Hello everyone and welcome to today's webinar with the webinar vet. My name is Catherine Bell and I am delighted to be your chair for the session today, which has very kindly been sponsored by Zaita. Before we get started, I just have a couple of quick housekeeping notes to run over.
So we're really pleased and very fortunate to have today's speaker with us live, which means you will have the chance to put your questions directly to her during the Q&A session at the end. So in the Q&A box at the bottom, if any questions come up as you're listening, please just type them in there and we will get to them at the end. As always, for those who are wondering.
Your certificates will be available once the session has been uploaded to the webinar vets platform, and we usually aim to do that within 48 hours, so definitely by the weekend they'll be ready for you. OK, so back to our session today. I am very pleased to be joined by Doctor Melanie Dobromilsky.
Melanie is a specialist in small animal diagnostic pathology, combining her clinical expertise with undergraduate and postgraduate teaching, as well as a range of research collaborations. She completed her PhD in molecular immology in 2009 and became a Fellow of the Royal College of Pathologists in 2014. Melanie also serves as an honorary senior lecturer in anatomic pathology at the Royal Veterinary College.
Her professional interests span feline pathology, immunohistochemistry, and diagnostic and prognostic research. She is particularly passionate about practical appliance education, empowering clinicians to make the most of pathology in improving patient outcomes. So welcome Melanie, thank you so much for being here live today.
Big thank you again to. Saya for sponsoring this session and I will pass over to you now. Thank you very much.
Thank you very much for that lovely introduction. So yes, today's webinar is all about PCR and you might be wondering why a histopathologist is talking to you about PCR because it's a very molecular technique, and I'll explain why. So before I became a pathologist, before I even thought about becoming a pathologist.
I did a PhD that involved a lot of PCR and a lot of that PCR was very much from scratch, so it was starting with genetic sequences, finding areas that you could target that were different, designing the primers, making sure they worked, and optimising. So there was a lot of PCR in it and it meant that I got a really thorough grounding in how PCR actually works on the ground in the lab. And also along with that, obviously all the ways in which it can be good or bad or have problems or not work, etc.
And then when I became a pathologist and I went into diagnostic work, obviously PCR is becoming more and more an important tool in our diagnostic toolbox. So a lot of us are using PCR based tests every day, but a lot of us don't have a good understanding of how it works. And also why knowing how it works, why that matters when it comes to understanding the results that you get from that test.
So that's why we're here today. So this talk is going to run through how PCR works and it's gonna be quite a significant part of the beginning of the talk and if you already know how PCR works then you're gonna be quite bored because we are gonna go back to very basic principles and we're gonna go through each of the steps and each of the components in that reaction mixture to get a really good understanding of how it works and why all of these things actually matter. So if you do understand PCR already, this probably isn't the webinar for you.
But we're also gonna look at the different types of PCR and some of the commonly used terms so that you have a good understanding of those. We're gonna look at the pros and cons of PCR testing and importantly what those results that you get from your PCR tests, what they actually mean and what you should be doing with them in the clinical world. We're gonna look at a few case studies just to look at where PCR is already being used in, in our clinical practise on a day to day basis.
And then we're gonna finish off with some questions about services, platforms and the the future trends of PCR in clinical practise. So I told you it was gonna start from very basic principles. This is as basic as I can make it.
So we're gonna have a really quick recap on genes and messenger RNA and all of that stuff. So genes are obviously made up of DNA. Hopefully you will remember that from either undergrad or even your A Level biology.
So each gene is made up of different segments, and they're called exons and introns. Exons are the parts that actually have the sequence that encodes for what's going to become the protein downstream. And the exons are interspersed with things called introns, which are actually non-coding regions of DNA acting a bit like a spacer.
It used to be thought that these were just junk DNA and we weren't really sure what they did, but I think we're finding more and more now. They might actually be doing something. But for the purposes of this talk, we can just think of them as bits of spacer between the axons.
Every gene has a so-called go button at the beginning. This is the promoter region. This is where the enzyme is going to bind, that makes the transcription happen.
But it's also really important because it controls whether a particular gene is going to be expressed at a certain point of time or not. So it's a really important part of the gene. And then at the end you, you literally have a stop codon, and that is just a signal.
It's a certain sequence of nucleotides which tells the transcription to stop at this point and to cast off. A bit like if you get to the end of a row of knitting, for example. So the process by which we go from DNA to messenger RNA is called transcription.
There's also another another step called splicing which takes you from pre-messenger RNA which has both the exons and the introns in it, down to messenger RNA, whereas just the exons, so the bits that actually are encoding. And then the process of translation that happens at the level of the ribosome, that's where we go from messenger RNA to a string of amino acids. I don't have time to go into the triplet code and all of that today, but that is an important part of it.
And your amino acids along chains of those, those are what go to make up the proteins. So that's a very basic recap. If we think about the DNA and the RNA molecules themselves, you'll probably know that these are made up of a sequence of molecules called nucleotides.
And those come in 4 different types. So you have adenine, which I've displayed here as an A. You have cytosine, which is the C, and guanine, which is a G.
And then you have thymoidine, if it's DNA, but in RNA this is actually replaced with uracil. So if you see a U in a sequence, you know, in a nucleotide sequence, that's, it's RNA and it's uracil. So I've denoted them here as these little cartoon molecules, you've got your C, your A, your G, and your T, and then you've got this sugar phosphate backbone as well, and that that's really important for the structure.
One of the really neat things about DNA and it's one of the things that makes PCR possible is that we know that DNA is made up of two strands. And these two strands are what we call complementary. And this is because when the DNA molecule forms, we know that the G always pairs with a C and the T always pairs with an A.
So it's what we call complementary. So if you know that the first strand is T CAG. You'll know that the complementary strand is going to be A, G, T, C.
And these will all line up and as I say, they're supported by the sugar phosphate backbone. The T and the A are joined together by 2 hydrogen bonds, and the C's and the G's are joined together by 3 hydrogen bonds. Now that is important and we will touch on that again later, but there's a difference between those two pairs.
