B-246 Innovative Controls for Molecular Diagnostics: Development of Recombinant Phage-like Particles that Encapsulates Target RNA viral gene segments

Background Developing diagnostic assays swiftly for emerging RNA viruses has been challenging , primarily because acquisition and propagation of these viral pathogens is often difficult and requires specialized laboratory set up such as BSL-3 or BSL-4. Methods We developed a non-infectious recombinant phage like particle containing RNA segments of Influenza A (M gene) and SARS-CoV2 (N gene) encapsulated in a viral capsid. A pETDUET plasmid vector was used to construct the His-tag recombinant phage capsid by inserting the phage coat and maturase genes along with the target RNA segments. The plasmid construct was transformed into E. coli-BL21 cells cultured in LB broth, and induced with Cinnabar Autoinduction Media (Teknova and Hollister, CA) to produce phage-like particles containing target RNA segments. The induced culture was treated with Benzonase, DNAse and RNAse and purified using TALON Cobalt Resin His-tag affinity method (Cytvia, Uppsala, Sweden). Results Droplet digital PCR assay showed that the final product (NATrVC-FC) contains 2.1E+11 copies/mL with 99.999% purity. The phage like particles remained stable for at least 12 months when stored at 2-8°C. Conclusion This method generates stable, recombinant phage-like particles that can be deployed to develop controls for emerging and or highly pathogenic RNA viruses quickly.