To improve test stability and accuracy of SARS-CoV-2 RNA and protein structures, Truckee Applied Genomics proposes COVID-19 test developers use TAG-NGPM™ (Next Generation Pathogen Media) a viral culturing transport media and TAG-NGPM+, a virus inactivating transport media as a replacements for all current specimen collection and transportation media including: Universal Transport Media (UTM), Viral Transport Media (VTM – including the CDC DIY media) and Zymo DNA/RNA Shield, liquid amies-based media (e.g., COPAN Eswab), nucleic acid transport media (e.g., Primestore MTM), 6 M guanidine-HCl, and saline solutions (normal saline and phosphate-buffered saline – PBS). 

TAG-NGPM is patent-pending and based on a subset of the broadly patented TAG-1 Class 1 general reagent that was designed as a tissue stabilization system to improve the quality and yield of Next Generation Sequencing (NGS) tests. This includes maintaining better cellular viral RNA integrity of both intracellular and extracellular substances and structures, enzyme activity, protein structures, and other metabolic functions, while also mediating pH and thermal stress in samples.

We propose that TAG-NGPM could serve as an excellent SARS-CoV-2 buffer reagent media for collection and transportation of samples to be used in PCR, rtPCR, RAA, and antigen tests. We propose that TAG-NGPM stabilized samples could provide better stabilization of RNA sequence and protein structures under more conditions.

Truckee Applied Genomics is now providing samples of TAG-NGPM to SARS-CoV-2 test developers on request. Contact NGPM@tagreagents.com.

Background

The initial use of TAG-1 was as a non-toxic direct replacement for formalin in processing solid tumor tissue. It was designed to require no pathology workflow changes, provide equivalent results in anatomical pathology tests, and improved results in current and emerging molecular pathology tests.

TAG-NGPM is based on a subset of the unique chemistry of TAG-1 and should support excellent stabilization of SARS-CoV-2 samples as follows:

1. Sodium Thiocyanate: Inhibits exogenous and endogenous proteases responsible for destruction of SARS-CoV-2 target RNA sequences. Sodium Thiocyanate combines with DMSO to produce an efficient pre-viral lysis step allowing for a more complete final sample lysis step resulting in better target amplification.
2. EDTA: Inhibits metalloproteases that have a direct negative effect on the nucleic acid target
3. DMSO: Drives cellular penetration and modulates viral cellular metabolic function when combined with EDTA. Concentrations can be varied to optimize PCR
4. Glycerol: Kosmotrope that stabilizes cell contents against thermodynamic stress and protects cellular proteins against damage when combined with aa-Trehalose. Additionally, adds strength to cellular components against environmental
5. Sodium Phosphate Buffers: Provides a biological pH system allowing for optimal cellular metabolic attenuation by components of TAG-NGPM
6. aa-Trehalose: Second kosmotrope that combines with glycerol to protect cellular regulatory proteins allowing increased cellular stability and preventing cellular autolytic activity.

Notes

1. Metabolically Intact Cells:  The key concept is that more metabolically intact cells improve diagnostic outcomes. Cellular components including proteins, surface antigens, DNA, RNA, mRNA, and regulatory enzymes, which may be destroyed or significantly degraded by environmental factors, are protected by TAG-NGPM.
2. Non-Toxic/Simple-to-Use:  TAG-NGPM is non-toxic, is not a bio-hazard, and therefore does not require special disposal protocols. The TAG-NGPM chemistry uses very low molar concentrations of components to accomplish the final endpoint of stabilized antigenic and RNA sequences for clinical analysis of SARS-CoV-2. 
3. Sample Integrity and Stability:  Sample integrity and stability are key to successful SARS-CoV-2 testing. TAG-NGPM and custom formulations have been specially designed to provide excellent sample integrity and stability across a broad spectrum of pathology
4. Virus Inactivation Option:  TAG-NGPM can also be supplied with optional features (TAG_NGPM+) to inactivate virus and prevent bacterial and fungal contamination of samples.

