Human Alphacoronavirus Universal Primers for Genome Amplification and Sequencing

Area of Science:

• Virology
• Genomics
• Molecular Biology

Background:

• Accurate viral genome sequencing is crucial for identifying genetic variations in pathogens.
• Low viral titers in clinical samples necessitate amplification for sequencing.
• Current lack of universal primers for alphacoronaviruses requires continuous development for diverse strains.

Purpose of the Study:

• To develop a universal primer set for all human alphacoronaviruses.
• To enable sequencing from samples with trace amounts of virus.
• To facilitate rapid diagnosis of emerging alphacoronavirus strains.

Main Methods:

• Design of overlapping primer pairs (AC primer set) covering the entire alphacoronavirus genome.
• PCR amplification of viral genomes from HCoV-229E and HCoV-NL63 strains.
• Evaluation of sequencing performance across various platforms.

Main Results:

• The AC primer set successfully amplified entire alphacoronavirus genomes.
• Complete genome sequences were assembled using different sequencing strategies.
• Sequencing error rates were below 0.01% across platforms.
• Detection limit in mock specimens was 10⁻³ PFU/ml (10² copies/ml).

Conclusions:

• The AC primer set is effective for sequencing all human alphacoronaviruses.
• The optimized procedure allows for sensitive detection and sequencing.
• This approach can support rapid diagnosis of mutant alphacoronaviruses during epidemics.