Tracking BA.2.12.1 🦠 by partial ORF1ab gene 🧬 target failure (pOGTF)

Scientists from the United States have recently identified a novel approach to rapidly and accurately detect the omicron BA.2.12.1 sub-lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples.

They have described that partial ORF1ab gene target failure (pOGTF) in molecular diagnostic assays is 97% sensitive and 99% specific for detecting the omicron BA.2.12.1 sub-lineage, an emerging variant in the United States. The study is available on the medRxiv* preprint server.

Study: Partial ORF1ab Gene Target Failure with Omicron BA.2.12.1. Image Credit: Corona Borealis Studio / Shutterstock

Background

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen of coronavirus disease 2019 (COVID-19), is an enveloped, single-strand, positive-sense RNA virus with a genome size of around 30 kb. Throughout the COVID-19 pandemic, amplification and detection of viral RNA in respiratory samples using reverse transcription-polymerase chain reaction (RT-PCR) has been considered to be the gold standard for disease diagnosis.

The emergence of mutations in the viral genome can lead to partial or complete PCR target failure. For instance, the H69/V70 amino acid deletion mutation in the SARS-CoV-2 spike protein leads to complete loss of spike gene detection in PCR. This phenomenon is termed Spike gene target failure. This approach has been considered by the Centers for Disease Control and Prevention (CDC), USA, to identify newly emerging viral variants with the characteristic H69/V70 deletion mutation. The widespread application of the spike gene target failure in PCR-based assays has been very successful in identifying the alpha and omicron variants.

In the current study, the scientists have described the efficacy and accuracy of partial ORF1ab gene target failure approach in rapidly identifying the SARS-CoV-2 omicron BA.2.12.1 sub-lineage, which has rapidly emerged in the US between March and April 2022. This sub-lineage contains spike L452Q and S704L mutations that may potentially increase its transmissibility, infectivity, and immune evasion ability.  

Study design

The upper respiratory samples collected from patients undergoing COVID-19 testing between March and April 2022 were used in the study. Two PCR-based molecular diagnostic assays targeting SARS-CoV-2-specific ORF1ab gene and pan-Sarbecovirus-specific envelop gene were considered for the qualitative detection of SARS-CoV-2 in clinical samples.

The PCR cycle threshold (Ct) values for ORF1ab and envelop targeted genes were compared to identify any abnormal differences between them. The ORF1ab gene target failure was defined as detection of the ORF1ab gene being delayed in comparison with the envelop gene.          

Important observations

A total of 428 samples with partial ORF1ab gene target failure were detected during the study period. The whole-genome sequencing analysis of the samples revealed the presence of omicron BA.2.12.1 sub-lineage. A correlation was also observed between the emergence of samples with partial ORF1ab gene target failure and the induction in omicron BA.2.12.1 circulation in the US. Based on these observations, partial ORF1ab gene target failure may serve as a proxy marker for BA.2.12.1 detection.

The analysis conducted using publicly available SARS-CoV-2 genome sequencing data revealed that the omicron BA.2.12.1 sub-lineage contains two synonymous mutations in the ORF1ab gene. The reduced efficiency of ORF1ab gene amplification in molecular diagnostic assays is associated with one or both of the ORF1ab mutations.

Considering a Ct value difference cut-off of ≥2 for the ORF1ab gene target compared to the envelope gene target, the partial ORF1ab gene target failure approach showed 97% sensitivity and 99% specificity for detecting omicron BA.2.12.1 sub-lineage in clinical samples.

Further validation of the partial ORF1ab gene target failure approach involved determining its association with the emergence of the sub-lineage and overall test positivity.

Considering all SARS-CoV-2 sequences and the sub-lineage-specific sequences uploaded to the Global Initiative on Sharing All Influenza Data (GISAID) database from North America, a significant correlation was observed between the frequency of partial ORF1ab gene target failure in tested samples and the frequency of BA.2.12.1 sequence submission in the database.    

Additionally, a significant correlation was found between the increase in partial ORF1ab gene target failure detection rates and the increase in overall test positivity rates.

Study significance

This study highlights the importance of considering partial ORF1ab gene target failure approaches for the rapid detection and monitoring of the omicron BA.2.12.1 sub-lineage in clinical samples. Compared to whole-genome sequencing, this method is less time-consuming and less expensive. 

*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.

Written by

Dr. Sanchari Sinha Dutta

Dr. Sanchari Sinha Dutta is a science communicator who believes in spreading the power of science in every corner of the world. She has a Bachelor of Science (B.Sc.) degree and a Master's of Science (M.Sc.) in biology and human physiology. Following her Master's degree, Sanchari went on to study a Ph.D. in human physiology. She has authored more than 10 original research articles, all of which have been published in world renowned international journals.