Highly Sensitive Lineage Discrimination of SARS-CoV-2 Variants through Allele-Specific Probe PCR.

Area of Science:

• Virology
• Molecular Biology
• Public Health

Background:

• Accurate detection of SARS-CoV-2 variants is crucial for managing the COVID-19 pandemic.
• Next-generation sequencing (NGS) is the standard for variant discrimination but has limitations with low viral loads or RNA quality.

Purpose of the Study:

• To develop and evaluate an allele-specific probe PCR (ASP-PCR) method for detecting SARS-CoV-2 variants of concern.
• To compare the performance of ASP-PCR against NGS for SARS-CoV-2 lineage discrimination.

Main Methods:

• Developed an ASP-PCR assay targeting lineage-specific single nucleotide polymorphisms (SNPs).
• Screened 1,082 clinical samples using ASP-PCR and compared results with 1,771 NGS results.
• Utilized probit regression models for performance comparison.

Main Results:

• Individual SNPs accurately identified specific SARS-CoV-2 variants of concern.
• ASP-PCR demonstrated higher accuracy than NGS in discriminating lineages, especially in samples with cycle threshold values between 26-30 and degraded RNA.
• ASP-PCR and NGS showed 99% concordance in results.

Conclusions:

• ASP-PCR is a valuable tool to augment, not replace, NGS for SARS-CoV-2 variant detection.
• ASP-PCR is particularly effective for screening samples with low viral loads or degraded RNA, supporting public health efforts.
• Further research into oligonucleotide chemistry could enhance ASP-PCR performance.