Infectious Disease Diagnostics and Differentiation of Viral vs. Bacterial Infections for Point of Care Applications

The following Technical Objectives (Objective 4 and a portion of Objective 6) are from a contract in budget negotiations with DTRA that is expected to be funded in October 2023. A rapid turnaround on the cost volume has been requested (by September 1, 2023.)

[Other portions of this new project include using our current Material Transfer Agreement with UTMB’s World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) to obtain de-activated viral extracts of various Dengue strains, and we may request an additional MTA with WRCEVA for similar viral extracts of other viral families.]

Importantly, this contract involves a sub-contract with UTMB’s Gregory Gray, MD [Professor in Infectious Disease Epidemiology in the Departments of Internal Medicine (Infectious Diseases), Microbiology & Immunology, and Global Health & Emerging Diseases] to collaborate on developing a pan-viral detection system for the viral family Orthomyxoviridae, as well as work to refine GeneCapture’s previously developed pan-viral system for Coronaviridae.   

The work requested from the Gray lab will also require an MTA for the shipment of prepared viral particles from various virus strains from the families Coronaviridae or Orthomyxoviridae that have been de-activated and RNA-preserved by the addition of TRIzol reagent, or the shipment of purified de-activated viral RNA.  

3.3.4 TECHINCAL OBJECTIVE 4

Broad Pan-Viral Screening Performed by Principal Investigator (PI), GeneCapture’s Assay Team (Molecular Biologist, Assay Engineer, and Lab Technician) and the UTMB sub-contractors.

GeneCapture proposes to develop the design requirements for pan-viral REVEAL testing and specifically design degenerate primer sets for pan-viral detection of the viral family Orthomyxoviridae and to continue to refine its pan-Coronaviridae set.  Further funding will be required to extend this work to the other important viral groups to create a broad panel pan-viral screen.

3.3.4.1 Develop Broad Pan-Viral REVEAL Primer Sets

During this initial phase, GeneCapture shall modify the Gray lab’s conventional RT-PCR/PCR primer sets to design appropriate degenerate REVEAL primers or find new potential primer sets more suited for REVEAL for the respiratory viral family Orthomyxoviridae, which includes Influenza viruses. GeneCapture shall also refine the pan-viral Coronaviridae set previously developed.

Highly conserved regions across the viruses within each family shall be determined using proprietary GeneCapture software or commercially available software, such as Geneious, and candidates for REVEAL primers shall be selected based on minimizing degeneracy while producing amplicons between 100 and 250 nucleotides in length. Primer sets shall be tested against a single reference strain for each group and redesigned as required to achieve success.  Combining degenerate sets shall also be explored to determine the maximum coverage a single reaction well of REVEAL testing can accommodate.

In addition to viral family primers, GeneCapture shall design and revise positive control primers to a common control gene in mammalian cells, GAPDH or ß-tubulin, as the positive control for the assay.

3.3.4.2 Test Broad Pan-Viral REVEAL+CAPTURE Platform

The Gray lab shall prepare and ship either de-activated RNA extracts and/or viral particles from various virus serotypes de-activated and RNA-preserved by the addition of TRIzol reagent of four to six strains from each of the two viral families Coronaviridae and Orthomyxoviridae, for a total of 8-12 viral extracts at this stage of testing.

For de-activated and purified RNA extracts, 0.5 mL to 1.0 mL of extracts is requested.

For viral particles de-activated and RNA-preserved by the addition of TRIzol reagent, 2 mL to 6 mL of TRIzol mixture is requested.  

GeneCapture shall challenge the primer sets one family/genus at a time with the RNA from these strains using a manual version of the REVEAL assay with a Limit of Detection (LOD) goal of 10 GEq/µL for each virus within a 60 min test. The accuracy and precision of detection for each family shall be determined at 2-3x LOD.

3.3.6 TECHINCAL OBJECTIVE 6  

Preclinical and Mock Field Testing  Performed by Principal Investigator (PI), GeneCapture’s Engineering Manager (COO), and GeneCapture’s Assay Team (Systems Engineer, Assay Engineer, Biomedical Engineer, Product Engineer, and Lab Technician), UTMB Sub-contractor, and 3rd Party test site.

3.3.6.1 Bacterial versus Viral Testing

In house, GeneCapture shall challenge its novel pan-viral screen with up to four additional viral strains of importance to the DoD to extend the capability testing of that panel.  These strains will be provided to GeneCapture by the Gray lab as a set of six de-activated RNA extracts and/or viral particles from various virus serotypes de-activated and RNA-preserved by the addition of TRIzol reagent and shall include at least one negative sample.  The negative sample shall be either a true negative containing no RNA, or a sample of a viral agent that is not a member of the families Coronaviridae and Orthomyxoviridae.  Four of the positive samples should be strains that the test was not previously tested against.   The samples should contain RNA copy numbers in the range that GeneCapture has determined is detectable for each viral family.