Liquifies saliva, lyses virus, extracts RNA for direct RT-qPCR testing in one step
High sensitivity reduces false negatives >30%
Liquified saliva enables automation
1-pipette protocol saves incubation time, consumables and kit costs
Hazard-free chemistry increases safety at reduced cost
To realise the advantages of Saliva Extraction Reagent (ER1) plus add viral inactivation, stabilisation and ambient transport at point of collection, see alternative PCR DIRECT Extracting Transport Medium
Collect saliva samples according to laboratory procedure, in dry tubes without transport media. For best results, we recommend freezing samples while in storage prior to processing.
Add 100 µl of sample to a 1.5ml low binding micro tube
Add 231µl of PCR DIRECT ER1 to the sample with the same pipette, mix ten (10) times
Heat sample for five (5) minutes at 70°C (Optional)
Sample is ready for downstream molecular analysis or processing
Note: Inadequate mixing in step 2 will result in lower sensitivity. Mixing by pipetting is preferred
Nucleic acid yield and processing efficiency may vary depending on the virus extracted. Samples prepared with the PCR DIRECT ER1 should be used in the intended downstream processes within 4 hours of preparation or frozen if intended for use within 24 hours. Freeze-thaw cycles after adding PCR DIRECT ER1 should be limited to 2 or fewer. For longer term storage of RNA, we recommend the addition of an RNase inhibitor in the amount recommended by the manufacturer to ensure long term stability. PCR DIRECT Saliva Extraction Reagent is applied in the lab.
This protocol may be adjusted for larger sample volumes by scaling the volume of PCR DIRECT ER1 in proportion to the increase in sample volume.
Saliva Extraction Reagent liquifies saliva on contact
PCR DIRECT Saliva Extraction Reagent is added to saliva that was freshly collected via the spitting method. Initially, the saliva is viscous, filamentous in nature and adheres to the sides of the tube. These properties result in inaccurate pipetting, increased contamination risk and drive manual-only processing. After the addition of PCR DIRECT, the saliva is transformed into a low-viscosity fluid.
Improved saliva pipetting accuracy
The volume transferred was measured relative to the target volume. Results are averages of 32 replicates for saliva mixed with PCR DIRECT and 8 replicates for raw saliva.
Saliva samples were mixed by pipetting a ratio of 100 μL saliva to 231 μL reagent
An automated liquid handler (OT-2, Opentrons) attempts to aspirate saliva that was freshly collected via spitting. Before the addition of PCR DIRECT, long filaments remain attached to the pipette tips. After the addition of PCR DIRECT, the automated pipettors easily aspirate the liquified saliva.
To quantify the impact on accurate pipetting, the volume of aspirated saliva and saliva treated with PCR DIRECT was measured by increasing volumetric aspirations with a micropipettor. This was then converted into a percentage of target volume pipetted and is shown in the chart to the right. Without treating raw saliva, there is an approximately 30% reduction in aspirated versus target volume of saliva. This significant reduction in aspirated saliva would result in under sampling and subsequent losses in sensitivity.
PCR DIRECT Saliva Extraction Reagent delivers superior sensitivity in direct RT-qPCR
Saliva harbors nucleases, proteases and macromolecules that are inhibitory to PCR. Previous methods to liquify saliva have reduced this inhibition but result in poor downstream sensitivity, thus reducing utility for pathogen detection.
PCR DIRECT Saliva Extraction Reagent inactivates and sequesters PCR inhibitors resulting in best-in-class direct RT-PCR testing. As seen below, and PCR DIRECT resulted in a limit of detection of 4200 SARS-CoV-2 genome copies/ml, which is up to 30% more sensitive than the next most sensitive alternatives. This improvement in sensitivity would reduce false negative calls by greater than 33% based on observed viral load in SARS-CoV-2 positive patients.
Limit of detection for PCR DIRECT Saliva Extraction Reagent was established using heat-inactivated SARS-CoV-2 (BEI Resources) spiked into raw saliva. 5 µl of PCR DIRECT Saliva Extraction Reagent was added to the RT-PCR reaction (Taqman® Fast Virus 1-Step Master Mix). Data for non-Arcis methods is in public domain and taken from IFUs, EUAs or other manufacturer produced studies.