And then that that double strand of DNA then twists around and that that forms what we know as the classical, the double helix of the DNA. So let me reiterate this whole thing about complementarity because it's really important to understanding PCR. So if you've got a single strand of DNA with all of its base pairs kind of exposed, that's gonna act as a template.
So we take that template strand and if we know the sequence of that, we can predict what's going to bind to it and what the complementary strand, what the sequence of the nucleotides is going to be. So you will know that the T is going to bind to an A, the D to a C, and so forth all the way along. And this complementarity is really, really important when you think about how PCR works.
Which is what we're going to look at next. So if you type some interesting words into AI this is what the AI image it generates. This is what it thinks of the molecular photocopier looks like.
Certainly when I was in my lab, I, I don't remember anything looking quite like this. I love the way that all of the AI images always have a really cheerful face on them, but I don't remember, I don't even know what these are, these little blue octopuses running around doing something. Anyway, so what is PCR?
It's a molecular technique and it's designed to detect a very specific sequence of that genetic code within a much larger pool of genetic information. And then when it's found that very specific sequence, it then amplifies it up until it gets to a level where we can detect it in different ways. So this is where we get the concept of the molecular photocopier from.
And in fact when I was doing my er PhD this was the type of machine that we were using and I I don't remember it having a smiley face sadly. So now let's get into the nitty gritty of how PCR works and to understand how it works, you first need to think about what all the different elements are in that reaction mixture. So in each of these little tubes, there's going to be a mixture of things and that's where the reaction actually takes place.
So this is our ingredients list, it's a bit like a recipe for a cake or something. So in there we've got a mixture of a buffer and some salts. Er, the level of salts will vary depending on each particular PCR and as part of that optimisation and validation process, they will have played and and varied the level of salts to get the the reaction to be absolutely optimal.
Most of it is made up of water, but you have to be very, very careful, you can't just use any old water, it's got to be really, really sterile and clean. You mustn't have any contaminating DNA or RNA in there obviously cos then you're gonna get false positives. You need your free nucleotides, so that's your G's and your C's and your T's and your A's.
These are the building blocks that are going to go and make up that new DNA that you're going to produce in that reaction. You need an enzyme that's going to catalyse that reaction, and that's called tac polymerase, and we're gonna talk a bit more about that in the next slide because it's important. You obviously need the sample that you're going to test and that may or may not include the sequence that you're targeting with your particular PCR.
And then you need a pair of things called primers. The primers are actually really, really important, they're a critical part of the PCR and and to understand how PCR works and why some PCRs are better than others, you really have to understand what primers are and what their role is because each PCR test is going to have its own unique pair of primers that have been specifically designed for that particular test. So let's have a look in a bit more detail at tac polymerase.
So the name tac actually comes from the name of the bacteria that it comes from, which is Themis aquaticus, that's tack, and this is what AI thinks that tack looks like, that's quite cute. So this bacteria actually handily for us is a thermophilic bacterium, which means it's evolved to work at very high temperatures. So for example, in this picture here, this is, Yellowstone National Park.
This is one of the hot springs and this yellow area here is where this bacterium likes to hang out. So it's evolved to work at very high temperatures. And this enzyme, its primary function is to synthesise new DNA so that's very handy for us.
So it takes individual nucleotides from your reaction mixture, so that's your G's and your T's and your C's and your A's, not, not the B, which is up here in the, never heard of a B nucleotide. It takes those individual nucleotides and it assembles them into a brand new strand of DNA. And it does that by using another DNA strand as a template, you remember what I was saying about it being complementary.
So the DNA already in there is what acts as the template. So as I said, luckily for us it works at high temperatures and it's stable even at very, very high temperatures that we need to denature that DNA which again is really handy for us in the lab because it means that we don't have to add new enzyme in every time we go through a cycle of the PCR reaction. So that's tack The other really important thing that I want to spend a bit of time looking at is these primers.
So every PCR reaction will have a pair of primers and you will have to have designed those primers to bind to very specific bits of DNA. And in order to make your PCR specific, your primers must buy into a unique sequence that you can see in the area of interest. So for example, if this .
Sequence of nucleotides is what say you're you're targeting, this is what you want to look for. This is the two different strands, so we've denatured it and we've got two strands. One of your primers needs to bind to this end of this strand and to nowhere else, and the other of the primer pair must bind to the opposite end of the other strand and nowhere else.
And that's why primers are so important when it comes to designing your PCR. So they're chemically synthesised, they're just short sequences of nucleotides, so you might hear them called oligonucleotides or oligos. And when you're designing a a new PCR you have to design your primers very carefully.
So that they only bind to very specific areas of your DNA sequence. So when you're designing them, and I did lots of this in my PhD, you have to find a section of DNA you want to detect. So it has to have unique sequences in both of those areas where your primers are going to bind.
It's got to be at one end of one section and the other strand of the other end of the section. And not only that, but the, the bit in between, the length that you are going to replicate has to be a good length for your PCR reaction as well. So not only have you got to find two unique sequences to design your primers to, they've got to be the right distance apart as well.
And as I say, it's really important those primers bind to those areas and nowhere else because that's what gives PCR its specificity. So anyone who like me er has done a lot of this sort of er designing primers for PCRs and validating them, you'll know there's a list of rules that are really important when it comes to designing those primers. So your primers, they must bind to the right places, obviously, otherwise your PCR isn't going to work.
And they must bind really well and that's what gives the test its sensitivity. Just as importantly, they mustn't bind anywhere else and not just in, you know, that that particular sequence, but they mustn't bind to any DNA anywhere that could possibly be in your sample. So that's quite a big ask.
And they mustn't bind anywhere else because that's what gives you your specificity. If they did bind to other areas, then you'd be getting false positives. So you might be feeling really happy that you found two unique sequences where you can design your primers and they're the right distance apart.
And then you need to look at those primers and make sure that they're, they're OK. And there's lots of other things you have to check for. So, it's really important they don't do something weird like forming secondary structures like primer dimers.
