COVID-19 Variant Detection with RT-qPCR SARS-CoV-2 Mutation Assays
Matthew Moser1, David Gauthier2, Angelica Olcott3, Steven Okino3, Charlie Rzadkowolski4, Jason Ma3, Raf Podowski3, Wei Vivian Fu3, Alan Wu1, Jeffrey Whitman1
1. University of California at San Francisco (UCSF), Department of Laboratory Medicine– San Francisco (United States)
3. Bio-Rad Laboratories, Life Science Group – Hercules (United States)

Introduction:

Method:The COVID-19 pandemic has been our most significant global health crisis in modern times. Continuous
molecular surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is necessary
to understand how the virus is evolving over time in association with infectious characteristics. Efficientmethods of identifying mutations are vital to public health efforts to detect when a new variant of interestor concern begins circulating.

Wet lab assay validation of specimens contrived in negative nasopharyngeal (NP) swab matrix with synthetic
SARS-CoV-2 wildtype and variant (Alpha/Beta/Gamma/Delta/Epsilon/Kappa/Mu/Lambda/Omicron) RNAs, athigh or low inputs (Cq≈25.0, Cq≈35.0, respectively), was first conducted at Bio-Rad to determine the accuracyof the assays. RNA extraction of the contrived sample was followed by testing 10 µL of sample using themanufacturer’s procedure (see assay protocols). Following wet-lab validation, the assays were received atUCSF for the following performance evaluation study with remnant clinical samples.

The sequencing of SARS-CoV-2 variants is a costly and time-consuming operation that requires
specialized expertise and methods. As such, sequencing is a major bottleneck for public healthsurveillance. Real-time quantitative polymerase chain reaction (RT-qPCR) has been utilized to overcome
these limitations and has the potential to develop novel workflows for variant screening (Dikdan RJ,2022).
To address this, Bio-Rad has developed a novel reverse algorithm RT-qPCR SARS-CoV-2 mutation assay
called the “Multiple Mutation Assay” (MMA). This assay allows for the detection of eight of the most
relevant SARS-CoV-2 mutations (Alpha/Beta/Gamma/Delta/Epsilon/Kappa/Lambda/Omicron). Singlemutation assays have also been developed which target individual variant-associated mutations, as wellas the Delta-Omicron variant discrimination assay which can distinguish between both the Delta andOmicron variants.
Here we report a study to verify the performance of these single and multiple mutation assays forresearch use, targeting variant-associated mutations found in the spike gene of key SARS-CoV-2 variantsof concern (VOCs) in remnant clinical samples. The single and multiple mutation assays from Bio-Rad canidentify up to four key SNPs and an internal reference gene in a single reaction, and both require <2 hours
to perform on standard real-time PCR instrumentation.

RNA extracted from remnant NP swab specimens from the University of California, San Francisco ClinicalLaboratory at Zuckerberg San Francisco General Hospital (ZSFG) were tested using Bio-Rad’s RT-qPCR SARSCoV-2 single- and multiple-mutation assays. The following assays were utilized: MMA 2, MMA 3, MMA 4 (todistinguish Delta/Omicron BA.1/Omicron BA.2), and a Delta-Omicron Single-Mutation Assay (Delta-Omicron
Variant Discrimination Assay). Several cohorts of samples which varied based on the date of infection wereanalyzed using the single and multiple mutation assay types. The study included nucleic acid amplification test(NAAT) negative samples, NAAT positive samples, NAAT positive samples collected mid-2020 (European variant
typed by PCR) and samples from fall and winter 2021 with epidemiological predominance of Delta and Omicronvariants, respectively. All assay PCR plates included wells that were spiked with synthetic RNA positive controls
corresponding to each of the eight key variants, provided by Bio-Rad.
PCR plates were set up as described in the assay protocols for Bio-Rad’s PrimePCR SARS-CoV-2 Multiple andSingle Mutation Assays. To set up the RT-qPCR reaction, sample RNA was added to a master mix containing BioRad’s “Reliance One-Step Multiplex Supermix” and the primers/probes from the mutation assay, as described inthe protocol. Samples were run on a CFX Opus Real-Time PCR System, alongside variant-specific positivecontrols and negative controls designed for research use only. Specimens were blinded to variant status at thetime of testing. Reagents and method are intended for research use only.
Studies on remnant clinical specimens were approved by the institutional review board at UCSF and ZSFG
protocol approval.

