High-quality RT-PCR with chemically modified RNA controls.

Area of Science:

• Molecular Biology
• Biochemistry
• Infectious Disease Diagnostics

Background:

• Real-time reverse-transcription polymerase chain reaction (RT-PCR) is crucial for detecting infectious diseases like COVID-19.
• Effective quality assurance in RT-PCR relies on appropriate positive and negative controls.
• Current commercial kits often use DNA controls, which fail to validate the critical reverse transcription step, risking false negatives.

Purpose of the Study:

• To address the limitations of DNA-based controls in RT-PCR kits.
• To develop and evaluate novel RNA-based controls for enhanced diagnostic accuracy.
• To improve the reliability of infectious disease detection through better quality control.

Main Methods:

• Development of chemically modified RNAs: phosphoroselenaote RNA (Se-RNA) and phosphorothioate RNA (S-RNA).
• Assessment of Se-RNA and S-RNA as templates for reverse transcription.
• Evaluation of the stability (thermo-, bio-, chemo-) and specificity of the modified RNAs.
• Testing the efficacy of Se-RNA and S-RNA as positive and negative controls in RT-PCR kits.

Main Results:

• Chemically modified RNAs (Se-RNA and S-RNA) exhibit high thermostability, biostability, and chemostability.
• These modified RNAs demonstrate high specificity.
• Se-RNA and S-RNA serve as effective templates for the reverse transcription process.
• The modified RNAs can function as both positive and negative controls for RT-PCR kits.

Conclusions:

• Phosphoroselenaote and phosphorothioate RNAs are suitable alternatives to DNA controls for RT-PCR.
• These novel RNA controls enhance the quality assurance of RT-PCR kits by validating the reverse transcription step.
• The use of Se-RNA and S-RNA can reduce the risk of false-negative results in infectious disease diagnostics, improving patient sample testing.