End-point RT-PCR based on a conservation landscape for SARS-COV-2 detection.

Area of Science:

• Molecular Biology
• Virology
• Bioinformatics

Background:

• Reverse transcription quantitative PCR (RT-qPCR) is the standard for SARS-CoV-2 detection but is costly and complex.
• Developing countries require accessible and affordable diagnostic tools for widespread disease surveillance.
• Emerging SARS-CoV-2 variants necessitate adaptable diagnostic strategies.

Purpose of the Study:

• To develop a cost-effective, large-scale diagnostic test for SARS-CoV-2 using end-point PCR.
• To create a bioinformatics approach for designing conserved PCR primers.
• To evaluate the accuracy and variant detection capabilities of the developed end-point PCR test.

Main Methods:

• A bioinformatics pipeline was used to identify conserved regions in SARS-CoV-2 and other respiratory pathogen genomes for primer design.
• End-point PCR primers were designed based on kmer frequency analysis.
• The developed assay was validated on 871 nasopharyngeal samples previously diagnosed using RT-qPCR.

Main Results:

• The end-point PCR test demonstrated high accuracy in detecting SARS-CoV-2, including variants like B.1.1.7, P.1, B.1.427/B.1.429, and B.1.617.2/AY.
• The assay achieved a detection limit of 7.2 viral copies/µL.
• The test successfully differentiated SARS-CoV-2 from other viruses causing COVID-19-like symptoms.

Conclusions:

• End-point RT-PCR is a viable and economical alternative to RT-qPCR for SARS-CoV-2 diagnosis, especially in resource-limited settings.
• The bioinformatics approach is adaptable for designing primers against new viral strains and variants.
• This end-point PCR test can be a valuable tool for ongoing epidemiological surveillance of SARS-CoV-2.