N gene target failure method effectively detects SARS-CoV-2 Omicron in clinical samples

In a recent study posted to the medRxiv* pre-print server, researchers demonstrated that a commercial real-time polymerase chain reaction (RT-PCR) assay rapidly detects the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant.

Study: N Gene Target Failure (NGTF) for detection of Omicron: a way out for the "stealth" too? Image Credit: G.Tbov/Shutterstock

The assay used N-gene amplification failure to produce sequence-level evidence for Omicron detection in clinical samples of coronavirus disease 2019 (COVID-19), simultaneously distinguishing it from the Delta variant.

Since December 2020, several SARS-CoV-2 variants of concern (VOC) have been identified, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). Based on genome sequencing, most recently, the Omicron variant (Nextclade strain 21K) was identified in South Africa in November 2021 and was designated as the PANGOLIN lineage B.1.1.529.

There is an urgent need to clinically diagnose the Omicron variant and distinguish it from other SARS-CoV-2 variants. Although the definitive evidence of strain evolution is possible by genome sequencing, it is a time-consuming technique worsened further by the shortage of commercially available kits that can specifically detect the Omicron variant. Although S-gene amplification failure on RT-PCR is a surrogate method to suspect Omicron, it does not deliver 100% accurate results, raising the need for other methods.

About the study

In the present study, researchers performed RT-PCR using a commercially available SARSCoV-2 assay. They tested 15144 samples from March 2020 to mid-Jan 2022 (almost 23 months) to detect E (Envelope) and N (Nucleocapsid) genes of the SARS-CoV-2 RNA.

The study cohort included both hospital staff and cancer patients. The researchers selectively chose 11 RT-PCR-confirmed COVID-19-positive cases from December 2021 for amplicon-based viral whole-genome sequencing, of which five showed N-gene amplification failure.

The researchers performed library preparation using the QIAseq FX library kit and then paired-end sequencing using an Illumina MiSeq platform at a targeted depth of 0.3 million reads per sample. For genomic data analysis, the raw fastq reads were checked for quality and construed through the bioinformatics pipeline for the synthesis of consensus fasta sequences, which were processed using

Next clade web application for the identification of clade and mutation calling.


The researchers observed an overall SARS-CoV-2 RNA positivity rate of 16.2% for the last two years. Towards the end of December 2021, as Omicron (BA.1 lineage) emerged worldwide, a sharp rise in the number of positive cases was observed, hence the positivity rate in the first half of January 2022 escalated to 47.24% (386/817) from 3.89% in December 2021 (26/667). SARS-CoV-2 cases diagnosed from December onwards showed the amplification of the E gene and NGTF, a clear deviation from the trend of the past two years.

NGTF was observed in 402 out of the 412 positive cases between 1 December 2021 and 16 January 2022, while the remaining 10 showed amplification in both E and N genes. The examination of N-gene primer and probe sequences confirmed that the ERS31-33 deletion, characteristic of Omicron variant, overlapped with the N gene probe used, resulting in NTGF. All five cases with NTGF were confirmed on genome sequencing to be consistent with the Omicron variant, whereas all six COVID-19 cases with both N and E gene amplification belonged to Delta.


Most commercial kits identify Omicron by SGTF resulting from the S gene HV69-70del, that is also observed in the Alpha (B.1.1.7) and Eta (B.1.525) variants, whereas the N gene ERS31-33del results in NTGF. The N gene probe overlapped with the ERS31-33del observed in Omicron, thus explaining the NTGF. Moreover, these mutations also affect ORF1ab, E, and N genes in the Omicron variant. These findings indicated that the cases showing N gene failure on RT-PCR were related to the Omicron, BA.1 lineage. Therefore, Omicron was behind the COVID-19 cases showing NGTF by the kit used in the study.

On the other hand, dual amplification in the E and N gene (or in some cases N gene alone) most likely represented the presence of the Delta or non-Omicron variant. The ability of this test to distinguish variants holds clinical value because of the reported differences in clinical severity and communicability between them.

The BA.2 lineage Omicron variant, ‘stealth’, is genetically distinct from the BA.1 lineage and escapes detection on routinely used PCR kits as it does not harbor HV69-70del, therefore not resulting in SGTF. The gene sequencing could not detect it either, although the NTGF detected by the RT-PCR method could be used to detect the BA.2 lineage as well, as the ERS31-33del was present in the N gene in both Omicron lineages. The authors emphasized that the preservation of the N gene or its dropout depends on viral genetic mutations and the probe design used for the gene in individual kits.

*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:
  • Shivangi Harankhedkar, et al. (2022). N Gene Target Failure (NGTF) for detection of Omicron: a way out for the "stealth" too? medRxiv. doi: https://doi.org/10.1101/2022.01.28.22269801 https://www.medrxiv.org/content/10.1101/2022.01.28.22269801v2

Posted in: Medical Research News | Medical Condition News | Disease/Infection News

Tags: Assay, Bioinformatics, Cancer, Coronavirus, Coronavirus Disease COVID-19, covid-19, Evolution, Gene, Gene Sequencing, Genes, Genetic, Genome, Genomic, Hospital, Illumina, Library Preparation, Mutation, Omicron, Polymerase, Polymerase Chain Reaction, Respiratory, RNA, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Syndrome

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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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