Development of highly specific singleplex and multiplex real-time reverse transcription PCR assays for the identification of SARS-CoV-2 Omicron BA.1, BA.2 and Delta variants
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Abstract
The Omicron variant of SARS-CoV-2 (B.1.1.529), first identified during November 2021, is rapidly spreading throughout the world, replacing the previously dominant Delta variant. Omicron has a high number of mutations in the spike gene, some of which are associated with greatly increased transmissibility and immune evasion. The BA.1 sublineage has been most prevalent but there is recent evidence that the BA.2 sublineage is increasing in proportion in many countries. Genome sequencing is the gold standard for Omicron identification but is relatively slow, resource intensive, of limited capacity and often unavailable. We therefore developed a simple, rapid reverse transcription PCR (RT-PCR) method for sensitive and specific detection of the Omicron variant, including both the BA.1 and BA.2 sublineages. The assay targets a total of 5 nucleotide mutations in the receptor binding domain of the spike gene that give rise to 4 amino acid substitutions at G339D, S371L, S373P and S375F. The forward primer was designed as a double-mismatch allele specific primer (DMAS) with an additional artificial mismatch located four nucleotides from the 3’ end to enhance binding specificity. Assay specificity was confirmed by testing a wide range of previously-sequenced culture-derived viral isolates and clinical samples including the Alpha, Beta and Delta variants and ‘wild type’ SARS-CoV-2. Respiratory syncytial virus and influenza A were also tested. The assay can be run in singleplex format, or alternatively as a multiplex RT-PCR to enable Omicron and Delta variants to be detected and distinguished within the same reaction by means of probes labelled with different fluorescent dyes. Sublineages BA.1 and BA.2 can be differentiated if required. The methods presented here can readily be established in any PCR laboratory and should provide valuable support for epidemiologic surveillance of Omicron infections, particularly in those regions that lack extensive sequencing facilities.
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SciScore for 10.1101/2022.04.07.22273168: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Clinical samples: Residual, anonymised nasopharyngeal and oropharyngeal swab samples in virus transport medium were obtained from staff and students at Imperial College London, and from household contacts of individuals with COVID-19 (Research Ethics Committee reference Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Cell cultured viral isolates: SARS-CoV-2 RNA extracted from Vero cell supernatants was kindly provided by Professor Wendy Barclay, Department of Infectious Disease, Imperial College London. Verosuggested: …SciScore for 10.1101/2022.04.07.22273168: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Clinical samples: Residual, anonymised nasopharyngeal and oropharyngeal swab samples in virus transport medium were obtained from staff and students at Imperial College London, and from household contacts of individuals with COVID-19 (Research Ethics Committee reference Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Cell cultured viral isolates: SARS-CoV-2 RNA extracted from Vero cell supernatants was kindly provided by Professor Wendy Barclay, Department of Infectious Disease, Imperial College London. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)Software and Algorithms Sentences Resources Using alignments, performed by MEGA version 7.0.21, of SARS-CoV-2 spike gene sequences downloaded from the GISAID database (https://www.gisaid.org/), we searched for combinations of spike mutations which would permit the Omicron variant to be differentiated from all other variants of concern and from ‘wild type’ SARS-CoV-2. MEGAsuggested: (Mega BLAST, RRID:SCR_011920)Primers (Table 1) were checked by in silico PCR (https://genome.ucsc.edu/cgi-bin/hgPcr) to rule out cross reactivity with the human genome, and by NCBI BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cg) to exclude reactivity with other respiratory viruses including human coronaviruses 229E, OC43 and NL63. https://genome.ucsc.edu/cgi-bin/hgPcrsuggested: (In-Silico PCR, RRID:SCR_003089)BLASTnsuggested: (BLASTN, RRID:SCR_001598)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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Results from scite Reference Check: We found no unreliable references.
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