Viral inactivation and RNA/DNA extraction from swabs or saliva while in transit

  • Non-hazardous chemistry inactivates virus safely and avoids disposal costs

  • Nucleic acid extraction during ambient transport avoids costs of cold-chain, tips and tubes

  • Virus in swab or saliva arrives lysed, ready for direct RT-qPCR; compatible with bead or column-based isolation kits if concentration required

  • Liquifies saliva to enable automated liquid handling. Also boosts sensitivity, reducing false negatives >30% See images

  • Sequesters PCR inhibitors


    Apply at the point of collection with a dropper or pre-aliquot into tube of saliva collection device

ETM Product Packaging

UFL101 50 Preps £250
UFL105 500 Preps £1,785

Need

Sample preparation of nucleic acid from swab or saliva is a workflow spanning collection, pathogen inactivation, transportation, extraction and RNA/DNA isolation. These separate steps lead to:

  • Safety risks from live pathogens or hazardous chemicals
  • Less accurate results from nucleic acid degradation
  • Workflow inefficiencies due to added heat and purification steps

Solution

Extracting Transport Medium combines inactivation, transportation, and nucleic acid extraction into a safe single step  that takes out cost while increasing sensitivity

  • Viral inactivation with REACH-compliant, guanidine-free medium reduces risk from donor to bench
  • Lysis and stabilization at point of collection provides consistency
  • Liquifying saliva enables consistent, automated liquid handling.
  • For use with direct RT-qPCR or in nucleic acid isolation, with swab or saliva
  • Compatible with nucleic acid isolation kits

Use PCR DIRECT Extracting Transport Medium (ETM) with swabs or saliva with protocols below.  For use with direct RT-qPCR or in nucleic acid isolation.

Swab Protocol

Swabs, immediately after collection, should be placed into sterile collection tubes containing aliquoted PCR DIRECT ETM. We recommend preparing sample collection tubes ahead with 300 µl of PCR DIRECT ETM per tube. For maximum sensitivity in direct RT-qPCR PCR, the reaction may be prepared with up to 50% sample by volume (e.g., up to 12.5 µl sample in a 25 µl reaction). If a bead- or column-based isolation kit will be used, 200-300 µl of sample in PCR DIRECT ETM may be used for subsequent processing.

  1. Collect oral or nasal swabs according to laboratory procedure
  2. Place swab head into PCR DIRECT ETM; swirl and rotate swab head against the tube wall 10 times to extract the maximum amount of liquid from the swab head
  3. Begin to remove swab, press swab head against the side of the collection tube to maximize the amount of prepared lysate
  4. Shake tube vigorously at least ten times
  5. Sample is ready for transport and subsequent downstream molecular analysis or processing

Note: the head of the swab should be fully submerged in a minimal volume of PCR DIRECT ETM. Complete immersion will ensure that the entire collected sample has been lysed. Minimizing the volume of PCR DIRECT ETM ensures the highest concentration of nucleic acids and supporting direct PCR testing methods.

Saliva Protocol

For saliva samples, donors should be instructed not to consume food or drink, or use gum, toothpaste for at least 30 minutes prior to sample collection. Saliva should not be mixed with other transport media or lysis reagents prior to the addition of the PCR DIRECT ETM as this may affect results. This protocol may be adjusted for larger sample volumes by scaling the volume of PCR DIRECT ETM in proportion to the increase in sample volume. If a bead- or column-based isolation kit will be used, 200-400µl of sample in PCR DIRECT ETM may be used for subsequent processing.

  1. Add 100 µl of saliva to a 1.5 ml low binding micro tube
  2. Add 235 µl of PCR DIRECT ETM to the sample with the same pipette, mix ten (10) times
  3. Sample is ready for transport and subsequent downstream molecular analysis or processing

Note: Inadequate mixing in step 2 will result in lower sensitivity. Mixing by pipetting is preferred.

PCR DIRECT inactivates ≥ 99.99% coronavirus

Test Agent Negative Control PCR DIRECT
Contact Time (minutes) 0 30

Average Log recovery ±SD

(Log10TCID50mL-1)

5.96 ±0.10 ≤1.90 ±0.00

Average Log reduction ±SD

(Log10TCID50mL-1)

N/A ≥4.06 ±0.00
Percent reduction (%) N/A

≥99.99

PCR DIRECT ETM protects RNA for up to 4 days at ambient temperature

PCR DIRECT ETM lyses virus and donor cells, protects released RNA and DNA, and inhibits nucleases at ambient temperatures. To measure RNA stability, heat inactivated virus (BEI) was spiked into raw saliva and tested over four days by RT-qPCR. This stabilized samples can be transported without cold-chain to laboratories, where samples can safely reside within automated liquid handlers while RT-qPCR tests are assembled.

Improve automated liquid handling of saliva by removing viscosity

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 percentage of target volume pipetted and in 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.

 

Assessment of PCR DIRECT ETM against Human coronavirus 229E was performed by incubation at room temperature for 30 minutes. Following contact time, the suspensions were purified, and ten-fold serial dilutions (in triplicate) were evaluated for virucidal activity using TCID50.

In negative control samples, following 0 minutes incubation, an average of 5.96 ± 0.10 Log10TCID50mL-1 coronavirus 229E was observed. Following a 30 minute exposure, an average 1.90 ± 0.00 Log10TCID50mL-1 coronavirus 229E was observed. An average Log reduction of 4.06 ± 0.00 Log10TCID50mL-1 and an average percentage reduction of 99.99% was observed.

Comparison of saliva before and after PCR DIRECT

Improved saliva pipetting accuracy

The volume transferred was measured relative to the target volume. Results are averages of 32 replicates for saliva mixed with Arcis reagent and 8 replicates for raw saliva

PCR DIRECT ETM’s non-hazardous lysis formulation delivers superior sensitivity in direct RT-qPCR and improves sensitivity when used with isolation kits

Swab samples extracted with PCR DIRECT ETM are compatible with Direct RT-qPCR or isolation kits

Contrived SARS-CoV-2 samples were generated by collecting swabs and spiking with heat-inactivated SARS-CoV-2. The spiked swabs were placed in ETM or Saline/PBS and tested directly by RT-qPCR or after processing with a bead-based and a column-based RNA isolation kit.

ETM effectively lysed the virus, and delivered improved sensitivity when paired with a bead-based RNA isolation. Equivalent sensitivity was observed with a column-based isolation kit.

Saliva extracted with PCR DIRECT ETM are compatible with Direct RT-qPCR or isolation kits

Contrived SARS-CoV-2 samples were generated by collecting saliva by active spitting and spiking with heat-inactivated SARS-CoV-2. The spiked saliva was mixed with ETM or Saline/PBS and tested directly by RT-qPCR (using Thermo Fisher Taqman Fast Virus 1-Step Master Mix) or after processing with a bead-based and a column-based RNA isolation kit.

In all cases, ETM effectively liquified the saliva, lysed the virus, and delivered improved sensitivity when paired to both bead- and column-based RNA isolation.

Highest levels of sensitivity among direct collection methods

Saliva harbors numerous 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 in table, PCR DIRECT resulted in a limit of detection of 4,187 SARS-CoV-2 genome copies/ml, which is greater than 30% more sensitive than the next closest methodology. This improvement in sensitivity would reduce false negative calls by greater than 33% for the next closest method, based on observed viral load in SARS-CoV-2 positive patients.

Product code

UFL101, UFL105

Sample type

Swabs (oral, nasal, nasopharyngeal), Saliva

Sample input volume

Varies

Reaction time

Varies

No. of reactions

50, 500

Storage

Store at room temperature, do not freeze

Shipping conditions

Room temperature