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A Novel Innovative Molecular Diagnostic Platform for SARS-CoV-2 Based on GenSaver Cards

The ongoing global pandemic of SARS-CoV-2 has created unprecedented needs for SARS-CoV-2 diagnostic testing, from sample collection to safe transport of the collected samples to RNA processing and analysis. Currently, RT-qPCR assays are the gold standard for diagnosing SARS-CoV-2.  A key bottleneck for SARS-CoV-2 testing lies at the RNA extraction step.  TASA aims to address the RNA extraction bottleneck by developing a new method to simplify and accelerate SARS-CoV-2 RNA processing.

 

TASA has developed a new method that uses Dried Saliva Spot (DSS) sampling techniques and FastEx RNA Dried Saliva Spot Kit for isolating of viral SARS-CoV-2 RNA directly from Ahlstrom-Munksjö GenSaver™ Color 2.0 cards, providing molecular testing efficiency, stabilization, safe sample collection, RNA extraction, long-term RNA storage, retrieval, and biobanking, while making SARS-CoV-2 testing easier and more accessible for laboratories and enabling substantial expansion of SARS-CoV-2 testing and screening capacity around the world.  

 

In this study, we evaluated GenSaver Color 2.0 cards as a novel collection device for SARS-CoV-2 (COVID-19) and investigated whether the viral SARS-CoV-2 RNA (COVID-19 RNA) was stable on GenSaver Color 2.0 cards for the long-term preservation of RNA at ambient temperature.

METHODS

For our research study, saliva was collected from eight anonymous volunteers, who were also tested positive for SARS-CoV-2 using other methods. 0.4 mL of saliva was pipetted on GenSaver Color 2.0 cards, then dried for approximately 2 hours at 70°C.  The samples were then stored in multi-barrier pouches at ambient temperature in the dry-keeper desiccator cabinet. After storing the cards for a period of 1, 21, 30, 75, 90, 210, 240 and 365 days, the samples were processed to investigate SARS-CoV-2 RNA stability on GenSaver Color 2.0 cards at ambient temperature.

 

SARS-CoV-2 RNA was isolated from GenSaver Color 2.0 cards using the following protocol:

TASA RNA Purification and Elution Protocol

  1. Punch 4 disks (approximately 6 mm diameter) or cut 1 strip (approximately 2.0 cm x 0.6 cm) from the Sample Collection Card and place them into a 1.5 mL microtube.  

  2. Add 1.0 mL of TASA Dried Saliva Spot Purification Solution 1 and vortex gently several times for approximately 3 minutes. Pipette the TASA Dried Saliva Spot Purification Solution 1 up and down to further mix, then remove as much solution as possible. 

  3. To wash, add 1.0 mL of Nuclease-free Water and vortex gently several times for approximately 2 minutes. Discard the water completely. Repeat this washing step one more time.

  4. Add 0.4 mL of TASA Dried Saliva Spot Purification Solution 2 and vortex gently several times for over 2 minutes. Pipette the TASA Dried Saliva Spot Purification Solution 2 up and down one time using the 1.0 mL pre-cut pipette tips or wide bore pipette tips, then remove the solution completely.

  5. Add 60 µL of TASA Rapid RNA Elution Solution 1.

  6. Set the heat block to 85°C. Incubate for 10 minutes to release the RNA. Centrifuge briefly.

  7. Add 6 µL of TASA Rapid RNA Elution Solution 2.

  8. Recover the RNA by pressing the punches or strips to the bottom of tube with 200 µL pre-cut pipette tips or wide bore pipette tips.

      RNA is ready for use. 

WORKFLOW

SARS-CoV-2 RNA DSS-01.png

Real-time PCR Analysis

SARS-CoV-2 RNA yield is measured by amplifying the SARS-CoV-2 nucleocapsid gene N1 region and nucleocapsid gene N2 region, together with human RNase P nucleic acid as an endogenous internal control.

