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Simultaneous detection of omicron and other SARS-CoV-2 variants by multiplex PCR MassARRAY technology
Supaporn Wacharapluesadee1, Piyapha Hirunpatrawong1, Sininat Petcharat1
1Thai Red Cross Emerging Infectious Diseases Clinical Center, King Chulalongkorn Memorial Hospital, Bangkok, Thailand.
Scientific Reports
|February 6, 2023
Summary
A new nucleotide matrix-assisted laser-desorption-ionization time-of-flight mass spectrophotometry (MALDI-TOF MS) assay, the point mutation array (PMA), rapidly identifies SARS-CoV-2 variants of concern (VOCs). This assay shows high sensitivity and concordance with Next Generation Sequencing (NGS) for variant detection.
Area of Science:
- Molecular Biology
- Virology
- Mass Spectrometry
- Infectious Disease Diagnostics
Background:
- Emergence of severe and transmissible SARS-CoV-2 variants necessitates rapid, multiplex molecular testing.
- Accurate identification of Variants of Concern (VOCs) is crucial for public health surveillance and response.
- Existing methods like Next Generation Sequencing (NGS) have limitations in speed and sensitivity for certain clinical scenarios.
Purpose of the Study:
- To develop and validate a novel assay for rapid identification of major SARS-CoV-2 VOCs.
- To assess the sensitivity and concordance of the developed assay compared to NGS.
- To evaluate the capability of the assay in detecting co-infections and differentiating Omicron subvariants.
Main Methods:
- Development of a nucleotide matrix-assisted laser-desorption-ionization time-of-flight mass spectrophotometry (MALDI-TOF MS)-based assay named Point Mutation Array (PMA).
- PMA panels (PMA-ABDO and PMA-Omicron) designed to target specific mutation sites in the SARS-CoV-2 spike gene.
- Validation using 256 clinical specimens, comparing PMA results with Next Generation Sequencing (NGS) and real-time PCR cycle threshold (Ct) values.
Main Results:
- The PMA assay accurately identified four major SARS-CoV-2 VOCs (Alpha, Beta, Delta, Omicron) and Omicron subvariants with a limit of detection as low as 10 viral copies/µl.
- 100% concordance was observed between PMA and NGS for specimens with real-time PCR Ct < 26.
- PMA demonstrated higher sensitivity than NGS for samples with Ct > 26, correctly identifying 25 out of 28 samples missed by NGS. The method can also detect co-infections/recombinations.
Conclusions:
- The PMA assay provides a rapid (<8 hours) and sensitive method for identifying known SARS-CoV-2 VOCs and Omicron subvariants.
- PMA offers advantages in sensitivity, particularly for samples with lower viral loads (higher Ct values), and can detect multiple variant infections.
- While PMA is highly effective for known variants, spike gene sequencing or NGS remains essential for detecting novel, emerging variants.
