Proteinase k

Upon collection, approximately 500 µl of saliva was aliquoted into a 1.5 mL Eppendorf microcentrifuge tube pre-loaded with 100uL of lysis buffer containing 10 mg/mL of Proteinase K (AmericanBio, Canton, MA) to release viral RNA and 2 M Guanidine hydrochloride (Sigma Aldrich, St. Louis, MO) diluted in sterile PBS. Once collected, saliva samples were lysed for 1 min at 1500 rpm. The lysed saliva samples were subsequently heat inactivated for 5 min at 95 °C. After cooled down, the treated samples were subjected to SaliVISION RT-LAMP and Thermo Fisher Scientific TaqPath COVID-19 RT-PCR assays immediately or stores at − 20 °C until further processing.

A B. thetaiotaomicron strain expressing an HA-epitope tagged allele of the periplasmic protein SusA (Shipman et al., 1999 (link)) was grown to OD600 ~0.8 in minimal media with methionine, pelleted and washed in 1x cOmplete EDTA-free protease-inhibitor cocktail (Roche, Basel, Switzerland) before being pelleted and stored at −80 ˚C. Pellets were thawed and resuspended in PBS with proteinase K (0, 10, 50 or 100 µg/mL; AmericanBio, Natick, MA, USA), and incubated at 37 ˚C aerobically under continuous agitation (250 rpm) for 8 hr. Cells were then pelleted and washed 3 times in 1x cOmplete EDTA-free protease-inhibitor cocktail, pelleted and stored at −80 ˚C. Thawed cells were lysed using BugBuster reagent (Millipore Sigma, Burlington, MA, USA), 20 µg of clarified protein lysate was loaded onto an SDS-PAGE gel, transferred to a PVDF membrane and probed with rabbit anti-BtuG2 and rabbit anti-HA (Santa Cruz Biotechnology, Dallas, TX, USA).

For SARS-CoV-2 detection in saliva samples with the FDA EUA approved SalivaDirect assay, saliva specimens were aliquoted into 96-well plates, in the amount of 50 µl, and subsequently treated with 2.5 µl of 50 mg/mL proteinase K (AmericanBio, Canton, MA). Sample plates were shaken for 1 min, wherein they were then incubated at 95 °C for 5 min for proteinase K inactivation. After that, the RT-qPCR was performed using TaqPath 1-Step RT-qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA) with SalivaDirect primer and probe set in a total volume of 20 µl per reaction, of which 5 µl was for the saliva sample. The samples were run using a Bio-Rad CFX96 Touch qPCR cycler. SalivaDirect’s RT-PCR was used for viral nucleic acid detection with two probes: FAM probes for the presence of N1-gene amplicons, and Cy5 probes for the RNase P housekeeping gene. The threshold for positive samples was set at a Ct value of ≤ 40, in line with the SalivaDirect platform; however, exceptions were made based upon other result characteristics.