qPCR, Nested PCR, and the Search for Bacterial Serotypes

Tracking Streptococcus pneumoniae and Hidden Bacterial Reservoirs

What is Streptococcus pneumoniae, and why is it important to study?

Donna Paznar: “Streptococcus pneumonia is, it’s a bacteria that typically infects the respiratory tract of any age, but it’s very, very dangerous, or can be life-threatening in the younger and the older population. So, in infancy, and then, of course, the geriatric population, it becomes quite the, quite a very severe disease in the end. Since we’ve had all of these kind of precautionary measures with masks and things like that, we definitely saw a decline in things in these respiratory illnesses, but since then, not only have immune responses been declining for certain bacteria like Streptococcus pneumonia, but it has also been a huge, huge issue for us specifically with the fact that now there’s very, very colonized strains of this bacteria. So, the issue that we’re facing right now is more so the fact that children, specifically, are these kind of really, really special reservoirs for the bacteria itself, and they don’t actually show any signs or symptoms, and they’re not seriously affected by the bacteria, but they’re carriers, and then so that is then progressing further and further.”

What does it mean when someone carries the bacterium without showing symptoms?

Donna Paznar: “Thankfully, it almost always, if, if you are colonized for the bacteria, you almost always will test positive. And so the reservoir is very similar to what we have with the COVID-19 virus, where it’s in that nasopharyngeal tract. So it basically, the bacteria kind of just stays there. So again with these latent phases and these active phases, it doesn’t necessarily mean that the bacteria is active within its infection period, so to say, which basically means that when it’s in these latent phases, it’s not replicating, it’s not doing, it’s not affecting, it’s not causing any, it’s not causing any interference, so to say, with the area that it is in. But it just means that you essentially are a carrier.”

Why is it difficult to determine when colonization began?

Donna Paznar: “Because you can’t exactly tell when, that’s the problem, you can’t tell when with the research, you can’t say, ‘Okay, they acquired it on this day, at this hour, at this time, and they’ve had it colonized for this long.’ But it essentially just means that you have the bacteria within your body, but your immune system is strong enough to not allow it to spread, and usually it’s in some sort of latent phase of its replication cycle, where it’s not active at the moment, so it just, it’s just hanging around, basically.”

Using qPCR, Nested PCR, and Sequencing for Serotype Research

How does qPCR support the research workflow?

Donna Paznar: “Everything that I do is reliant first on we use qPCR. With Streptococcus pneumonia, we don’t actually have a threshold level, so to say. So we don’t have, ‘Okay, if the Ct value is below this, you know, we might have to think about whether it’s really positive or negative kind of situation.’ Anything that’s under the Ct of 40 we consider as positive, and then we always, if it’s very, very, very low Ct, we always do a retesting of it.”

Donna Paznar: “But the biggest thing that comes from that is again positivity or negativity. So, as soon as I have that information, which is huge, that’s when I then go to further down the line of the sequencing. So, from there I’ll usually run a nested PCR, so I’ll run a nested PCR with the extracted DNA from the subject sample. We will basically then send that off the product off to sequencing, or we’ll sequence it ourselves. And then that’s when the fun begins, because then it’s basically, you know, aligning sequences and figuring out all of exactly what serotype it is, and things like this, but that also is very difficult with Streptococcus pneumonia, because there’s over 100.”

What is nested PCR, and how does it differ from qPCR?

Donna Paznar: “Yeah, yeah, of course. So, um, basically, so it’s again a polymerase chain reaction. So, what’s different about a nested PCR compared to a qPCR is usually there’s two sets of primers that are used, so, and you do two successive PCR reactions. So you do your, you run your first round, so you have round one, so to say, with your first set of primers. And specifically with my research, that first round is actually meant to amplify a broader range of the DNA region of Streptococcus pneumonia. So we know, all right, we’ve got Streptococcus pneumonia from our qPCR, we found out, okay, we’re positive.”

Donna Paznar: “From there, that’s when it goes into the first round of the nested PCR, where we kind of amplify this broad region of the Streptococcus DNA. And then the second primer, this sometimes is like referred to as the inner primer, and it uses a small sample of the first round, so you actually use the product from your first round of PCR, and then you use that in your second master mix with your second set of inner primers, and this usually, or is meant to, which is like the fun part of designing your own primers and assays, is targeting a smaller sequence of the bacteria that you’re looking at.”

Why is nested PCR useful for identifying bacterial serotypes?

Donna Paznar: “Yeah, absolutely. And like another way you could like also say that is the fact that it’s so highly specific. It’s so, so, so highly specific compared to other methods, or just a normal conventional PCR, for example, where you’re just using one primer set. So because there’s over 100 serotypes, you can imagine there is very many genetic differences between each of the strains, but they’re very small, like they’re not jumping out at you, so to say.”

Donna Paznar: “And so nested PCR also, like the second part, is very, very specific to the serotype itself. So then you know that the product that you’re getting, not only are you reducing your chance of getting a false positive, but you’re also, you know, that your, your product that you’re getting is going to be very, very highly specific for that serotype, specifically.”

How could multiplex qPCR make future serotype surveillance more efficient?

Donna Paznar: “I think in the future the goal would be to not have to do the sequencing and be able to fully rely on the primer sets themselves. So that, and then again, I think part of my research will also encompass the fact that I’m going to group the serotypes based on those serotypes that are also covered within and with, like, those that are covered with the vaccines and those without.”

Donna Paznar: “The goal is to be much more cost effective for the healthcare system, but right now, of course, we’re spending the money to figure all these little pieces out. But yes, it is, it is definitely much more cost effective than running qPCRs or conventional PCRs with every single individual serotype.”

For Research Use Only. Not for use in diagnostic procedures.