So, primer dimers happen when one primer sticks to another primer rather than to your target sequence. And obviously if they're sticking to each other, they're not going to be detecting the thing that you want. So, you know.
Your 5 prime primer could bind to another 5 prime primer, so that'll be a self dimer, or your 5 prime primer could bind to your 3 prime primer, cross dimer, or an individual sequence or primer could bind to itself and could form one of these things called a hairpin. And that's incredibly frustrating if you think you've managed to find the right place for your primers, and then you run it through the checks and you find, no, these are no good, and you have to go back to the drawing board. There's a couple of other things that your primers need to kind of tick on the list, they must be just the right length.
So obviously the longer your primer is, the more specific it's going to be. So you know, the more nucleotides you've got, the better it's going to be in terms of binding to the right place, which is what we really want. But if you have primers that are too long, then that binding process, that reaction isn't very efficient.
So they have to be just the right length. They also have to be, rich in GCs and as I said earlier, G and C have the three hydrogen bonds between them, so that makes it really nice and a good stable bind as opposed to just the two hydrogen bonds between the A and the T. So we like our primers to have a high GC content because that makes them really stable when they bind and particularly at the 3 prime end, something we call a GC clamp, just means that once they bind, that's it, they're bound and they're nice and stable.
And then after all of that, your pair of primers obviously has to work under the right under the same conditions as each other because obviously they're both going to be in in that same tube. So after all of that, you've got to make sure that they've got similar melting temperatures and that they're going to anneal to the target under the same temperature, for example. So there's lots of validation and optimisation to be done as well.
So. Designing primers isn't necessarily that easy a thing to do, and testing them and getting all of those conditions just right is quite a thing to do. If you're interested in this particular aspect of PCR, I did find a really nice blog that had lots of nice interesting articles in it, and I've got the link to that at the end as well.
But the reason I'm going into this is to explain why not all PCRs are equally good. So you might have 3 different PCRs that all claim they're detecting the same organism, for example. But they're all likely to have different primers and different conditions and so they're not all necessarily going to be equally good.
And that's why this really really matters that you have an understanding of this. So let's talk through the actual process of PCR itself. So here I've got a double strand of DNA.
This is going to be our area of interest. So the first step of the PCR is to heat that up to about 95 degrees C, and that causes the double strand to break apart. And instead of that double strand, you have two separate strands.
And all of these nucleotides are then exposed, so they're not bound to another nucleotide. So at this stage, effectively you have two template strands, but they are running in opposite directions. So hopefully at the next stage, you then cool that reaction down to your annealing temperature and that's going to be the temperature at which your primers are going to bind hopefully to that site of interest.
So obviously we've got one binding at this end of this strand and then the opposite the opposite end of the other strand. That annealing temperature again is going to depend on your particular pair of primers and it can be anything from sort of 50, 60 degrees. But again, that will have been optimised while the PCR was being developed and tested.
So at that stage, we've then got all of our free nucleotides in that reaction mixture as well. Our enzyme tag is gonna come in and it's gonna find the end of this and it's gonna start bringing in these free nucleotides and using the template strand as a as a template. It's gonna look at what's here and it's gonna say, OK, there's a G, we need a C, there's a C, we need a G, C, G, and it's gonna keep moving along the strands, basically synthesising new DNA using this as a template.
It's gonna do it for this strand, but it's also gonna do it in the opposite direction for the other strand as well. And that basically continues until that whole strand has been replicated. So you know we started with one copy of this area of interest and at the end of that cycle we've now got 2.
So let's just recap that. So PCR has multiple steps. The first step is denaturation, so that's where we heat up to about 95 degrees, and that separates our double strands into two separate single strands which can both act as templates.
Then we have the second step, which is the annealing step, we cool that reaction down to anywhere between 50, 60, 65, and that's so our primals will come in and they will bind to that area of interest and hopefully nowhere else. And then the third step is extension. That's where we warm it up again to about 72 degrees because that's the temperature that our tac enzyme likes.
And then that basically starts to build a new, strand of DNA using this one as a template. It makes the complementary strand. So at the end of each cycle, we have effectively doubled the amount of our target DNA that's in our reaction tube.
And then essentially all that happens is that we repeat this cycle many, many times until that fragment of DNA becomes detectable. And there's several different ways in which we can detect it. But that is essentially PCR.
So when we think about how we detect it, this is mostly what I was doing for my PhD, so we use a technique called Agarose gel electrophoresis. So you pour these gels and they have little wells in here. And you take your PCR reaction with a loading dye so you can see what you're doing and you very carefully pipette it into little wells in this gel.
You then apply an electrical field to that, so DNA, the backbone is negatively charged, and your fragments of DNA will migrate towards the positive electrode which should be at the bottom of the gel. Unless you make, make a common mistake, which is to put the lid on the wrong way round, in which case, your DNA fragments will go off the top of the gel and into your buffer. Or if you run it too fast and you forget that you're running a gel and it might just run off the bottom and into the buffer as well.
But with any luck that won't happen. And your DNA fragments will migrate towards the positive electrode, and the important thing to know is that the smaller the fragment of DNA, the faster it will travel in the gel and the further it will travel. And that's what helps us separate out all the DNA that's in that particular sample, depending on the size of the fragment.
And of course when you designed your primers, you will know how far away, how far apart they were from each other. So you will know how big that piece of DNA is that you are hoping to replicate is meant to be. And so you can look at your gel and you can see is it positive or not, and is that fragment the right size.
So in order to visualise that DNA we add a fluorescent dye to our gel. It's called thidium bromide and this dye intercalates with molecules of DNA. And then they, they light up when you view that under UV light.
So this is a gel from my own PhD. Probably not a very good one, but it will do. So you also have what we call a molecular weight ladder down the side.
So this is made up of a mixture of fragments of DNA of which we know the size. So we know that this is say 50 base pairs, 100 base pairs, 200, 250, all the way up. So we can compare the bands in our reaction mixture to the molecular weight ladder.
