Armored RNA as virus surrogate in a real-time reverse transcriptase PCR assay proficiency panel

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Biosafety

Background:

• Decentralization of high-consequence pathogen testing necessitates reliable, non-infectious training materials.
• Existing diagnostic networks require standardized surrogates for proficiency testing and capacity building.

Purpose of the Study:

• To develop and validate a noninfectious, quantifiable armored RNA surrogate for four high-consequence animal viruses.
• To assess the stability and performance of armored RNA in diagnostic workflows.

Main Methods:

• Engineered armored RNA containing targets for classical swine fever virus, foot-and-mouth disease virus, and two vesicular stomatitis virus serogroups.
• Spiked armored RNA into oral swab fluid and evaluated performance through RNA recovery, RT-PCR, and real-time detection.
• Assessed armored RNA stability under various shipping and storage conditions.

Main Results:

• Armored RNA successfully mimicked virus-positive samples through all RT-PCR stages, including RNA extraction and detection.
• Consistent performance was observed across four different commercial RNA extraction methods.
• Armored RNA demonstrated stability under simulated shipping and storage conditions (24h at 37°C, 2 weeks at -70°C).

Conclusions:

• Armored RNA serves as a viable noninfectious, quantifiable, and stable surrogate for assay training and proficiency testing.
• This development supports enhanced disease surveillance and surge capacity in decentralized laboratory networks.
• Validated armored RNA enhances the reliability and safety of diagnostic procedures for high-consequence animal pathogens.