2. Bio-Rad Laboratories, Clinical Diagnostics Group - Irvine (United States)
4. Exact Diagnostics (a Bio-Rad Company) – Fort Worth (United States)

Performance of Single and Multiple Mutation Assays
Bio-Rad Wet-lab Validation of Multiple Mutation Assays
The results of wet lab validation for a series of PrimePCR SARS-CoV-2 single mutation assays for research use
provided correct allelic discrimination calls when analyzing the wildtype reference, Alpha, Beta, Gamma and Epsilon
variant RNAs. In total, 90 out of 90 low input and 328 out of 328 high input samples gave correct calls. Similarly,
wet-lab validation of a multiple mutation assay showed that for all samples tested, 90 out of 90 high input and 90
out of 90 low input reactions identified the correct mutations.

Results of multiple mutation assay #1 with high and low input

Table 1: European Variant Samples
Remnant NP samples (n=27) collected from April to May 2020 were analyzed by the MMA 2 assay. The set included20 samples PCR positive for the European strain (mutation D641G) and 7 negative samples. The mutation assay wasable to detect the SARS-CoV-2 virus in all positive samples but did not detect this variant mutation since the MMA
assay lacks the D641G primer/probe set.

Single Mutation Assays
Identifies presence of specific SARS-CoV-2mutation: over 20 assays are available• 3-plex assay includes reference gene and internal
human RNAseP control
Variant Discrimination Assays
Discriminates between two SARS-CoV-2 variants• Assay distinguishes between Delta and Omicron(BA.1 and BA.2)• Assay that discriminates between Omicron BA.1
and Omicron BA.2
Multiple Mutation Assays
Multiplex assay contains internal control, detects fourSARS-CoV-2 mutations in a single well*.
• Four Assays available• Detect Pre-Delta, or distinguish between
Delta/Omicron BA.1/Omicron
BA1.1/Omicron BA.2

The Multiple Mutation Assay (MMA) is a novel technique for profiling SARS-CoV-2 variants based on their associatedmutations. Multiplexed primer/probe combinations can detect up to 4 different mutations plus the MPC control in a single
well. The use of the following reverse algorithm approach may aid in triaging samples for further sequencing by NGS. Resultsfrom each MMA for SARS-CoV-2 positive samples are compared to its mutation map to determine the probability that a known
variant is present. If a SARS-CoV-2 positive sample does not correspond to any known mutation profile, then it may indicate a
new emerging variant, and encourage sequencing by NGS.

Assay Analysis• Single mutation and variant discrimination assays
can use allelic discrimination in CFX Maestro.
• Multiple mutation assays can use Cq and/or
RFU analysis in CFX Maestro.
*Patent pending, Bio-rad Laboratories

In-house Bio-Rad wet lab assay validation using contrived samples with synthetic SARS-CoV-2 RNA
showed that at both the low and high inputs, the single and multiple mutation assays were able to identify
the correct mutation profile >99% of the time. The accuracy of these results encouraged theperformance evaluation of these research use assays in a UCSF study using remnant NP samples. As
described below, clinical samples analyzed using RT-qPCR to validate Bio-Rad’s multiple mutation assays
demonstrated >99% accuracy in detecting the variant-associated mutations of samples in known
cohorts tested.
A collection of 20 SARS-CoV-2 European variant positive samples from April to May 2020 were run using
MMA 2. The assay was able to correctly identify the presence of the virus in infected samples and did notshow any false-positive results in the negative samples (Table 1). MMA 2 was not able to evaluate thespecific variant as the European strain mutation D641G was not included in this assay. However, theability to accurately determine the presence of the SARS-CoV-2 virus that does not match a variant ofconcern is important to demonstrate.
A small set of 20 SARS-CoV-2 negative samples were tested using MMA 2 and MMA 3. Both assays
determined that all the samples were negative (Table 2). MMA 3 did have a borderline positive sample in
one of the replicate tests however, this was by <0.1 C1 of the cutoff.
A set of 40 specimens containing 29 NAAT positive samples collected between September and October of2021 were evaluated using MMA 2, MMA 3, and MMA 4. Delta was the predominant circulating variant
during this time period. All the assays were successful in detecting SARS-CoV-2 virus and typing asDelta variant. All negatives came back as expected with no false-positives being detected (Table 3).

