PCR inhibitors are a hidden but pervasive challenge across environmental testing, wastewater surveillance, and gene therapy workflows. In this first Science Snapshot episode, the Absolute Gene-ius team revisits insights from multiple experts to explain how digital PCR helps overcome inhibition and deliver more reliable quantification.
PCR inhibitors are everywhere and they can definitely mess with your data. From muddy lake water to complex viral vector preparations, inhibition is a reality that can’t be ignored.
In this inaugural Absolute Gene-ius: Science Snapshot, hosts Jordan Ruggieri and Lisa Crawford revisit standout moments from past seasons to explore how scientists across disciplines deal with PCR inhibition. Through clips from experts working in wastewater surveillance, environmental microbiology, and gene therapy manufacturing, the episode highlights why inhibitors distort qPCR results and how sample preparation alone doesn’t always solve the problem. Guests including Ray Ketchum, Sarah Philo, Patrick Hanington, Dave Bauer, Kimberly Gomez, and Min Jin Kim explain both the biological sources of inhibition and the technical reasons digital PCR is more resilient. Together, these perspectives paint a clear picture of why dPCR is becoming the method of choice when overcoming inhibitors is required.
Jordan Ruggieri00:00
Do we want to, do we want to do "Welcome back to Absolute Gene-ius. We're your hosts, Jordan Ruggeri", okay, and then, then you go "And Lisa Crawford".
Lisa Crawford00:08
Got you, I got you.
Jordan Ruggieri00:08
And you need a southern drawl for that one too. All right?
Lisa Crawford00:12
No. Okay, no problem.
Jordan Ruggieri00:26
Welcome back to Absolute Gene-ius. We're your hosts, Jordan Ruggieri
Lisa Crawford00:30
And Lisa Crawford.
Jordan Ruggieri00:31
Today, we're kicking off something new for the series. This is the first of what we call Absolute Gene-ius: Science Snapshot. These Science Snapshot episodes will cover specific themes and topics that are relevant to dPCR and qPCR, and we'll be using clips from past interviews to share perspectives from multiple scientists.
Lisa Crawford00:49
Today, we'll be digging in on PCR inhibitors across wastewater, environmental samples and even gene therapy labs, PCR inhibitors are present and can distort PCR results. But scientists are finding ways to overcome them, either with sample preparation or with dPCR.
Jordan Ruggieri01:04
Let's talk about the sample prep aspects and approach. First, almost all of our guests use some sort of sample prep to extract, isolate and enrich DNA or RNA before doing their PCR. This is definitely one way to try to remove or reduce the presence of PCR inhibitors. However, we did hear from Dr. Ray Ketchum that you also need to be aware and cautious of DNA extraction bias. Here's our exchange on this topic from season one, episode five.
Ray Ketchum, PhD 01:31
We haven't actually done the real time PCR versus the digital PCR comparison to see if there's, there's a difference there. I don't suspect that there is simply because of the way the DNA is extracted. Most of those inhibitors should be removed. But there is another issue that we have run into recently, and I think it's something that the industry as a whole is starting to notice a little bit more, and that is that, you know, when you take this complex collection of bacteria and you're doing a consortium on them, whether it's from a gut, human gut microbiome, the soil microbiome, or whatever, there has been this assumption that your DNA extraction is extracting the DNA from all of the bacteria equally well. And that's simply not the case. In our case, what we found is that a lot of our bacteria are spore forming. And if they've formed spores, we just have a heck of a time really getting the DNA out representative of what's actually in the, in the product. It turns out that we've been way under counting what our product actually has. So those are some of the, some of the issues that we run into.
Lisa Crawford02:43
That one was before my time, but I listened to past episodes, and I believe that this topic of inhibitors came up several times in the first season alone. Right, Jordan?
Jordan Ruggieri02:50
Oh yeah, it actually came up in our very first episode ever, which was Dr. Sarah Philo, who was doing research in wastewater surveillance. Here she identifies the issue and talks about how her experiment had higher detection via dPCR, which helped to address the impact of those inhibitors.
Sarah Philo, PhD 03:07
We tested for some different antimicrobial resistance genes with qPCR and dPCR. And pretty much all of them had a higher quantification with dPCR than with qPCR, which makes sense, at least, knowing all of the inhibitors in wastewater and environmental samples tend to make qPCR a little bit less efficient. I will say that the gene copies that we detected in digital PCR were higher than what we detected with qPCR. So this is a big problem with wastewater. It's just like when you extract everything, a lot of times, the inhibitors are also still present. Just because there's a lot of things in wastewater that can be kind of hard to clean up. It's pretty well established that like PCR and qPCR do have some effects from those inhibitors, but with digital PCR, that tends to be less of an issue.
Jordan Ruggieri04:05
Also from our first season. Here's a clip from Dr Patrick Hannington sharing how dPCR has helped his team confidently quantify targets in environmental water samples that included cyanobacteria and sediment.
Patrick Hanington, PhD 04:17
We have a lot of experience using qPCR, quantitative polymerase chain reaction. And it was a just a few years ago now that we, we realized, you know, that some of the limitations of using qPCR in an environmental context, a lot of them have been addressed by digital PCR. The big one that we often struggle with is PCR inhibitors in a sample. Because we're often working with either water samples. And I mean, you guys probably haven't been to Alberta before, but the lakes here are pretty gunky. I don't want to, like, I don't want to bash them too badly. We have cyanobacterial blooms, like blue green algae blooms in every one of our central Alberta lakes. We're always wrestling with PCR inhibitors, cyanobacteria. And you know, we can do cleanup procedures of the DNA. When we do the DNA extraction, we can try to clean that sample up a little bit. And often those work pretty well to get rid of inhibitors. But it doesn't take much to impact the qPCR reaction and digital PCR, one of the big advantages from our perspective, anyways, is that it does a much better job of minimizing the impact of inhibitors on your reaction. That is the number one big advantage for us in the environmental space I think.