So we can see whether the band that we can see is roughly the right size or not. When you're establishing a new PCR and you want to check that it is amplifying the correct thing, not only can we look at the size, but you can also actually cut this DNA out of the gel and you can sequence it to make sure that your primers really have detected the sequence that you wanted them to, so that's an extra step that you can do. The more intense the band is when you view it under UV, the more DNA is in the gel.
But, you know, this is semi-quantitative at the very best. So it's not really a quantitative technique. So here we have the molecular ladder.
You'd always have a positive control in every PCR run, so this you would include a sample that you know had the area of DNA that you were testing for, and that's just to ensure that everything in your reaction mixture has worked, the PCR machine has worked for example, so that you don't get any false negatives. And then you would also have a negative control, so you'd have a sample which didn't have your target sequence and you would run that through and hopefully you wouldn't see any bands at all. If you see a band in this one, this means you've got contamination and that means that you have to discard all the other results.
So it's very important, every PCR run would have a positive and a negative control included as well. I said I'd talk a bit about terminology, sometimes people can get a bit confused. So the PCR that I've just been talking about, the standard form of PCR basically the DNA gets measured but only at the end of your PCR reaction.
So you'd run it through a number of cycles and you'd run it on the gel at the end. That's very different to something called quantitative PCR, which is much more common these days, or QPCR. That's a technique that actually measures the amount of DNA as the reaction happens, so you can see pretty much at the end of every cycle how much DNA is in your, particular reaction.
Because of that, it can also be known as real-time PCR because you're watching it in real time or quantitative real-time PCR. And I put this in partly so that you don't get confused with something called RTPCR cos real time RT you'd be very easy to get those mixed up. But RTPCR tends to refer to something different, which is reverse transcriptase PCR, and that's where we use another enzyme to convert RNA into DNA and then we do the PCR on the DNA.
So I just wanted to put that in just to clarify if anyone was a bit confused about all the RTs and the Q's. So quantitative PCR is really cool because as I say, it's quantitative, but also it's real time and you can see how much DNA is being produced as the reaction goes on. There's two broad types of QPCR.
One of them is a dye-based method, so within your mixture along with all of your free nucleotides, you'd also have some dye molecules and basically as that PCR reaction progresses and as you're forming new DNA. The dye gets integrated within that new DNA at regular intervals. So obviously as more and more DNA is formed, you're gonna get more and more fluorescence, and you can detect the fluorescence in special machines and that's what helps you quantify the amount of DNA in your reaction.
The second type of QPCR is what we call probe-based Q QPCR, and this is basically a schematic of the probe. So in these cases, as well as having a primer pair, you also have a probe which is designed to be specific to a sequence in the middle of the area of interest. And that is combined with something called a quencher molecule and a reporter molecule.
And basically when your probe is intact, the quencher molecule stops the reporter from reporting. Hang on, there we go. So as long as your probe is bound and intact, the quencher stops the fluorescence from being released.
So in a reaction, I've only shown one strand here, but this is your primer binding to one end and your probe binding to the middle of your sequence of of interest, your target. And at this point, this is intact, so the quencher is stopping any fluorescence from being released or reported. As that reaction progresses and you have new DNA being synthesised, once it comes up to the level of the probe, it actually starts to destroy the probe, and what happens is that the quencher becomes separated from the reporter.
And once that happens, obviously the reporter is free, and it can fluoresce away as much as it likes, and that's what's being detected in the machine when we're looking at the levels of fluorescence. So two slightly different techniques. QPCR gives us a bit extra information, obviously, it gives us something called a CT value, and I just wanted to talk a bit about what a CT value is and why it's important.
And those lovely curves that you see, what, what do those mean? Now, some people will say that CT values aren't that important and PCR is more of a black and white thing, but I think that's, that's not correct. So a, a CT value is actually a really important piece of information to have and to understand.
So when you're doing QPCR there is a kind of background level of fluorescence, and you have to get above that background fluorescence level before we can say that's definitely a real signal, so we're definitely seeing some new DNA being built. So that's our baseline. And the CT value is the number of cycles of PCR that's needed for the fluorescence to go across that threshold.
So basically a CT value is is in is . Is proportional, it's indirectly proportional to how much target is in that test sample. So, the lower the CT value, the higher the amounts of target were in the test sample in the first place.
And it's also important because this is the logarithmic, relationship as well. So basically the fewer cycles you need to get above that threshold means that the fewer cycles you needed to get the target above detectable levels. So if you're thinking about something like a pathogen, for example, the lower the CT value, the more of that pathogen was present in the sample at the beginning of the reaction because you needed fewer cycles to amplify it up to a point where it's detected.
And for things like potential pathogens, that's really important because it's gonna tell you something about, you know, viral load for example or how significant is that particular pathogen. Could it be actually responsible for the clinical signs. So CT values do do matter in my opinion.
And so this is how we represent that, so this is a nice quantitative PCR curve. So on this axis you have obviously the amount of fluorescence that's being detected. Along this axis here you have the number of cycles that that PCR reaction has gone through.
And obviously this is what's being measured, by the machine. So in each of these different colour curves, basically you started with a different amount of targets in that reaction. So you can see for the, the, the sample that had the most target at the beginning needs the fewer number of cycles to cross that fluorescence threshold.
So. This one crosses here sort of somewhere around 12 cycles. Whereas, you know, at the other end, you're talking about say 30, 35 cycles before you can even detect anything significant in terms of fluorescence.
So when you're thinking about interpreting results, it's really important to bear the CT threshold, the the CT number in, in, in mind. And personally, you know, if you've got a reaction where they needed 35, 40 or more cycles to be even able to detect the pathogen. I'm not gonna take, I'm not gonna put as much, value on that as I am as something that only say 12 cycles before it was, detectable.
So that's a little bit about CT values and I, I hope that makes sense and also explains why they are important. So that's PCR we're gonna move now into looking at the pros and cons of PCR testing, and I don't really feel like pros and cons is the right terminology to use, it's really the things that you need to bear in mind when you're doing PCR testing and interpreting the results. So when you think about what does PCR actually tell you, when you strip it right back, fundamentally, all it's telling you is that a particular sequence of genetic code has been detected in your sample.
