RT-qPCR half-reaction optimization for the detection of SARS-CoV-2

Area of Science:

• Molecular Biology
• Virology
• Diagnostic Testing

Background:

• Reverse transcription real-time polymerase chain reaction (RT-qPCR) is the standard diagnostic test for COVID-19.
• The high cost of RT-qPCR, due to reagent and testing volume, presents a barrier to widespread implementation.
• Developing cost-effective diagnostic alternatives is crucial for managing the pandemic.

Purpose of the Study:

• To evaluate a modified RT-qPCR assay using half the recommended reaction volume for SARS-CoV-2 detection.
• To assess the economic viability and diagnostic performance of the reduced-volume assay.
• To determine if a lower-volume RT-qPCR can maintain accuracy in identifying SARS-CoV-2 RNA.

Main Methods:

• Analytical limit of detection (LoD) and reaction efficiency were assessed using synthetic RNA for N1 and N2 targets with half-volume RT-qPCR.
• Clinical sensitivity and specificity were determined using a panel of 76 SARS-CoV-2-positive and 26 negative samples.
• Comparison of half-volume reactions against standard-volume RT-qPCR was performed.

Main Results:

• The half-volume RT-qPCR assay demonstrated high efficiency (101-105%) for both N1 and N2 targets.
• The limit of detection for the half-reaction was 20 copies/µL for N1 and 80 copies/µL for N2.
• Clinical sensitivity and specificity reached 100%, with a decrease of up to 5.5 cycle thresholds compared to standard reactions.

Conclusions:

• The half-volume RT-qPCR assay is a feasible and economical alternative for SARS-CoV-2 RNA detection.
• This optimized assay maintains high diagnostic accuracy, offering a cost-effective solution for COVID-19 testing.
• Reduced reaction volumes in RT-qPCR can significantly lower diagnostic costs without compromising performance.