Lisa Crawford05:38
Nice clip choices, Jordan. And really nice hearing different scientists essentially share the same experience of dPCR helping them work around PCR inhibitors to produce results they trust. As the resident nonscientist, can you remind me how or why dPCR is so good at managing the effects of inhibitors?
Jordan Ruggieri05:53
I could Lisa, but why, when we had Thermo Fisher Scientific's Dave Bauer break down the technical side in season one, episode eight. Here he covers just how dPCR helps manage PCR inhibitors and how they can result in Ct value shifts in qPCR.
Dave Bauer, PhD06:09
There's a handful of, kind of the bullet points I like to think of, as where it's valuable, and that's one of the key ones. Is if your samples have inhibitors, if they're dirtier samples, that might compromise the PCR enzymes for real time PCR, you're dependent on the efficiency of that reaction from start all the way until the signal crosses your Ct threshold. Any changes to that efficiency down that pathway is going to change your answer, change the concentration. However, with digital PCR, because it's that binary, "do we have signal or not?" I don't care if you know, if you were seeing an amp curve in your individual mini reaction, you might see it super sluggishly growing really poor efficiency. That's fine, as long as by the end of cycling, it gets above the cloud of the background noise that still counted as a positive either way. And so that gives you a lot of robustness against inhibitors. And the other value it adds is multiplexing, because a lot of times with PCR, with real time PCR, if you're multiplexing, you might compromise the efficiency of the different primer and probe assays in there as you mix more and more together. So it's hard to make a four-plex quantitative assay with real time PCR, but with digital PCR, it's so much easier to have multiplex assays, multiple targets with different color dyes, all in the same reaction, because we don't care about the efficiency.
Lisa Crawford07:28
Thanks for reminding me of that episode, and Dave's great explanation. Let's look at one more example, specifically gene therapy workflows where inhibitors from buffers and proteins can be problematic. Kimberly Gomez and Min Jin Kim explained in season two, episode six, how digital PCR can provide accurate and reliable results, particularly when using the Applied Biosystems QuantStudio Absolute Q dPCR system.
Kimberly Gomez07:52
With qPCR, you would usually run a standard curve. You would make a dilution series and run that alongside your sample of unknown concentration and derive your concentration from that standard curve. Big issue with that is sometimes your standard curve could be made from reference material, so a different AAV. Or it could be made from a plasmid. So a lot of the preparation and making that standard curve can really affect the concentration that you get, that you derive from that standard curve. Another thing to consider is that with qPCR, it's not as robust against inhibitors. We see with dPCR that we have a lot more robustness with different matrices. So that's a big advantage, especially when you're pulling your sample from different points of the, from production of the virus.
MinGin Kim, PhD08:48
So basically the summary would be like for qPCR, although it is like, very commonly used, and everybody nowadays in our field at least knows how to use a qPCR. But there are limitations on requiring a standard curve like Kim mentioned, which may affect and impact the actual results, accuracy and precision. Another one is the inhibitory effects that is more prevalent in qPCR than digital PCR, just because it's in a more bulk sample prep situation. In that case, sometimes for AAV applications, you would pull samples from different stages of the production and which would include different proteins, different buffers, which have different inhibitory effects. In that case, qPCR would be a little bit more limited into neglecting or mitigating that effect, as opposed to digital PCR. And also for precision wise you, it is recommended for qPCR to do at least a triplicate of your sample which increases the sample volume. Digital PCR, especially our Absolute Q, we do have lower sample volume input as well as almost no dead volume, which helps preserve your samples. On top of that, since it is an absolute quantification, it technically, in theory, does not require any replicates to be run, so you could run your one sample in one array. Another advantage is that there are some other analyses that you could do on digital PCR that you can't on qPCR, but it's about the molecular integrity of the gene of interest that is being delivered to the subject.
Jordan Ruggieri10:32
That is another good one. Lisa. I think these clips confirm that PCR inhibitors are everywhere, from wastewater, to plants to gene therapy labs. I think the lesson here is to be aware of this omnipresence of PCR inhibitors and choose methods and technologies that minimize their effect when quantification is critically important. The gene-iuses showcased here can attest to dPCR being a great option in these instances.
Lisa Crawford10:57
Okay, let's call that a wrap for our first Science Snapshot. Thank you to our listeners for joining us today and remember to stay tuned this season as we dive into conversations with new guests and revisit themes and topics from past conversations in more of these Science Snapshot episodes.
Jordan Ruggieri11:11
And don't forget to subscribe to Absolute Gene-ius so that you don't miss a single episode. Stay curious and we'll see you next time. This episode was produced by Sarah Briganti, Matt Ferris, and Matthew Stock.
Lisa Crawford11:23
And they'll say, "I'm Lisa Crawford", and you can say "I'm Jordan Ruggeri. Today we're doing something different.” And then I can come back in. Does that work? Or I can just do it all and you can just leave.
Jordan Ruggieri11:34
All right, see you guys later.