And then I put probably in brackets because obviously that depends on how specific your PCR primers are as well. And then if you're doing QPCR it's also going to give you a good idea of how much of that sequence was present in your reaction at the start. And that's really it, that's what it's telling you.
Maybe more importantly to bear in mind is what PCR is not telling you. So for example, if you're looking for an organism that might be a pathogen, it doesn't tell you that that pathogen was intact. I mean, it could be fragmented and all of the genetic code could be in little bits and pieces and it just so happens the bit that the PCR is looking for is intact and it's been found.
Doesn't mean that the rest of the organism was intact. Also often can't tell you whether that organism was dead or alive, although some of the more up to date ones are trying to factor in whether, you know, markers that will tell you whether it was alive or not, but it, it might not be alive. And even if it is alive, it might not necessarily be causing the clinical signs or the pathological lesion that you're trying to investigate.
It won't tell you where that organism, or well, that genetic sequence was in the lesion itself. It's not going to tell you which cells or tissues it's associated with and it's not gonna tell you whether there was any associated tissue or immune response to it either. So there's quite a lot that it's not telling you.
And I think that's really important to understand that when it comes to interpreting your PCR results, it's very much important to put it in the wider clinical context. And there is no such thing as a perfect test. If anyone tells you a test is perfect, then I'm afraid they're pulling your leg.
There is no such thing as a perfect test, and PCR is not an example. So it's possible to have false positives and false negatives, like any test. So for example, you might get a false positive if there's a cross reaction.
So perhaps that particular pair of primers is detecting something else that's very relat closely related to the thing it's targeting, but it's picked up the wrong thing. There might be contamination in that sample, for example, and again, that's why you would have a negative control in there to try and, you know, detect that if it happens. You might detect an organism, but it might be dead or non-viable or fragmented, or it might just be a commensal or otherwise not particularly significant from a clinical point of view.
You might get a false negative due to sampling error, for example. Or if your primers aren't good, so you know, if the PCR you're running doesn't have the best primers, you might get a false negative. It's, you know, the, the, the, the, the sequence you're looking for is there, but it just hasn't been detected.
The reaction might fail in some reason, of course that's why we have our positive controls, so hopefully that would be picked up in your test. Or maybe there's a new sequence. So we heard a lot during COVID about new mutations and new strains coming up and certain organisms, certain viruses in particular are very good at coming up with new mutations all the time.
So it's just possible that if you're looking for something like that, there's been a mutation, and if that mutation happens where the primers are meant to be binding, it might mean that your primers don't work anymore. Seems quite unlikely, but it's, it's possible. So these are all things to consider.
So I'm now gonna move on and look at some examples where PCR is already being used in veterinary medicine, just to give you some examples of where it could be, you know, potentially useful. And I'm gonna give some examples of where it's used in genetic screening. I'm going to look at tumour diagnostics and prognostics and I'm going to look at detection of potential pathogens.
So for genetic screening, I've just picked a couple of examples which I think are, you know, relatively well known. Obviously, there are some genetic mutations that we've identified, in cats with hypertrophic cardiomyopathy. So we found these two particular mutations in these two particular, breeds of cats.
And I put this picture in of my Maine Coon cross cat and then only afterwards realised that actually my rag doll, funnily enough, is hiding in the box underneath. So that was, that was very appropriate. And then the second example which I think is really good is polycystic kidney disease, that we can screen for in Persians and related breeds because we know that there's a single nucleotide polymorphism there that we can detect in the PKD1 gene.
So hypertrophic cardiomyopathy is something that I'm particularly interested in, and we do know about these two particular genetic mutations, one of them in the Maine Coon and another one, same gene but different mutation in the rag doll. So we can screen for these using PCR, and it's really helped in terms of trying to reduce the prevalence of these mutations in the breeding cat population. So homozygous cats are not used for breeding now, but even if you're going to screen non-breeding cats of these breeds, it also gives you an idea of the relative risk of that individual developing HCM later in life.
And Maine Coon cats that are homozygous do tend to develop clinically relevant HCM. But it's not perfect, and obviously there are other genetic mutations out there as well, so it has been found that cats negative for that mutation can also develop HCM. So it's a good screening tool, but it's not the whole answer.
And also interestingly, these two mutations are almost completely breed specific, so there's no point in screening non-Mine Coon or non-rag doll cats for these mutations, so it, it's not recommended, but it's a useful tool. And the other genetic disease where PCR testing has been really, really beneficial is polycystic kidney disease in Persians and related cats. Now when I graduated back in 2004, we used to see these cats all the time.
So I was a Bristol, graduate, so I was, I saw a lot of these cats being undergoing ultrasound, for example, looking for those classical cystic lesions in the kidneys. And I pulled this graph from the Langford vet's website. I think it's really, really nice because it shows that by using this screening for this genetic defect, they've managed to radically reduce the percentage of cats that test positive over time.
So you can see starting in 200. It was really quite common and then now the latest data was for 2018. So using that screening to obviously select, which cats you breed from has meant that they've managed to reduce the prevalence of this disease in that particular breed, which I think is great.
So another area where we already use PCR quite a lot in our day to day clinical work is tumour diagnostics and prognostics. I'm gonna just talk about a few of the more common examples, but also I think we are very much just at the beginning of this and in the future I wouldn't be surprised if we were doing a lot more sequencing of tumours, looking for different mutation signa signatures. And I think as we learn more and more about tumours.
In humans and in animals, we'll start looking for specific mutations that may be present, and that might help us with tumour diagnostics, with prognostics, but also in determining which treatments are most likely to be effective in any individual or particular case by looking at what mutations are present in that particular animal's tumour. So, you know, this is gonna become more personalised treatment planning and I think PCR will play a major part in that. But let's have a look at some of the common examples that already happen, so one that you're probably familiar with is a PCR that looks for mutations in the CIT gene.
