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

In a study posted to the medRxiv* pre-print server, a team of researchers evaluated the performance of two different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic assays, both of which used one of the US Centers for Disease Control and Prevention (CDC) 2019-nCoV_N1 primer-probe set. The researchers aimed to detect the genomic material of SARS-CoV-2 and its variants, such as the recently 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/ShutterstockStudy: 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/Shutterstock

Most of the currently used coronavirus disease 2019 (COVID-19) diagnostic tests employ quantitative polymerase chain reaction (qPCR) assays to detect the SARS-CoV-2 genome in patient samples. In February 2020, the CDC released a qPCR-based laboratory test called the CDC 2019-Novel Coronavirus (2019-nCoV) that targets two sites 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 incorporated in several SARS-CoV-2 diagnostic assays. Notably, the 2019-nCoV_N1 probe overlaps an Omicron mutation within the sequence targeted by the fluorescent probe. 

Omicron has around 50 mutations within its RNA genome which could adversely affect the ability of diagnostic assays to detect the SARS-CoV-2 in COVID-19-infected patient samples, leading to inconclusive or false-negative results. It carries a C to U mutation at gene position 28,311, corresponding to the third nucleotide from the 5’ end of the 2019- nCoV_N1 probe target sequence. 

The study 

The researchers of the present study evaluated how the 28,311 mutation impacts N1 gene detection to avoid potential false-negative results from samples with the Omicron variant. They used an in vitro transcribed (IVT) N gene RNA representing the wild type and VOC of SARS-CoV-2 Omicron. Further, they confirmed the size and purity of these RNA samples via agarose gel. Then these RNA sequences were amplified using two different amplification protocols, and the amplification efficacy of N1 and N2 targets were determined using the NEB SARS-CoV-2 qPCR multiplex assay kit that simultaneously detected the N1 (HEX), N2 (FAM), and the human RNase P (RP) targets. 

Results

The researchers observed that the amplification was unperturbed by the Omicron mutation at position 28,311. The N2 primer-probe set served as a control to correct differences in RNA template input as the Omicron variant does not have a mutation within the region targeted by the N2 probe. 

Cq values are inversely proportional to the amount of target nucleic acid in a sample. The researchers observed that the mutant RNA crossed threshold one cycle faster than wild-type RNA due to a slightly higher input amount. After correcting the RNA input amount based on the N2 target, the difference in the average Cq values for the N1 target amplification between the mutant and wild-type RNA was less than 0.2, well within normal day-to-day and user-to-user variation suggesting equivalence in amplification speed. The N gene mutation did not decrease the assay sensitivity, as apparent from the results detecting 10 RNA copies per reaction for both the wild-type and mutant RNA in 27 out of 27 samples.

Next, the researchers used SalivaDirect, a simplified, and non-invasive SARS-CoV-2 diagnostic platform that also uses the CDC 2019-nCoV_N1 target and human RP. The assay results showed efficient amplification of both the mutant and wild-type RNA, and mutant RNA was detected slightly faster with an average Cq value of 0.71 due to a higher input amount of the mutant RNA. Moreover, the assay was highly sensitive for the mutant N gene and the 10 copies of input RNA detected in 27 out of 27 samples.

Conclusions 

The study data suggest that the diagnostic assays that use the 2019-nCoV-N1 primer-probe set efficiently detected the mutant N1 sequence as efficiently as the wild-type sequence. Since the Omicron variant contains a C to U mutation corresponding to the 3rd nucleotide position within the CDC 2019-nCoV_N1 probe target sequence, its mutation impacts assay sensitivity.

The present study used the Primer Monitor tool to evaluate several mutations in SARS-CoV-2 variants, overlapping with the 2019-nCoV panel. Some of these mutations decreased assay sensitivity, whereas others, such as the mutations witnessed in the Delta variant, did not impact N gene assay sensitivity. In addition, they identified a new variant circulating in Gauteng, South Africa, with a genetic mutation at position 28,320, which also overlaps the CDC 2019-nCoV_N1 probe. Currently, this mutation does not match with any published Omicron sequences, and additional testing is warranted to determine the impact of this mutation on probe detection. Consequently, the CDC has designed an alternative flu SC2 multiplex assay that can simultaneously detect the N gene from SARS-CoV-2, influenza A, and influenza B.

To summarize, the study recommends continual monitoring of the emergence of new SARS-CoV-2 variant mutations and testing the performance of probes to ensure that COVID-19 diagnostic tests continue to yield accurate results in the future.

*Important notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Neha Mathur

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Neha Mathur

Neha Mathur has a Master’s degree in Biotechnology and extensive experience in digital marketing. She is passionate about reading and music. When she is not working, Neha likes to cook and travel.

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