J Clin Virol Plus . Detection of SARS-CoV-2 VOC-Omicron using commercial sample-to-answer real-time RT-PCR platforms and melting curve-based SNP assays

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J Clin Virol Plus


. 2022 Aug;2(3):100091.
doi: 10.1016/j.jcvp.2022.100091. Epub 2022 Jun 17.
Detection of SARS-CoV-2 VOC-Omicron using commercial sample-to-answer real-time RT-PCR platforms and melting curve-based SNP assays


Brian H M Sit ¹ , Kathy Hiu Laam Po ¹ , Yuk-Yam Cheung ¹ , Alan K L Tsang ¹ , Patricia K L Leung ¹ , J Zheng ¹ , Alison Y T Lam ¹ , Edman T K Lam ¹ , Ken H L Ng ¹ , Rickjason C W Chan ¹



Affiliations

• PMID: 35761832
• PMCID: PMC9213017
• DOI: 10.1016/j.jcvp.2022.100091

Abstract

Objectives: The World Health Organization (WHO) had designated the SARS-CoV-2 lineage B.1.1.529 as the new Variant of Concern Omicron (VOC-Omicron) on 26^th November 2021¹. Real-time reverse transcription polymerase chain reaction (RT-PCR), single nucleotide polymorphisms (SNP) and whole genome sequencing (WGS) tests were widely employed to detect SARS-CoV-2 and its variant. Yet, the SARS-CoV-2 Omicron detection performance of commercial real-time RT-PCR platforms and SARS-CoV-2 spike SNP assays remain to be elucidated.
Methods: In the first part of this study, we evaluated the VOC-Omicron detection performance of three commercial RT-PCR sample-to-answer platforms i.e. Roche cobas® 6800/8800, Roche cobas® Liat®, and Cepheid GeneXpert® systems. The detection performances were compared to one commercial conventional real-time RT-PCR assay (TIB MOLBIOL LightMix Modular SARS and Wuhan CoV E-gene) and one in-house real-time RT-PCR assay targeting RNA-dependent RNA polymerase (RdRP) gene of SARS-CoV-2 in the WHO COVID-19 Reference Laboratory at Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, The Government of the Hong Kong Special Administrative Region. In the second part of this study, we evaluated the SNP detection performance of four TIB MOLBIOL melting curve-based assays (1. Spike S371L/S373P, 2. Spike E484A, 3. Spike E484K and 4. Spike N501Y) in clinical samples obtained from hospitalized COVID-19 patients in Hong Kong. The SNP results were compared to whole genome sequences generated by Illumina platform.
Results: The VOC-Omicron detection limits of three commercial sample-to-answer assays were tested to be ≤ 2.35 Log₁₀ dC/ml. The detection performances of the sample-to-answer platforms were comparable to the two tested conventional real-time RT-PCR assays. The test sensitivities of TIB MOLBIOL VirSNiP SARS-CoV-2 Spike S371L/S373P assay and the Spike E484A assays were 100% and 96.6% respectively and the test specificities of both assays were 100%. An aberrant melting peak at Tm 42-44°C was observed when the specimens with Omicron variant were tested with the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike E484K assay. Notably, the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike N501Y assay failed to detect the spike N501Y mutation of Omicron variant in the tested specimens.
Conclusions: The SARS-CoV-2 detection sensitivity of three commercial platforms, Roche cobas® 6800/8800, Roche cobas® Liat®, and Cepheid GeneXpert® systems were shown not to be impacted by the large number of mutations of VOC-Omicron. Also, the signature mutations i.e. Spike S371L/Spike S373P and Spike E484A in VOC-Omicron were correctly identified by the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike S371L/S373P and VirSNiP SARS-CoV-2 Spike E484A assays. Unexpected findings including a shifted melting peak or absence of amplification curve/melting peak were observed when specimens with Omicron variant were tested with the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike E484K assay and Spike N501Y assay, suggesting a potential alert for Omicron variant, prior confirmation by whole genome sequencing.

Keywords: COVID-19; Coronavirus; Limit of Detection; Polymerase Chain Reaction; SARS-CoV-2; Single Nucleotide Polymorphism.