B-217 Effective Diagnostic Platform and Method to Differentiate the Infectious From Non-Infectious Viruses With Nucleic Acid Detection Workflow
Authors: Zhang W , Boey E , Ma B , Phua D , Lim S 
Published: October 2024

Abstract

Background
While qPCR has demonstrated high sensitivity, specificity, and ability to deliver reliable quantitative data in clinical samples, such results do not differentiate between infectious and non-infectious viral particles. In view of the principle of the qPCR method itself, the amplification detects the nucleic acid (DNA/RNA) of the virus, not the virus itself. In many cases, it is still possible to detect DNA/RNA fragments from dead viruses in recovered patients, while they are not infectious anymore. This will lead to prolonged quarantine period or treatment for the patients, which could cause great psychological pressure to the patients and increased healthcare burden. Therefore, an effective detection method that can discriminate viable and inviable pathogens is of great significance.

Methods
SARS-CoV2 viral RNA, SARS-CoV-2 viruses, or clinical samples with SARS-CoV2 infection were diluted in Delta MTM (Molecular Transport Media) to the respective testing concentrations. The samples were incubated with intercalating dye (PMAxx 100nM) with or without Triton X-100 for 10 min. The mixture was then exposed to the photo-activation device for 5-15 min (@ temperature below 37° C). The samples were then subjected to the standard nucleic acid extraction & purification steps with Qiagen viral RNA extraction kit. The extracted RNA was subjected to amplification with SARS-CoV2 Resolute 2.0 & Fortunate 2.1 assays on BioRAD CFX96.

Results
The viability PCR (vPCR) efficiency was almost 100% for all SARS-CoV2 RNA input tested suggesting that without viral membrane protection, PMAxx dye effectively bound to the naked RNA after photoactivation process, and the bound RNA was not able to be amplified. The vPCR efficiency was further enhanced with addition of surfactant Triton X-100 suggesting the improved permeability of dye through the viral envelope facilitated the PMAxx binding to the nucleic acid. COVID19 positive clinical samples with viral load ≈ Ct 30 treated with or without PMAxx and photo-activation procedures showed distinctive ΔCt value ranging from 9.27±0.18 to 15.01±0.14. The Ct after dye treatment were all non-numeric (non-detectable) if cut-off Ct sets @ 42.0, indicating the complete inhibition after vPCR pretreatment. Since the clinical samples were undergone heat-inactivation pre-treatment, the result shown here indicating that all samples were non-infectious, which was consistent with the samples tested.

Conclusions
We developed an effective viability PCR detection method and proprietary vPCR device, which was specifically designed for effective dye photo-activation. In addition, the method takes less than 15 min and could be integrated into the existing standard nucleic acid extraction and amplification workflow. Comparing to the standard PCR test result, this method could provide the information not only on the positivity of the tested pathogen, but also its viability, which could be very essential information for public surveillance and diagnostic decisions for healthcare providers.