So in particular in canine mast cell tumours, although we also can use this for gastrointestinal stromal tumours, and there is work also looking at this in canine oral melanomas. So for canine mast cell tumours, if the tumour has a mutation in exon 11 of this gene, it's been shown to be associated with a shorter disease-free interval and survival time and with an increased risk of dying and of local or systemic recurrence. Interestingly, if you have a mutation in the X1 8, it doesn't correlate with more aggressive behaviour, but having a mutation in either of these means that a particular type of, chemotherapeutic agent is more likely to be effective against that particular tumour.
So if you have a mutation in X1. 11 or exon 8 of the, the, the canine mast cell tumour, it's more likely to respond to therapy with tyrosine kinase inhibitors because it's it is actually targeting that particular protein that the gene encodes for. So it's very helpful when it comes to choosing which treatments to use for a particular individual animal.
BRAF is another one which is commonly being used now, so a mutation in the BRAF gene, this occurs in transitional cell carcinomas or also known as urothelial carcinomas, so arising in the urinary bladder and also in prostate carcinoma. It's a really specific test if it comes back positive because you don't find this mutation in, for example, a hyperplastic lesion or a chronic inflammatory process which may be presenting with a similar clinical signs. Unfortunately if it's negative, it doesn't fully exclude the tumour because not all of these tumours carry this particular mutation, but it's a very, very useful test.
It can be used for early detection of these tumours in breeds that we know are at increased risk of developing them, or if you're investigating. Clinical signs where a tumour is one of the possible causes, it's a nice way to try and rule it out, and it's a non-invasive test sample because it's really neat. The tumour cells are shed in the urine and we can actually look at those tumour cells and because PCR is so sensitive, we can look for the mutation in just those tumour cells.
So that's another example of where PCR is already being used. I haven't really got time today to go into the ins and outs of how Par works, it's quite complicated and it would be a a presentation all of its own. But PAR also involves PCR.
It literally stands for PCR for antigen receptor rearrangements. And it's really important as part of diagnosing lymphomas in particular cases. So obviously lymphoma is characterised by having large numbers of lymphocytes, but we can also see large numbers of lymphocytes in a reactive, lymphoid hyperplasia or in chronic inflammation.
So if you're trying to determine whether. A particular lymphocyte population is neoplastic or just reactive and inflammatory, you can look to see if they're clonal or not. So if all of those lymphocytes in your lesion have come from a single clone, then it's very likely to be neoplastic.
Whereas if in that population of, of lymphocytes you have a mixture of different clones, it's more likely to be inflammatory. So PAR is a test that helps us look for that. And so it's particularly important if you're trying to differentiate something, you know, between is this a lymphoma or is this atypical reactive lymphoid hyperplasia or chronic inflammation.
You would do immunohistochemistry for your lymphoid markers and then you may or may not also include PAR looking for clonality as part of that wider investigation. So that's another time where PCR is already being used a lot in our clin clinical diagnostics. So the last examples I'm gonna look at are detection of potential pathogens.
And just generally speaking, I think PCR is particularly useful if you have pathogens which are very difficult to culture, potentially dangerous to culture, maybe you don't want to be culturing these particular agents or if they take a long time to grow, or if they might be overgrown by other agents in the same culture. And another good thing about PCR is, you can do it on fixed tissues. So occasionally, sometimes we have a case where you want to try and diagnose a particular pathogen and you don't have fresh tissues available.
Obviously you can't culture anything from fixed tissues cos everything's been killed, but if you've got fixed tissues, PCR is a potential option. It's better done on fresh tissues because, formalin fixation does cause some damage to the DNA, but you can still do the PCR on the fixed tissues, which you definitely can't culture. But remember what I was saying earlier on, interpretation is the key.
So you know, yes, PCR will tell you whether that bit of sequence is there or not, but it won't tell you if the organism was intact, it won't tell you if it's alive, it won't tell you if it's causing the clinical signs that you are interested in. It doesn't tell you where it is in the lesion, which cells, tissues, what it, what associated tissue response. And then of course you've got all the questions about what do you do about multi-agent conditions, and then it's very important that you, you know, you look at things like your CT value, but you also look at the clinical signs and you use your clinical expertise and you use that PCR result as part of the, the bigger picture.
And this is why I say interpretation is really important. So talking about infectious diseases, anyone who knows me knows that I'm really interested in mycobacterial disease. And this lovely guy here, this is Brian.
So Brian was a patient of Professor Danielle Gilmour up at the University of Edinburgh and she very kindly lets me use him for all of my lectures where I'm talking about mycobacteria. So this is Brian. And I particularly like to use them when I'm teaching undergrads, and I say to the undergrads, OK, what, what are your diagnoses?
What, what are the differentials for this mass on this cat's nose? And they're all like tumour, tumour, tumour. So Brian says not all masses are neoplastic because Brian actually has a mycobacterial infection on his nose.
And mycobacterial disease is really important. It's surprisingly common. Not everyone realises this, but actually previous studies showed that just over 1% of all routine histopath submissions from UK cats are diagnosed with mycobacterial infection.
That's probably an underestimate, but it is surprisingly common. Also has quite variable clinical presentations, er, depends a bit on the immune status of the patient, depends on the species of microbacteria and that's why it's useful to be able to speciate. Also depends on the route of entry.
They can be quite challenging to definitively diagnose. They obviously have zoonotic potential, which is really, really important and some of the mycobacteria species more so than others. So again, it's important to know what type of bacter mycobacteria you are dealing with in any particular case.
And it is potentially treatable as well, as Brian likes to show. So when I'm thinking about Mycobacteria in cats, obviously there's lots of different species. So the way I like to think about it makes most sense to me is to break it into two broad groups.
So you have the Mycobacterium tuberculosis complex or MTB and you have the non-TB Mycobacteria complex as well. And I find it useful to split them because they have slightly different characteristics. So if we start with the Mycobacterium tuberculosis complex, this is about 1/3 of all of the infections that we see in cats, and it causes a tuberculosis type disease as you would expect from the name.
The strains are species adapted, which is really important when you think about the pathogenesis, but they're not associated with immunosuppression of the host. There's also a geographic prevalence, so if you're in certain parts of the UK there's an increased likelihood that you'll see a particular type of Mycobacterial infection in your area, and it's worth being aware of that. And it tends to affect males, outdoor cats and hunters.
