RNA viruses often cause disease and impact public health including H1N1 influenza, Ebola, Zika, and COVID-19, all of which have caused recent pandemics. Therefore, rapid and accurate detection of these viruses in a fast, reliable, cost-effective manner is critical for early infection detection, surveillance of disease spread, and effective treatment. Our objective was to develop a highly sensitive, inhibitor-tolerant, and sustainable one-step RT-qPCR reagent for accurate RNA quantification with resistance to common inhibitors and compatibility with unpurified samples.

Methodology: To achieve our goal, we combined a novel, engineered reverse transcriptase (RT) possessing high thermostability and an innate tolerance to inhibitors with accessory enzymes and an optimized buffer system to deliver a highly sensitive and robust one-step RT-qPCR reagent system. eQo 1-Step ToughMix is delivered in a convenient, lyophilized format (eQo Beads) that can be shipped and stored at ambient temperatures, eliminating the cost and environmental impact of dry ice and foam packaging. To evaluate the performance of eQo 1-Step ToughMix, we tested the stringency of the warm-start RT, carryover contamination amplicon elimination, and inhibitor tolerance capabilities. Finally, we used CDC’s Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay and H5N1 assay to test the sensitivity and limits of pathogen detection for the eQo 1-Step ToughMix.

Results: eQo 1-Step ToughMix is stable at ambient temperature for up to one year in the lyophilized format and works as a standard 4X liquid mix after rehydration. Fully assembled reactions were stable at room temperature for 24 hours without causing off-target cDNA synthesis. Heat-labile UDG included in the master mix effectively eliminated carryover amplicons and the reagent showed improved resistance against common PCR inhibitors including mucin, heparin, hematic, humic acid, formaldehyde. Using several model sample systems and assays including the Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay from CDC, we demonstrated that eQo 1-Step ToughMix accurately quantifies pathogens in a reaction with a six-fold log dilution series and the limit of detection was as low as two copies of the target. In addition, pathogens were successfully detected directly from saliva and other sample types without traditional extraction processes. Finally, this reagent exhibited superior performance compared to other reagents for detecting H5N1 and several other pathogen RNAs

Conclusions: Overall, eQo 1-Step ToughMix offers a sustainable, robust, and highly sensitive solution for RNA pathogen detection, whether from pure RNA samples or directly from unpurified lysates.