Thermolabile proteinase k

Q5 DNA Polymerase, Q5U DNA Polymerase, Vent (exo-) DNA polymerase, Sulfolobus DNA Polymerase IV, E. coli RNA Polymerase Holoenzyme, RNase I_f, Thermolabile Proteinase K, Thermolabile USER II Enzyme, T4 DNA Ligase, ET SSB, and Lambda Exonuclease were purchased from New England Biolabs (NEB). UltraPure BSA was purchased from Invitrogen. GreB was a gift from R. Landick (University of Wisconsin, Madison).

Clinical swab and saliva specimens were previously collected under Johns Hopkins IRB #00246027. Specimens were de-identified and blinded before testing. Nasopharyngeal swabs were eluted in 3 mL of Universal Transport Medium. Passive drooled saliva specimens were collected into an empty vessel. Five microliters of saliva was lysed with 50 μL of aqueous buffer containing 1% Triton X-100 and 1.2 units of Thermolabile Proteinase K (P8111S, New England Biolabs). 50 μL of swab eluate or 55 μL of saliva with lysis buffer was mixed with 150 μL magnetic bead binding buffer as previously described followed by loading the entire mixture into the sample port of the cartridge. The comparator assay for evaluation of clinical samples used a modified CDC testing protocol³² for swabs (Supplementary Methods, Supplementary Fig. 8) and FDA EUA authorized SalivaDirect protocol⁴³ for saliva were employed to test all the clinical specimens on a Bio-Rad CFX96 Touch Real-Time PCR System as reference.
Testing of SARS-CoV-2 variants used RNA from clinical samples extracted using a chemagic 360 instrument (PerkinElmer) with clades of each sample previously characterized by sequencing³¹ . 2 μL of extracted RNA was mixed with 150 μL magnetic bead binding buffer following by loading into the sample port of the cartridge.