Stealth Omicron: A Novel SARS-CoV-2 Variant That Is Insensitive to RT-qPCR Using the N1 and N2 Primer-Probes

Area of Science:

• Virology
• Molecular Biology
• Public Health

Background:

• The COVID-19 pandemic, caused by SARS-CoV-2, has necessitated robust diagnostic strategies.
• Standard diagnostic methods include real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR).
• The Centers for Disease Control and Prevention (CDC) recommends specific primer-probe sets (N1 and N2) for SARS-CoV-2 detection.

Observation:

• A SARS-CoV-2 variant, later identified as BA.5, was detected in Osaka, Japan.
• This variant was identified using polymerase chain reaction (PCR) with E primers.
• However, the same variant was not detected by RT-qPCR using the CDC-recommended N1 and N2 primer-probe sets.

Findings:

• Genetic analysis of the S and N gene regions was performed using reverse-transcription and nested PCR.
• DNA sequencing revealed point mutations within the probe sequences of both the N1 and N2 primer-probe regions in the BA.5 variant.
• These mutations likely explain the failure of the CDC-recommended RT-qPCR assays to detect this specific variant.

Implications:

• The findings underscore the critical need for ongoing genomic surveillance of emerging SARS-CoV-2 variants.
• Continuous monitoring allows for the timely identification of mutations that may impact diagnostic test performance.
• Adapting diagnostic assays based on genomic data is essential for maintaining effective COVID-19 detection and control strategies.