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Omicron can be quickly and reliably detected and distinguished from the Delta variant of SARS-CoV-2 based on N-gene amplification failure (NGTF) using commercial tools, according to a new study posted to the medRxiv pre-print server.  Researchers at Tata Memorial Centre in India used a real-time polymerase chain reaction (RT-PCR) assay for this study. The authors suggest this approach “can be used as a low cost, rapid-screening strategy for detection of Omicron.”

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). Most recently, the Omicron variant (Nextclade strain 21K) was identified in South Africa in November 2021.

As compared to the original Wuhan strain of SARS-CoV-2, the Omicron variant harbors a higher number of mutations and is highly transmissible resulting in a higher infectivity rate. This has led to a sharp spike in the number of global COVID-19 cases. By January of this year 95% of sequenced COVID-19 cases sequenced in US were caused by Omicron, according to US Centers for Disease Control and Prevention data.

The Omicron variant is characterized by more than thirty mutations (including single nucleotide variants as well as insertions and deletions) in the spike protein-coding regions which has also raised concerns regarding efficacy of vaccines. Furthermore, recent evidence suggests that therapeutic monoclonal antibodies have a minimal response against the Omicron variant, presumably due to mutations in the spike protein-encoding regions. This makes rapid and accurate diagnosis even more urgent.

Genome sequencing is a time-consuming way to track and diagnose strains and there are a shortage of commercially available kits that can specifically detect the Omicron variant. Although S-gene amplification failure (SGTF) on RT-PCR is a surrogate means of at least suspecting Omicron, this test is not 100% accurate, raising the need for other methods.

The study was carried as the third wave of the pandemic struck India.  Nasopharyngeal and oropharyngeal swabs were used to gather the samples, of which the researchers tested 15144 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 hospital staff and cancer patients. The team detected N-gene target failure (NGTF) in 402 out of 412 SARS-CoV2 positive cases from December to mid-January 2022 using a commercially available assay. 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.

Library preparation was done using the QIAseq FX library kit. Then paired-end sequencing was carried out with an Illumina MiSeq platform at a targeted depth of 0.3 million reads per sample. Lastly, Nextclade web application was used to process the fasta sequences for identification of clade and mutation calling.

The team sequenced the genomes of the five samples with NGTF and compared these results with six cases where NGTF was not seen. The team was able to confirm that cases with NGTF were the Omicron lineage, while cases with preserved N-gene amplification belonged to Delta lineage. They also found that the ERS31-33 deletion (nucleotide 28362-28370del) overlaps with N gene probe used, explaining NGTF. As the ‘stealth’ Omicron variant also harbors the ERS31-33 deletion, they suggest this approach will work for the detection of ‘stealth’ Omicron variant as well.


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