An Open One-Step RT-qPCR for SARS-CoV-2 detection.

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• The COVID-19 pandemic highlighted the need for accessible diagnostic tools.
• Real-time reverse transcription polymerase chain reaction (RT-PCR) is the gold standard for SARS-CoV-2 detection.
• Reagent restrictions and centralized production of diagnostic kits pose challenges for global accessibility.

Purpose of the Study:

• To develop a standardized, open-source One-Step RT-qPCR protocol for SARS-CoV-2 RNA detection.
• To evaluate the performance of purified recombinant enzymes and a non-proprietary buffer for this assay.
• To provide a cost-effective and accessible diagnostic solution for resource-limited settings.

Main Methods:

• Purification and testing of M-MLV RT and Taq DNA pol enzymes.
• Development of a Taqman probe-based One-Step RT-qPCR assay.
• Validation using synthetic RNA and 40 clinical samples (20 positive, 20 negative).

Main Results:

• The developed One-Step RT-qPCR protocol demonstrated sensitivity comparable to a commercial diagnostic kit.
• Diagnostic accuracy on clinical samples was confirmed to be equivalent to established methods.
• The protocol successfully utilized recombinant enzymes and a non-proprietary buffer system.

Conclusions:

• This study presents a proof of concept for an open-source approach to viral diagnostic kit development.
• The developed protocol offers a potentially cost-effective and accessible alternative for SARS-CoV-2 detection.
• This open approach could significantly benefit less developed countries in managing viral infections.