So in this group, we have some important species, we have M.mcoti, and this is actually a rodent adapted strain, so obviously if your cat's a hunter and it's going out and it's catching rodents that are infected, then it's at risk. And this is quite a common form to be identified.
We have Mbovis, which is obviously the cattle adapted form, and that's also quite common, particularly in certain parts of the UK. And again it's very important to be aware of that because of zoonotic potential, etc. Etc.
And then we have the M. Tuberculosis. This is the human adapted one.
Now actually cats display really good natural resistance, so it's incredibly rare to get an infection with M. Tuberculosis, but it's, it is in that group. And then the non-TB mycobacteria group, these are just environmental, so they're all around us, they're in the soil, they're in the water, but they can be opportunistic infectious agents and they can get into contaminated wounds and they particularly like to target immunosuppressed patients.
So it's important if you're dealing with one of these particular species and any infection that you look for any underlying cause of immunosuppression. And these will include things like the, the feline leprosy, the Mac complex, some of the slow and rapidly growing types as well. So in terms of diagnosis, on histopathology, we can't actually see mycobacteria, so this is a hematoxin ein stain.
We see a very typical granulomatous to pyrogranulomatous inflammatory response, but we can't see the bacteria in H&E, but we can be quite suspicious when we see this pattern. What we do have is a special stain called Ziel Nielsen or ZN. So the blue is obviously the counter stain, so you can see all the nuclei, and then these thin red or magenta structures, these are the mycobacteria.
So if you're having a good day and you're being lucky, your case will have lots of organisms like this and it'll be quite easy to diagnose. But more often than not in cats, there are very, very low numbers of organisms present in these lesions, and you can spend a very long time hunting the ZN stain to find maybe even a single little infectious agent. So they can be quite difficult to definitively diagnose.
You can see something like this and be really suspicious. Sometimes the ZN stain is not always that helpful. But the other important thing to remember about the histopathology is I can't tell you the species of Mycobacteria by looking at it down the microscope.
So histopath is great as a first line of diagnosis. Although if your ZN is negative, you can't entirely exclude it. If you've got the reactive, the, the inflammatory lesion that you would be very suspicious of, it's still a differential.
And histopath can't speciate. There've been lots of studies trying to see a particular pattern on the histo links to a particular species and we haven't found anything yet. So if you have a positive diagnosis, you still need to do further testing in order to work out what kind of mycobacteria you're dealing with.
And as we've seen from the previous slides, this can be really important. So your options are culture. Now this is actually the gold standard.
You must have fresh or frozen tissue but not fixed. But it can be incredibly slow to grow. It can take up to 6 months to culture, and to actually get anything to grow.
And about 50% of cases where they've actually seen the organisms on the ZN won't grow anything at all. So often it, it doesn't give you an answer even if you wait 6 months. There's also a blood test that's available, so the interferon gamma release assay, that's really handy, you do need to take a blood sample for that.
It's pretty sensitive, so it is another option, particularly if, you know, the patient doesn't have any evidence of an actual lesion at that time, it's a very good screening tool. And it can give you a good indication of what type of microback you are dealing with. But we also have PCR.
And PCR is particularly useful because often with these cases, cat comes in with a mass, it goes to surgery, it has a complete excision. They're thinking it's a tumour, so they put all the tissue and formalin. And then we diagnose mycobacteria and we say, oh, have you got any fresh tissue?
And they say, no, we fixed it all. So PCR is actually an option in this case, fresh is preferred, as I've said, formalin fixation does cause some damage to the DNA but we can use PCR for on formalin fixed tissue to try and get you that species, and that's really important when you're deciding whether to treat, what to treat, and whether to look for immunosuppression, thinking about other intact animals and the and the source of the infection as well. So those are your kind of options and PCR is very definitely part of that.
Just a couple of other kind of clinical scenarios where PCR can be useful, but only as part of kind of a wider investigation. Feline conjunctivitis is a really nice example, so you can do a PCR panel for potential infectious causes, herpes virus, chlamydia, mycoplasma, but don't forget you've still got to exclude all of those non-infectious causes, and this comes back to your clinical context and your clinical skills. So you know, entropion, any irritants, foreign body trauma, etc.
Etc. And then you might do the panel and you might have more than one come back as positive, so then what do you do? And this is where things like the CT value become more important because if you've got one particular organism and it's present at very high levels, then you might be more suspicious that that is significant.
But also don't forget to correlate back with your clinical signs, you know, take that PCR result and look at your patient. So for example, if it has something like a dendritic ulcer, which is virtually pathoneumonic for herpes virus and your PCR is positive for herpes, then that's probably the one that you're going to need to focus on treating. So just to illustrate that PCR is a useful part of a wider diagnostic toolbox, but you've got to interpret it with your clinical signs and CT values, etc.
And then feline diarrhoea, I almost didn't put this one in because I know it's quite a controversial topic. There's lots of companies offering PCR panels, and they have different organisms in those and they may or may not be helpful, but I think it's a really nice example again of where PCR can be important as part of a wider diagnostic investigation. Which you also need to use your clinical skills and and interpretation as well, so you all know much better than me, you know, is it intestinal or is it extraintestinal, is it acute or is it chronic, is it small intestinal, large intestinal, mixed?
Could it be noninfectious, you know, dietary causes for example, even something like an interception. There's lots of things that the clinician has also got to think about. But if you do think it's potentially infectious then again there are a number of potential causative agents that you can screen for using a PCR panel in this instance.
But again it's really important when you get those results back, link them back to, you know, what is this patient looking like clinically. Is this positive relevant or is it just, you know, in the background, what do you do if you get more than one positive result back, you know, we do get co-infections with some of these agents as well, but also the importance of the sample, so what you put in, getting the best faecal sample in the first place is also really important when it comes to having confidence in the PCR results that you'll get back. So that's the end of my presentation.
