Journal: medRxiv

Article Title: COVID-19 Diagnostic Testing For All - Using Non-Dilutive Saliva Sample Collection, Stabilization and Ambient Transport Devices

doi: 10.1101/2021.01.20.20243782

Figure Lengend Snippet: Gamma-irradiated SARS-CoV-2 virus (BEI Resources) at 2 genome equivalents/uL was spiked in 1mL of saliva kept either in non-GTR STM (“Saliva”) (open circles) or GTR-STM devices (open diamonds), extracted with MagMAX Viral RNA Kit (ThermoFisher) and RT-PCR performed with CDC’s N1 primer. The pass/fail criteria set at 35.7 CT is 3 CT values more than the average CT value of the “Saliva” only samples. “Saliva” samples without GTR-STM gave a mean CT of 32.4 CT (Std Dev, ±0.3), and Saliva Samples in GTR-STM gave a mean CT of 32.7 CT (Std Dev, ±0.2). Study setup Experimental Sample : A contrived GTR-STM sample (n=9) was prepared by spiking 1mL of saliva with 2 genome equivalents/uL of -gamma-irradiated SARS-CoV-2 virus. Control sample : A contrived non-GTR STM (“Saliva”) (n=3) sample was prepared by spiking 1mL of saliva with 2 genome equivalents/uL of gamma-irradiated SARS-CoV-2 virus. Sample Extraction :RNA was extracted from 200uL of sample from both control and stressed samples following manufacturer’s instructions for MagMAX Viral RNA (ThermoFisher) manual protocol and eluted with 50uL of elution buffer. Quantification : Amplify 5uL of extracted RNA from each sample in triplicates with TaqPath master mix (ThermoFisher) and CDC’s N1 Primer (IDT).

Article Snippet: GTR-STM was tested by extraction with either QIAamp Viral RNA Mini Kit (Qiagen, cat #52906) or MagMAX Viral RNA Isolation Kit (ThermoFisher, cat #AM1939) and compared with either PBS or neat saliva as controls.

Techniques: Irradiation, Reverse Transcription Polymerase Chain Reaction

Journal: medRxiv

Article Title: COVID-19 Diagnostic Testing For All - Using Non-Dilutive Saliva Sample Collection, Stabilization and Ambient Transport Devices

doi: 10.1101/2021.01.20.20243782

Figure Lengend Snippet: Heat-inactivated SARS-CoV-2 virus RNA (BEI Resources) at 500 genome equivalents/uL was spiked into 1mL of saliva kept either in GTR-STM collection devices or non-GTR-STM (“Saliva”) tubes, and stored at 25°C for 36 days. Matched spiked control saliva samples in both kinds of tubes were stored at -80°C. The pass/fail criteria are set at 32 CT value. 200uL of the sample is used at each time point to extract viral RNA with the MagMAX kit with a final elution volume of 50uL. The CT value of the viral RNA extracted with MagMAX viral RNA kit is normalized to input volume of 200uL (volume of sample used for RNA extraction). All GTR-STM samples gave excellent recoveries when compared to their matched -80°C control. Both the -80°C and the 25°C samples for the non-GTR-STM (“Saliva”) is above the pass/fail line even in the -80°C control samples indicating that the viral RNA is degraded by the Rnase in the short time (less than half hour) that the saliva sample is defrosting before RNA extraction is performed. Study setup Experimental Samples : 1 mL aliquots of saliva contrived with SARS-CoV-2 at 500 genome equivalents/uL (BEI Resources) were placed into either GTR-STM devices or non-GTR-STM (“Saliva”) tubes and stored at ambient (25°C) for up to 36 days. Control sample : 1 mL aliquots of saliva contrived with SARS-CoV-2 at 500 genome equivalents/uL (BEI Resources) were placed into either GTR-STM devices or non-GTR-STM (“Saliva”) tubes and stored at -80°C for up to 36 days. Sample Extraction : RNA extracted from 200uL of experimental and control samples with MagMAX viral RNA kit at days 10, 15, 20, 25, and 36 and eluted in 50uL of elution buffer. Quantification : 5uL of RNA was quantified with CDC’s SARS-CoV-2 RT-qPCR assay for N1 primer.

Article Snippet: GTR-STM was tested by extraction with either QIAamp Viral RNA Mini Kit (Qiagen, cat #52906) or MagMAX Viral RNA Isolation Kit (ThermoFisher, cat #AM1939) and compared with either PBS or neat saliva as controls.

Techniques: RNA Extraction, Quantitative RT-PCR