Pretreatment strategies to eliminate free RNA/DNA signals from non-infectious microorganisms in molecular diagnostics

Overview

This solution addresses a critical challenge in molecular diagnostics for food safety, clinical testing, and environmental monitoring: false-positive signals caused by free or residual RNA/DNA from non-infectious microorganisms. Such false positives can trigger costly product recalls, damage brand reputation, and undermine confidence in diagnostic results. The proposed pretreatment strategies are applied prior to molecular testing (such as RT-qPCR) to selectively eliminate free genetic material associated with non-infectious microorganisms, enabling more accurate detection of only the active, infectious targets.

The research is grounded in deep expertise in food virology and bacterial biofilms, with a focus on detection, inactivation, epidemiology, and persistence. By removing the background signal from inactivated organisms, laboratories and food producers can achieve more reliable molecular testing outcomes and reduce unnecessary economic and operational impacts.

Technical specifications

Pretreatment approaches evaluated:

• Amicon centrifugal units (molecular cutoff-based separation)
• Silica beads (bind free RNA)
• PtCl4 (photoactivated RNA/DNA binding dye)
• PMA and PMAxx (photoactivated RNA/DNA binding dyes)
• DNase/RNase enzymatic treatments

Key findings from preliminary validation:

• Amicon, silica beads, and PtCl4 showed limited free RNA removal (less than 1 log reduction)
• PMA and PMAxx achieved significantly higher free RNA removal, with PMAxx reaching up to 6 logs of reduction
• PMAxx parameters including concentration, photoactivation time, and temperature have been optimized
• Viral plaque assays confirmed complete inactivation of infectious hepatitis A virus prior to RNA extraction
• The approach is compatible with standard RT-qPCR workflows

Adaptability:

The methodology can be extended to other microorganisms, including environmental and pathogenic bacteria, and other viruses. The research team has biosafety level 1 and 2 capabilities and is equipped to perform extraction and qPCR experiments across diverse sample types and matrices.

Technology readiness level

The technology is currently at an advanced validation stage. Preliminary data has demonstrated proof-of-concept for PMAxx-based removal of free viral RNA combined with inactivation techniques using hepatitis A virus as a model. Optimization of PMAxx parameters (concentration, photoactivation time, and temperature) has been completed. Future validation will extend the approach to environmental, clinical, and food samples, and to additional microbial targets including pathogenic bacteria and other viruses. The research team is actively seeking industry partnerships to co-develop new diagnostic strategies and improve safety testing workflows. The proposed study could be completed within approximately one year, depending on the number of microbial targets and sample types investigated.