How does allele-specific qPCR make variant detection possible?
A key component of qPCR is the primers. Primers are sequences that are complementary to the target DNA, which in our case is SARS-CoV-2 RNA that has been reverse transcribed into DNA.
Minor mismatches between the primer and the sequence are tolerated in qPCR reactions, however, in some cases, mismatches can strongly suppress the qPCR reaction.
Allele-specific PCR (AS-qPCR), which was developed in the 1990s, exploits this behavior and can distinguish subtle differences between the SARS-CoV-2 variant and the original sequence. Our AS-qPCR method works by using pairs of primers, one pair per target mutation. One of the primers in the pair targets the wild-type (which is the non-mutated version of the virus), the other targets the mutation.
What does your variant detection assay target?
Even though variants are defined by a combination of mutations, some of those mutations are very specific to that variant. For the assay we’ve developed, we target three mutations that are highly specific to B.1.1.7: HV69/70 del, Y144 del, and A570D.
What is the limit of detection (LOD) for your variant detection assay?
This assay requires a sufficient amount of overall SARS-CoV-2 to yield interpretable results for B.1.1.7. Based on our current dataset, we have established a limit of detection for the B.1.1.7 assay at 30 copies/mL of SARS-CoV-2 as measured by our standard N1/N2 primer based SARS-CoV-2 assay.
Will this assay be able to detect variants other than B.1.1.7?
We are in the process of adapting this assay to detect SARS-CoV-2 variants other than B.1.1.7. As new variant testing becomes available, we will be sure to update our community.
Where can I learn more about Biobot’s variant detection methods?
Biobot’s variant detection method is based on research conducted by our collaborators in Singapore. This research was supported by the National Research Foundation, Prime Minister’s Office, Singapore under its Campus for Research Excellence and Technological Enterprise (CREATE) programme. To read more about this research, please visit https://www.medrxiv.org/content/10.1101/2021.03.28.21254404v1