Diagnostic assays that use the 2019-nCoV-N1 primer-probe are unlikely to be impacted by Omicron variant

In a research posted to the medRxiv* pre-print server, a staff of researchers evaluated the efficiency of two totally different extreme acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic assays, each of which used one of many US Facilities for Illness Management and Prevention (CDC) 2019-nCoV_N1 primer-probe set. The researchers aimed to detect the genomic materials of SARS-CoV-2 and its variants, such because the lately emerged variant of concern (VOC) Omicron.

Study: The Omicron variant mutation at position 28,311 in the SARS-CoV-2 N gene does not perturb CDC N1 target detection. Image Credit: Fit Ztudio/ShutterstockExamine: The Omicron variant mutation at place 28,311 within the SARS-CoV-2 N gene doesn’t perturb CDC N1 goal detection. Picture Credit score: Match Ztudio/Shutterstock

A lot of the presently used coronavirus illness 2019 (COVID-19) diagnostic exams make use of quantitative polymerase chain response (qPCR) assays to detect the SARS-CoV-2 genome in affected person samples. In February 2020, the CDC launched a qPCR-based laboratory take a look at referred to as the CDC 2019-Novel Coronavirus (2019-nCoV) that targets two websites on the SARS-CoV-2 Nucleocapsid (N) gene – 2019-nCoV_N1 and 2019-nCoV_N2. After being granted Emergency Use Authorization (EUA), these CDC probes have been included in a number of SARS-CoV-2 diagnostic assays. Notably, the 2019-nCoV_N1 probe overlaps an Omicron mutation inside the sequence focused by the fluorescent probe. 

Omicron has round 50 mutations inside its RNA genome which might adversely have an effect on the power of diagnostic assays to detect the SARS-CoV-2 in COVID-19-infected affected person samples, resulting in inconclusive or false-negative outcomes. It carries a C to U mutation at gene place 28,311, comparable to the third nucleotide from the 5’ finish of the 2019- nCoV_N1 probe goal sequence. 

The research 

The researchers of the current research evaluated how the 28,311 mutation impacts N1 gene detection to keep away from potential false-negative outcomes from samples with the Omicron variant. They used an in vitro transcribed (IVT) N gene RNA representing the wild sort and VOC of SARS-CoV-2 Omicron. Additional, they confirmed the scale and purity of those RNA samples through agarose gel. Then these RNA sequences have been amplified utilizing two totally different amplification protocols, and the amplification efficacy of N1 and N2 targets have been decided utilizing the NEB SARS-CoV-2 qPCR multiplex assay equipment that concurrently detected the N1 (HEX), N2 (FAM), and the human RNase P (RP) targets. 


The researchers noticed that the amplification was unperturbed by the Omicron mutation at place 28,311. The N2 primer-probe set served as a management to right variations in RNA template enter because the Omicron variant doesn’t have a mutation inside the area focused by the N2 probe. 

Cq values are inversely proportional to the quantity of goal nucleic acid in a pattern. The researchers noticed that the mutant RNA crossed threshold one cycle quicker than wild-type RNA on account of a barely greater enter quantity. After correcting the RNA enter quantity based mostly on the N2 goal, the distinction within the common Cq values for the N1 goal amplification between the mutant and wild-type RNA was lower than 0.2, properly inside regular day-to-day and user-to-user variation suggesting equivalence in amplification pace. The N gene mutation didn’t lower the assay sensitivity, as obvious from the outcomes detecting 10 RNA copies per response for each the wild-type and mutant RNA in 27 out of 27 samples.

Subsequent, the researchers used SalivaDirect, a simplified, and non-invasive SARS-CoV-2 diagnostic platform that additionally makes use of the CDC 2019-nCoV_N1 goal and human RP. The assay outcomes confirmed environment friendly amplification of each the mutant and wild-type RNA, and mutant RNA was detected barely quicker with a mean Cq worth of 0.71 on account of a better enter quantity of the mutant RNA. Furthermore, the assay was extremely delicate for the mutant N gene and the ten copies of enter RNA detected in 27 out of 27 samples.


The research information counsel that the diagnostic assays that use the 2019-nCoV-N1 primer-probe set effectively detected the mutant N1 sequence as effectively because the wild-type sequence. For the reason that Omicron variant incorporates a C to U mutation comparable to the third nucleotide place inside the CDC 2019-nCoV_N1 probe goal sequence, its mutation impacts assay sensitivity.

The current research used the Primer Monitor instrument to guage a number of mutations in SARS-CoV-2 variants, overlapping with the 2019-nCoV panel. A few of these mutations decreased assay sensitivity, whereas others, such because the mutations witnessed within the Delta variant, didn’t influence N gene assay sensitivity. As well as, they recognized a brand new variant circulating in Gauteng, South Africa, with a genetic mutation at place 28,320, which additionally overlaps the CDC 2019-nCoV_N1 probe. At present, this mutation doesn’t match with any revealed Omicron sequences, and extra testing is warranted to find out the influence of this mutation on probe detection. Consequently, the CDC has designed an alternate flu SC2 multiplex assay that may concurrently detect the N gene from SARS-CoV-2, influenza A, and influenza B.

To summarize, the research recommends continuous monitoring of the emergence of recent SARS-CoV-2 variant mutations and testing the efficiency of probes to make sure that COVID-19 diagnostic exams proceed to yield correct outcomes sooner or later.

*Necessary discover

medRxiv publishes preliminary scientific studies that aren’t peer-reviewed and, due to this fact, shouldn’t be thought to be conclusive, information scientific apply/health-related habits, or handled as established data.

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