 

Primers and probe for N1:

  • N1-F1: GAC CCC AAA ATC AGC GAA AT

  • N1-R1: TCT GGT TAC TGC CAG TTG AAT CTG

  • N1-probe: FAM-ACC CCG CAT TAC GTT TGG TGG ACC -BHQ1.

 

Primers and probe for N2:

  • N2-F1: TTA CAA ACA TTG GCC GCA AA

  • N2-R1: GCG CGA CAT TCC GAA GAA 

  • N2-probe: FAM-ACA ATT TGC CCC: CAG CGC TTCAG-BHQ1.

 

Real Time PCR assay was performed with the Promega GoTaq® Probe 1- Step RT-qPCR System and the 7500 Fast Real-Time PCR System (Applied Biosystems). PCR conditions comprised of reverse transcription step at 45 °C for 5 minutes followed by an initial denaturation step at 95°C for 30 seconds and 45 cycles of denaturation at 95°C for 3 seconds, and annealing/elongation at 62°C for 30 seconds.  When performing quantitative real-time PCR analysis, mean CT value was calculated using the average of 3 x PCR replicates.

 

The Ct (threshold cycle) of the test sample was compared against a standard curve constructed using dilutions of the Twist synthetic SARS-CoV-2 RNA (1,000,000 copies/µL).

RESULTS

Eight saliva samples from anonymous SARS-CoV-2 positive volunteers were successfully processed and detected using TASA FastEx RNA Dried Saliva Spot Kit.

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SARS-CoV-2 RNA stored on GenSaver Color 2.0 cards remained stabilized and detectable up to 365 days at ambient temperature.

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Table 1 shows the Ct values of the released RNA from GenSaver card at day 1 compared to day 365 in which ΔCt values for samples 599, 600, 681 and 682 were 0.91, 1.12, 1.72, and 4.29, respectively, indicating strong RNA stability and preservation on GenSaver Color 2.0 cards.

Table 1:

P2 Table 2.png

Figure 1 and Figure 2 show SARS-CoV-2 RNA stability on GenSaver Color 2.0 cards at ambient temperature. In Figure 1, Ct values were obtained by Real Time PCR analysis during a period of 365 days. In Figure 2, Ct values were obtained by Real Time PCR analysis during a period of 90 days. There were insignificant changes in Cycle threshold values over a period of 365 days, suggesting strong stability of SARS-CoV-2 viral RNA on GenSaver Color 2.0 cards at ambient temperature and indicating consistent recovery of the viral RNA using TASA FastEx RNA Dried Saliva Spot Kit.

Figure 1:

F1 P2 SARS-CoV-2 RNA Stability.png

Figure 2:

F2 P2 SARS-CoV-2 RNA Stability.png

CONCLUSION

This study is the first evaluation of GenSaver Color 2.0 cards as a collection device for SARS-CoV-2 saliva samples.  TASA FastEx RNA Dried Saliva Spot Kit and GenSaver Color 2.0 cards together provide high quality RNA recovery, stability, and PCR sensitivity for detection of SARS-CoV-2 (COVID-19).  We have successfully detected SARS-CoV-2 RNA after 1 year of storage at ambient temperature.  

 

TASA’s novel, compelling approach of combining sample collection and RNA purification in one step makes molecular testing easier, cost effective, automation capable, and more accessible for laboratories of all sizes anywhere in the world, including urban as well as in rural and remote areas by enabling easy collection, transporting, rapid extraction and detection of SARS-CoV-2.  SARS-CoV-2 dried saliva samples are stable, preserved and protected from degrading and can be stored at ambient temperature for at least 1 year. 

 

TASA’s new method of rapid extraction and purification of RNA samples, combining with GenSaver Color 2.0 cards, is ideal for any research applications, including future emerging viruses, and is an excellent, reliable alternative to conventional methods for RNA collection, stabilization, and isolation.

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