A new qualitative RT-PCR assay detecting SARS-CoV-2

Area of Science:

• Molecular Biology
• Virology
• Infectious Diseases

Background:

• The SARS-CoV-2 pandemic created an urgent need for reliable COVID-19 diagnostic tests.
• Shortages of commercial diagnostic kits due to high demand posed a significant challenge for microbiology laboratories.
• Existing real-time PCR assays required careful consideration of material availability and personnel training.

Purpose of the Study:

• To develop a novel qualitative RT-PCR probe system for COVID-19 diagnosis.
• To overcome limitations associated with commercial diagnostic kit availability and closed-system designs.
• To provide a flexible and scalable diagnostic solution for global distribution.

Main Methods:

• Development of a qualitative RT-PCR probe targeting three SARS-CoV-2 genes: RNA-dependent RNA polymerase (RdRp), envelope (E), and nucleocapsid (N).
• Inclusion of the β-actin gene as an endogenous internal control for assay validity.
• Validation of the assay against a commercially available kit and determination of the limit of detection (LoD).

Main Results:

• The developed assay demonstrated complete agreement with a commercial kit, achieving a coincidence rate of 0.96.
• The limit of detection (LoD) was 140 copies/reaction for the N gene and 14 copies/reaction for RdRp and E genes.
• Two samples failed the endogenous internal control, highlighting its importance in assay interpretation.

Conclusions:

• The novel RT-PCR probe offers a reliable and accurate method for COVID-19 diagnosis.
• The open-system design allows for flexibility in nucleic acid extraction and RT-PCR platforms, facilitating broad instrument compatibility.
• The assay is designed for large-scale distribution, addressing global diagnostic needs and overcoming supply chain limitations.