1 Vs 2 gene COVID-19 Assay… Find out from our experts which is best for testing during the COVID-19 pandemic?

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Welcome to our latest blog!

There has been a lot of talk about two gene targets on the market for COVID-19 tests? Are we right? In this blog, we will clear up the questions and answers you have and what truly is better; a 1 gene target or a two-gene target in light of the pandemic we face today.  Read more and find out what our expert scientists in R&D have to say.

Multi-gene target rationale origin

A multi-target rationale is a traditional method that enabled the opportunity to mitigate against mutations in the virus which previously was prudent as sequencing technology was in its infancy.

With the SARS outbreak in 2003, whereby commonly used was the multi-gene target given that the total sequences available were a tiny fraction of those available for SARS-CoV-2.  A lot less was known about the genomics of the SARS virus, so it was necessary during that time to provide multiple assays until more sequence information was provided to provide a specific one gene assay.  Molecular capability and design capability has come a long way since then.

Why then, did the World Health Organisation (WHO) originally publish guidance for testing 3 targets?  The rationale behind this is that any 1 design target the WHO published wasn’t specific enough to exclusively detect SARS-CoV-2, the virus causing COVID-19, due to lack of available sequence information at the time. In an attempt to improve this, 3 designs were chosen as the protocol to improve confidence in the test.  On January 17th, this was reduced to 2 targets due to the 3rd having lack of sensitivity. On 17th March 2020, the WHO issued interim guidance on laboratory testing for coronavirus disease (COVID-19) in suspected human cases, within which states: “In areas where COVID-19 virus is widely spread a simpler algorithm might be adopted in which, for example, screening by rRT-PCR of a single discriminatory target is considered sufficient” Once a virus is well established in a population, variation within a virus is less likely to occur.  As is now the case with COVID-19 being so widespread, one, specific sequence, in one gene target, is preferential.

Our Bioinformatics Scientist talks on designing a 1 gene COVID-19 assay

“Early in the SARS-Cov-2 outbreak, there wasn’t very much genome sequence available for this novel coronavirus and no  positive samples to validate diagnostic qPCR assays with, so a 2-3 assay design strategy was adopted by many labs/organisations seeking to provide diagnostic testing solutions for the merging health crisis.

One set of primers and probe were designed with a broad detection spectrum to include other beta-coronaviruses and second,  more specific assay was designed to detect only the SARS-CoV-2 virus, although the specificity of this second assay is dependant not only the number of mismatches but also their position within the primers.

The two-assay approach also provides some redundancy so that if one assay starts to accumulate mutations,  which is a particular risk with an RNA genome, the other one should still give a positive result in the presence of the target.

Our strategy was to design one assay which was specific to the SARS-CoV-2 coronavirus, using the expertise of our qPCR assay design scientists, which was both sensitive and robust, to enable detection of the virus without the need to use a dual assay approach. This is a strategy that we have previously used in our CE-IVD kits and the vast number of our RUO qPCR kits.”

Surveillance Program

We have an ongoing surveillance program that tracks the mutation of the virus every day by our expert bioinformaticians (genomics data scientists) who know that every virus sequence published is detected by our kit. Each new sequence presents a risk to any COVID -19 kit detection and false negatives without surveillance programs in place.

To ensure the COVID-19 primers and probe remain specific to detect SARS-CoV-2 genomes, Primerdesign’s Bioinformaticians review daily the SARS-CoV-2 sequence submissions on the GISAID
EpiCoV database. As of 17th April 2020, our bioinformaticians can confirm the COVID-19 assay primers and probe still show 100% homology with the 6,384 full length, good quality SARS-CoV-2 sequences published on the GISAID EpiCoV database.

Each new sequence presents a risk to kit detection and false negatives for competitors without surveillance programs.  The potential risk of false negatives increases with 2/3 target assays meaning a patient with a false negative will potentially be back in the community affecting the general population by spreading COVID19.

“Unlike the SARS outbreak of the early 2000s, the amount of sequencing conducted by diagnostic laboratories around the world is unprecedented. The GISAID database EpiCoV currently contains over 6,000 sequences, of which are good quality full length genome sequences. It enables us to check our assay design against the database daily, to ensure that mutations are not accumulating in the target region of the primers and probes. We are therefore confident that our assay will still detect 100% of the sequences available, giving our customers confidence that the easy to use one assay approach will not miss any positive cases due to mutations. It also won’t give conflicting results which may arise if mutations accumulate in one of two assays, leading to a decrease in sensitivity of that assay.” Gemma Stokes, Bioinformatician Scientist Primerdesign.

“If clinical diagnosis is being performed, complete confidence is needed in your test.  At Primerdesign our bioinformatics team check our COVID-19 CE design daily against all globally published sequence information for assurance in a comprehensive SARS-CoV-2 detection profile.  Ultimately, we have complete confidence in our unique design and product. We therefore do not need to target multiple genes. ” Adam Herridge, Product Manager Primerdesign.

Did you know? We release every week based on daily findings our COVID-19 assay specificity document that allows you to have 100% confidence in your testing. You can find this under the resources section on our product webpage – Click Here

Factors to consider when choosing a test

The vast majority of global molecular pathogen testing, that is a disease caused by an organism or virus, is conducted by searching for a single target that is specific to the organism in question.  This target should be selected on 3 main factors:

  • Its ability to detect all variants of strains desirable within a group (think breeds of cat)
  • Its ability to not detect closely related organisms (think a tiger)
  • Biochemically, for the test to work, the region should perform well under test conditions (in the PCR)
  • The gene within which isn’t the key definer of a good test, rather that the sequence chosen is specific to the organism/virus of interest and is unlikely to mutate (hence maintain specificity)

If the above criteria are achieved, you have an accurate and precise molecular qPCR test.  This 1 specific target approach is the gold standard for molecular pathogen detection worldwide.

Two Gene target requirements

Two target assays Iterative or sequential requires, double the reagents, set up time and resource.  With this in mind obtaining results is a lot lengthier and with additional procedures required increases the chances of inaccurate results. Extended handling of fragile RNA which is prone to degradation (could result in false negatives, meaning infected patient released into com).

Multiplexing two targets in 1 well interfering of both targets biochemically with each other.  Problems occur including the amplification of non-specific targets between oligos (synthetic components of test) which increases the risk of false negatives due to reduced sensitivity and subsequently meaning retests would be necessary again for patients.

When multiplexing if one target is present in much higher concentration than the other target; for example if a patient has a co-infection with a Common Cold causing Coronavirus, and has low levels of COVID-19, if the test uses a generic Coronavirus family sequence for one of the viruses, this could outcompete the COVID-19 specific part of the test, and again show a false negative.

If you would like to enquire about our COVID-19 single target assay specific to SARS-CoV-2 then please email enquiries@primerdesign.co.uk or simply contact us here.


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