A RT-qPCR system using a degenerate probe for specific identification and differentiation of SARS-CoV-2 Omicron (B.1.1.529) variants of concern

Abstract

Fast surveillance strategies are needed to control the spread of new emerging SARS-CoV-2 variants and gain time for evaluation of their pathogenic potential. This was essential for the Omicron variant (B.1.1.529) that replaced the Delta variant (B.1.617.2) and is currently the dominant SARS-CoV-2 variant circulating worldwide. RT-qPCR strategies complement whole genome sequencing, especially in resource lean countries, but mutations in the targeting primer and probe sequences of new emerging variants can lead to a failure of the existing RT-qPCRs. Here, we introduced an RT-qPCR platform for detecting the Delta- and the Omicron variant simultaneously using a degenerate probe targeting the key ΔH69/V70 mutation in the spike protein. By inclusion of the L452R mutation into the RT-qPCR platform, we could detect not only the Delta and the Omicron variants, but also the Omicron sub-lineages BA.1, BA.2 and BA.4/BA.5. The RT-qPCR platform was validated in small- and large-scale. It can easily be incorporated for continued monitoring of Omicron sub-lineages, and offers a fast adaption strategy of existing RT-qPCRs to detect new emerging SARS-CoV-2 variants using degenerate probes.

Discrimination of SARS-CoV-2 omicron variant and its lineages by rapid detection of immune-escape mutations in spike protein RBD using asymmetric PCR-based melting curve analysis

“…However, despite being promising approaches, these techniques require certain instrument platforms and have high requirements for the laboratory and the operators, which might be not applicable in many areas at present. Several multiplex quantitative RT-PCR assays were previously reported [ 23 , 24 , 26 , 29 ]. Jessen R. and colleagues used the degenerate probes to identify certain mutations, which required two probes for each mutation site to detect the wild type and mutant genotype, respectively.…”

“…Jessen R. and colleagues used the degenerate probes to identify certain mutations, which required two probes for each mutation site to detect the wild type and mutant genotype, respectively. The LOD of the multiplex PCR was around 50 copies/μL of RNA [ 26 ]. Xiong D. and colleagues combined the amplification refractory mutation system (ARMS) primers with TaqMan probes, and achieved an LOD as low as 1 copy/μL of RNA [ 29 ].…”

Tracking SARS-CoV-2 Omicron lineages using real-time reverse transcriptase PCR assays and prospective comparison with genome sequencing

“…Few studies have been published which describe rRT-PCR assays that detect and distinguish between BA.1, BA.2, BA.4, and BA.5. Jessen et al 50 developed two assays targeting S:ΔH69/V70 and S:L452R to detect these lineages and found that they had near perfect concordance with genome sequencing for 811 clinical specimens tested by both methods. However, these assays were unable to distinguish BA.4 and BA.5 50 .…”

“…Jessen et al 50 developed two assays targeting S:ΔH69/V70 and S:L452R to detect these lineages and found that they had near perfect concordance with genome sequencing for 811 clinical specimens tested by both methods. However, these assays were unable to distinguish BA.4 and BA.5 50 . Another assay that distinguishes the four main Omicron lineages targeting S:S371F/S373P/S375F, S:ΔG142/V143/Y144, S:ins214EPE, S:ΔH69/V70 and N:ΔE31/R32/S33, has been described and run as a pentaplex; evaluation of the assay was limited and performed using positive genomic RNA or gBlock material in duplicate for each lineage to confirm inclusivity and exclusivity for each marker 51 .…”

The Single-Nucleotide Polymorphism (SNP) Validity to Detect Omicron Variants

“…The result was placed into a clean wash tube (2 mL) and then rotated at maximum speed in microcentrifuges for 1 minute. The viral spin column results were placed into a 1.5 mL tube, and then, 10–15 μ L of sterile RNAse-free water was added [ 24 , 25 ].…”