Table 2: Negative Sample Testing
NAAT Negative NP samples (n=20) were tested using MMA 2 and 3; MMA 2 did not show any “False-Positive” results.
MMA 3 was borderline for a “Positive” indication (<0.1 Cq) just below the Cq assay cutoff of 40, and repeat runs
indicated “Negative.”

The final evaluation focused on samples from late December 2021, a time period when both Delta andOmicron were circulating. All three of the multiple mutation assays and a Delta-Omicron variant
discrimination assay were evaluated on a collection of thirty-two SARS-CoV-2 NAAT positive samples.
The MMA 2, 3, and 4 assays showed results consistent with the presence of Delta-associated mutationsin 3 samples, and results consistent with the presence of Omicron-associated mutations in 28 samples.
MMA 4 was able to distinguish the Omicron BA.1 sub-lineage; BA.2 was not detected. Omicron-consistent
results were seen in 27 of the samples from the Delta-Omicron variant discrimination assay (Table 4).
One false negative result was observed for MMA 2, 3, 4 while 2 false negative results were seen by theDelta-Omicron assay. We are unable to determine if this is due to pre-analytical errors, such as
specimen stability and freeze-thaw, or analytical sensitivity.

Table 3: Delta Variant Samples
NP samples (n=40) collected during fall of 2021 were analyzed using MMA 2, 3, and 4; All four assays were able todetect SARS-CoV-2 in the positive samples and type them as Delta variant, which is consistent with the predominantcirculating variant at that time. No false positive results were found..

Overall, we conclude that there is excellent accuracy of these multiplexed assays for discrimination ofSARS-CoV-2 positive and negative samples in addition to detection of variant-associated mutations in asingle well.

Design of Mutation Assays:
Utility of the Multiple Mutation Assay Map

Results and Discussion:

Conclusion:

Table 4: Delta/Omicron Variant Samples:
SARS-CoV-2 NAAT-positive samples (n=32) from late December 2021 were analyzed by MMA 2, 3, 4, and the DeltaOmicron variant discrimination assay. The MMA 2, 3, and 4 assays showed results consistent with the presence ofDelta-associated mutations in 3 samples and results consistent with the Omicron variant in 28 samples. MMA 4 wasable to distinguish the Omicron BA.1 sub-lineage; BA.2 was not detected, consistent with the time period. Omicronconsistent results were seen in 27 of the samples from the Delta-Omicron variant discrimination assay.

Streamlined RT-PCR SARS-CoV-2 variant assays have the potential to improve the speed andaccessibility of variant screening (Dikdan RJ, 2022). Bio-Rad has developed a research use single
mutation assay, variant discrimination assay and multiple mutation panel that have demonstrated
excellent correlation with molecular testing performed in the clinical laboratory. Such assays allow for
identifying SARS-CoV-2 specimens that do not type for known circulating variants of concern and triage
them for sequencing, providing the potential to optimize workflows for new variant detection for publichealth surveillance.

References:PrimePCR Single and Multiple Mutation Assay Protocols, bio-rad.com/SC2Variants
Exact Diagnostics SARS-CoV-2 Variant Controls Brochure Q-1684
Oikdan RJ, Marras SAE, Field AP, et al. Multiplex PCR Assays for Identifying all Major Severe AcuteRespiratory Syndrome Coronavirus 2 Variants, The Journal of Molecular Diagnostics, volume 24,
issue 4 (April 2022). https://doi.org/10.1016/j.jmoldx.2022.01.004