Rapid Detection of Predominant SARS-CoV-2 Variants Using Multiplex High-Resolution Melting Analysis.

Area of Science:

• Molecular Biology
• Virology
• Diagnostic Assays

Background:

• The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses a significant global health challenge.
• Accurate and rapid detection of these variants is crucial for effective public health interventions and disease control.
• Existing detection methods may be time-consuming, costly, or require specialized equipment like sequencers.

Purpose of the Study:

• To develop and validate a rapid, low-cost, and sequencing-free high-resolution melting (HRM) assay for direct SARS-CoV-2 variant detection.
• To assess the specificity, sensitivity, and clinical performance of the developed multiplex HRM assay.
• To provide a flexible and adaptable platform for identifying current and potentially novel SARS-CoV-2 variants.

Main Methods:

• A multiplex high-resolution melting (HRM) assay was designed to target specific mutations in SARS-CoV-2.
• Specificity was evaluated using a panel of 64 common respiratory pathogens.
• Sensitivity was determined through serial dilutions of viral isolates, and clinical performance was assessed using 324 patient samples, with results compared to reverse transcription-quantitative PCR (qRT-PCR) and Sanger sequencing.

Main Results:

• The multiplex HRM assay accurately identified SARS-CoV-2 and differentiated between mutations within approximately 2 hours.
• The limit of detection was below 10 copies/reaction for all targeted sites, demonstrating high sensitivity.
• The assay showed 97.9% agreement with Sanger sequencing for variant detection and exhibited no cross-reactivity with other pathogens.

Conclusions:

• The developed multiplex HRM assay is a rapid, reliable, and economical method for detecting SARS-CoV-2 variants.
• Its flexibility allows for potential adaptation to detect future emerging variants.
• This assay can aid in screening prevalent virus strains, monitoring epidemic trends, and informing prevention and control strategies.