FAQs  

1. Is TAG-NGPM toxic? TAG-NGPM is non-toxic. Compared to sample media including guanidine-HCl, TAG-NGPM is non-toxic, using extremely low molar concentrations of components. TAG-1™, on which TAG-NGPM is based, has been labeled as a non-toxic Class 1 general reagent.
2. Can TAG-NGPM VTM be configured to inactivate virus? Yes, TAG-NGPM can be configured with a viricide to inactivate virus.
3. Can samples collected in TAG-NGPM VTM be used on assay platforms that include a bleaching step, such as the Hologic Panther or Fusion Systems, and some Diasorin systems without the same risk of producing cyanide gas as has been reported when using the PrimeStore MTM VTM, Zymo DNA/RNA Shield, as well as that found in the Spectrum Solutions Saliva Collection Devices? Unlike PrimeStore MTM, the patented TAG-NGPM system does not use guanidine thiocyanate, but rather uses very low molar concentration of sodium thiocyanate as a chaotrope to degrade nucleases and endonucleases.  A third party analytical chemistry lab has completed a study and issued a COA confirming no hazardous gases are produced when using TAG-NGPM+ as directed and as are typical lab protocols.
4. How does TAG-NGPM protect samples from temperature variances from collection to processing? TAG-NGPM has a dual buffering system designed to impart significant thermal stability. The dual buffering system protects against temperature driven pH changes that greatly degrade RNA quality. In addition, the TAG-NGPM system integrates the thermal stability properties of aa-trehalose, DMSO and glycerol in a way that amplifies the benefits of each.
5. How does TAG-NGPM protect samples against lytic cellular enzymes that degrade RNA? TAG-NGPM addresses endogenous and exogenous enzymes produced by both lytic and cellular regulatory activity by stabilizing pH. The TAG-NGPM dual buffering feature neutralizes pH which both decreases the rate of cell lysis and manages the negative impact of regulatory enzymes on lysed cells components to better protect target RNA sequences for amplification.
6. How does TAG-NGPM protect samples from changes in pH? TAG-NGPM includes a proven dual buffering system which neutralizes pH.
7. How can TAG-NGPM VTM be used in COVID-19 tests that have already received an FDA Emergency Use Authorization (EUA)? On July 20, 2020 the FDA provided guidance on substituting VTMs in EUA-approved COVID-19 tests. In summary, manufacturers may substitute a new VTM and maintain their EUA by internally conducting a minor bridging study, documenting results, and filing an email report with the FDA. A bridging study typically consists of establishing equivalent performance of the test when using the new vs. original VTM through a parallel testing of the two components. If the Limit of Detection (LoD) using the new VTM (TAG-1) is the same or better than the LoD for the unmodified EUA test, then the FDA guides that the two tests can be considered to have equivalent performance. The FDA would like to see the laboratory validation data through an email to CDRH-EUA-Templates@FDA.HHS.GOV. Once validated, developers can refer to the Conditions of Authorization section in their Letter of Authorization for instructions on how to update their labeling and Instructions for Use.
8. Why would TAG-NGPM work well for saliva-based tests? TAG-NGPM should work well in saliva for the following reasons. Saliva is a very rich enzyme matrix. TAG-NGPM for the reasons stated above, protects the RNA sequence targets from degradation. It should be noted the viral titers in saliva tend to change in various stages of the COVID-19 infection.
9. What samples types are suitable for using a TAG-NGPM VTM? Nasopharyngeal, oropharyngeal, and saliva.
10. How might TAG-NGPM VTM chemistry impact amplification in molecular assay tests that don’t require RNA extraction? Laboratories should be able to use an aliquot from the TAG-NGPM VTM solution to directly conduct an RT-PCR assay without initial nucleic acid extraction. The target RNA sequences used in PCR and rtPCR, are subject to degradation due to several factors. TAG-NGPM has been designed to mediate these factors. These factors are a broad range of enzymatic, thermal and pH related issues in the sample. TAG-NGPM addresses endogenous and exogenous enzymes produced by both lytic and cellular regulatory activity. The TAG-NGPM chemistry has been designed and tested to have optimal activity against lytic cellular enzymes that degrade RNA. TAG-NGPM additionally has a dual buffering system to impart significant thermal stability to the sample as well as changes to the pH caused by modified redox gradients and sample debris. Significant changes in ambient temperature and or sample processing times can have a significant effect on assay LoD. In assays that do not require an extraction step, the issue of viable target RNA is addressed by delivering intact target for analysis.
11. TAG-NGPM chemistry and RNA amplification: TAG-NGPM uses a methodology of low molar concentrations of chemistry which enables the desired cellular outcomes with no negative activity on the TAQ polymerases used in the PCR master mix for amplification. TAG-NGPM has been shown to have no adverse impact on PCR and rtPCR amplification. TAG-1 has not interfered with the PCR amplification processes used by researchers who have tested it at UC Davis, University of Trieste, and a major biotech. Additionally, TAG-NGPM has no known interference with amplification in RAA isothermal tests.
12. What vials and swabs are recommended for use with the TAG-NGPM VTM? The recommended vial is a 10 ml polypropylene tube with a 1.5-3 ml fill of TAG-NGPM chemistry. Flocked, unflocked, or film-coated Dacron or rayon swabs are recommended for kits with swabs.
13. What is the expected improvement to COVID-19 tests that use TAG-NGPM as a VTM as measured by lowering test Limit of Detection (LoD)? Many factors impact test LoD, but the central idea is “garbage in garbage out.” The  better quantity and quality target RNA fragments preserved from collection to processing, the more sensitive the test should perform. TAG-NGPM is a next generation pathogen media designed to improve VTM performance by better preserving samples from variances in time-to-processing, temperature variations, and pH variations all of which degrade RNA. In samples with low levels of RNA to begin with (for example saliva) stabilization of RNA is even more critical. Given some test samples are held for 24 to 48 hours or more at varying temperatures before processing, we propose that RNA quality and yield from collection to processing can be improved by up to 15% or more when using TAG-NGPM as a VTM vs. some other collection and transportation media. Further, we propose that as testing moves to use more NGS methods, the benefits of TAG-NGPM as a VTM will be amplified and proven through research use applications.
14. Is TAG-NGPM commercially available and at what price? Yes, TAG-NGPM is commercially available for pathogen test kit developers. Kit developers are responsible for validating their tests using TAG-NGPM as a substitute VTM per FDA guidance. Truckee Applied Genomics is committed to making TAG-NGPM broadly available at highly competitive prices, and with higher performance, as compared to any other VTM, MTM, and UTM. Contact info@tagreagents.com for a quote.