA were stored in LoBind microcentrifuge tubes (Eppendorf, Hamburg, Germany) at –80°C.