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Sponsored by Diversigen, Inc.

Kegan Sunderland, PhD qPCR and Custom Initiatives Manager Diversigen, Inc.
Quantitative Polymerase Chain Reaction (qPCR) — also known as real-time PCR — is a molecular biology lab assay technique based on the polymerase chain reaction (PCR). The significant difference in qPCR is the monitoring of amplification on a targeted gene or taxon during the PCR. This allows qPCR to be used as a quantitative or semi-quantitative tool of measurement, whereas conventional PCR is generally a process completed to create a product. However, generic qPCR assays can fall far short in delivering the most valuable insights possible, as explained below by Kegan Sunderland, Ph.D., who is qPCR assay designer and Custom Initiatives Manager for Diversigen, Inc., a wholly owned subsidiary of OraSure Technologies, Inc.
What are the benefits of a tailored qPCR assay compared to a generic, pre-existing qPCR assay?
The primary benefit of tailored qPCR assays is that they are designed for the specific needs of a project or research objective. There are a few scenarios where a customized qPCR assay can be the right choice. For example:
- If a proprietary strain of a probiotic has no existing assay available, an assay can be designed for that specific strain.
- If a desired target lacks any existing primers or even lacks sequencing data, a custom qPCR assay can start with sequencing your target and designing primers based on the sequencing data
Another benefit is the quality of having a validated primer design pipeline compared to pre-existing assays. These primers are designed for specificity and are optimized to avoid unwanted off-target amplification. It is critical that these be thoroughly checked for primer dimers, secondary structures, and tertiary structures. Selected primers must also be optimized for melting point, GC content, and amplicon length to improve their amplification efficiency.
Depending on the source of a pre-existing assay, these design considerations may or may not have been assessed. Among academically sourced primer designs, these features are frequently not addressed. Furthermore, a dry lab design can only take a qPCR assay so far. It’s vital that primer sets be thoroughly tested in the lab including appropriate positive and negative controls to ensure specificity requirements are met.
Using generic, pre-existing qPCR assays can generate unreliable data — with severe downstream consequences. For example, if you’re running a clinical trial, unreliable data could translate to massive costs if the trial needs to start over. In other words, how far are you willing to go in a clinical trial before you figure out the data you based your trial on is useless? That could be the case if you use a pre-existing assay.
In addition, commercially developed primers may or may not have been thoroughly evaluated. And if they did, often they won’t provide you with the sequences. Diversigen™ will report everything necessary for you to recreate a qPCR assay including primer/probe sequences, thermocycling conditions, and master mix components.
How can a microbiome qPCR assay be designed to support ongoing research or a clinical trial?
qPCR assays provide highly sensitive, robust, and accurate quantitative detection of genomic targets. This means they can be used to track quantities of bacteria or fungi over the course of a treatment, which can then be leveraged for statistical analysis. qPCR assays can even be used to distinguish between microbial family, genus, species, strains, or even minor variations within a strain to provide an accurate quantity of your desired target. This quantitative information can be useful for:
- Inclusion/exclusion criteria for a clinical trial;
- Determining if a probiotic is present in a patient and at what quantities;
- Tracking concentrations of a beneficial or harmful taxa over the course of treatment; and
- Assessing the impact on unintended off-target microbes.
What if a target someone is interested in doesn’t have sequencing data in any databases yet to design primers. Can Diversigen™ still help?
Yes. Diversigen™ can leverage our sequencing platforms to generate new sequencing data for your target of interest. From this data, we can design primers specific to whatever taxa is of interest.
Why is it important to validate qPCR assays, and what are some of the pitfalls in this step?
Validated assays provide essential information on the amplification efficiency, repeatability, accuracy, precision, sensitivity, specificity, and matrix interactions of your assay. They are the gold standard for using qPCR data and reports to support FDA submissions. When assays are not validated or at a minimum, checked for the basic requirements of accuracy and specificity, the data produced can lead to many errors.
For example, taxa can be double-counted without knowing it, off-targets like the host can produce signal or the assay may not be repeatable and gives a different result every time it’s run.
From here, if you’re making decisions based on this data, it could be quite a bit of time down the line in your research before figuring out a treatment is not actually working. We prevent our clients from wasting time and money on assays that aren’t proven to be reliable. We’re always advising them to consider the potentially catastrophic downstream impacts of skipping an assessment of their qPCR assays.
Can you walk us through the process Diversigen™ uses to create a qPCR assay to support clinical trials?
- We meet with our clients to discuss upstream processes such as treatments or sample collection strategies that may impact the data they receive from a qPCR assay. After all, the data must be meaningful to them and to the goals of their investigations or clinical trials.
- Next, we use our validated pipeline to produce primer sets and determine thermocycling conditions.
- After that, we run initial pilot tests in the wet lab to generate data showing specificity, efficiency, and accuracy.
- Then we optimize assay conditions if necessary or try additional primer designs.
- Finally, we validate the most optimal assays generating all necessary data to meet FDA and MIQE requirements.
More companies enter the microbiome and genetic testing markets every day. What is unique about working with Diversigen™ and it’s approach to designing qPCR assays?
At Diversigen™, we can pair sequencing data with qPCR design to generate assays for targets that don’t yet have sequences. Our primer design and validation pipeline produce trusted data. We can tailor the assays to client needs, whether that data is for research only or to support submission to the FDA.
What kind of assurance does a designed qPCR assay give to the quality of the assay and results?
Designing an assay with Diversigen™ can lead to improved accuracy and repeatability of results. Our validated primer design pipeline produces assays that are specific to a client’s strain, species, or genus. Designing an assay with us enables clients to trust the results. We take their target and sample type into account when designing and validating assays so they know the assay will work for them. Other assays may have specificity issues or unknown matrix interference for their sample types that could skew their results.
What larger trends do you see emerging in microbiome research and related technologies?
Sequencing data and qPCR data are being used to complement each other. While sequencing data can answer the “who is there” within a sample, the data does not provide quantitatively how much of a taxon is present. It can only provide relative abundances. Pairing with qPCR provides quantitative information to link to the sequencing data. There’s also a trend to start with sequencing as an exploratory phase, but eventually move into qPCR for the clinical phase. qPCR assays and their data are more well-defined to support FDA submissions and approval, so it’s often the technique used during clinical trials. Once it’s known which taxa are of interest from the sequencing, a qPCR assay can be designed to track those taxa quantitatively.
Contact Diversigen to speak with a qPCR expert and discuss the development of a qPCR assay to deliver highly sensitive quantification of target taxonomic biomarkers. Learn more here.
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