If anyone's got any questions, I'm very happy to take those now. If you're interested in this and you want to do a bit more reading, this is the website that had all of the information about primer design and PCR that was, I found that very readable and very understandable and I'd recommend that. And then Emmy Barker a few years ago now, 2021, wrote a really nice article for In practise that looks at this topic and performing and interpreting them in dogs and cats and it's open access so anybody can get that if they want to have a bit more of a read.
We've also got the website of, Zaita, and this QR code if you want to scan it, that will take you to a site with lots more information about their particular products and, and PCR. And then if you want to reach out to me for any reason, this is my email address as well, so there's some extra, extra reading and information for everyone if you're interested. And that's the end of my talk.
Brilliant, thank you so much Melanie, that was really interesting and very thorough, so thank you so much. Lots of information there to digest. It's usually at this point I ask people to pop the questions in the Q&A box, but we have had loads, so, so I'll go straight in.
So first up we've got. Bit of a comment and then a question, so we've got PCR has traditionally been seen as something that lives in a lab, not in a clinic. But that's quickly changing.
Are there platforms now that make it practical and easy for general veterinary practise without losing accuracy or sensitivity? Yes, and that's one of the amazing things more recently. So back when I was doing my my PhD it was very much a lab based thing that we used in research, but more and more now, we're seeing.
These machines that you can use, you know, in the clinic in practise, and all of the hard work's already been done for you, so the optimising the primers and all of that testing has been done and it's been packaged so that you need minimal input, minimal expertise, and it's usually if you go for the right one, it's very simple to use as well. So yes, it is moving over to being more of a, a potential resource to have in your practise. Yeah.
Brilliant, thank you. We've got another question, so we all know that waiting on lab results can be a challenge in busy clinics, especially when quick decisions matter. How does the turnaround time compare between sending samples to an external lab and running PCR in the clinic, and also how does that affect case management?
Yeah, so I think you have to really think about those cases where having that quick answer really matters. So obviously if you're sending it to an external lab, even the best lab in the world, you're looking at a day, you know, and that's if they're running their PCR every day and if your couriers are all working and everything goes according to plan. But you know with these in-house ones, you know, to actually run a PCR takes, you know, less than an hour, so you could be getting a result back much, much quicker.
And for something that's very acute where you need to know that answer, say something like maybe you've got a dog that's come in with very acute hemorrhagic diarrhoea, you're worried about Parvo, you know, if you think you could get an answer back in an hour and you then know how to treat that patient, but more importantly, whether you need to quarantine that that patient. And think about the potential for for infection of other animals, you know, that that could make a huge difference. So I think for particular for particular cases, yes, there's definitely, you know, it could be very, very useful to be able to have that information very quickly.
Fantastic, thank you. When choosing a PCR platform, what should vets look for to make sure it's both clinically relevant and reliable? Yeah, so that's a really good question and probably there is no one correct answer.
It depends on your clinic. So I think all of the things I've gone through today, so you know, how robust is the PCR, how good are the primers, you know, is there positive control, negative control? Have they published data that shows that they've validated the test, that it works, you know, all of those things.
Are they gonna tell you things like CT values? That's all stuff that I would be looking for. But also, you know, is it going to integrate with your workflow in your practise, you know, do you have the space for it, can you set it up?
All of those things. So there's there's a lot to think about when you're looking at the different platforms but yeah, hopefully this webinar's given you the right questions to ask in that particular instance. I hope so.
Brilliant, thank you. We've just got a few more, are you OK for time if we just stay on? Yeah, OK, brilliant.
There's lots of buzz around PCR and with that can often come a few myths that can hold people back from adopting it. What would you say the most common misconceptions, that clinicians should be aware of are? I think, you know, I would encourage people to go and look at the platforms that are on offer if you're thinking about doing this in practise because probably you know you think of PCR as being done by people in white coats in a research lab somewhere, but with some of the machines that are becoming available, you know, you probably don't need that much kind of, you know, you don't need to be a molecular scientist to use them, so.
I would go and have a look at those platforms and ask those questions, you know, how easy is it to use? Is the, is the user interface really nice and it's clear cut? Because you probably don't need to have that much expertise.
Probably more important is the sample sample technique, and getting the right samples. So, you know, there's a particular platform, can you use things like faeces or serum or that those are the sorts. The questions that I will be asking and how much expertise do you really need to have cos probably not as much as you think.
Yeah. OK, brilliant. Thank you very much and then just one more, so David's asking, we see companies proposing PCR solutions and others er lamp solutions.
Both are molecular techniques, what is the major difference? Yeah, I don't know so much about lamp, but I think a lot of the things I've emphasised in this, presentation is having a good understanding of how the technique works so that you understand what the results are actually telling you. And it's the same with any molecular test.
If you understand how that test works, then you will know what those results actually mean for your particular patient. So like I was saying with the PCR, you know, if you get a positive, what does that mean? Well, it says that that particular sequence has been detected, but that doesn't mean that that, oh that's your diagnosis, that's what's causing the diarrhoea for example.
And I think there's a lot of hype around these PCR panels as well. For particular conditions like diarrhoea, for example, and I would look very carefully at what agents are included in those panels. They're not all the same, and some of the agents are included in those panels, not actually that useful, so I would be quite picky about which cases I used PCR for and which, which assays.
I was using in particular situations. Yeah, it's, it's important to understand the pathogenesis as well, and knowing, you know, if, if I do detect this particular organism, what does that actually mean? So there's a lot more to it than just, you know, oh, it's positive, it's negative.
And having that understanding hopefully will help you, interpret those tests. Fantastic, that's brilliant. I think we're gonna have to call it a day there, but we have got an awful lot of questions that are still coming in, so what we might be able to do is maybe pull them all together and do a little bit of a blog post on it and maybe get that out to everybody, just because we, we'll be here for another hour otherwise.
But I just wanted to say a, a really big thank you again, Melanie, for your presentation. A huge thank you to our sponsors ISAF for supporting this session today. Just a quick reminder.
That that this session will be edited and uploaded to the webinarve platform within 48 hours and your attendance certificate will also then be available. As always, this will automatically be added to your CPD records, which I know everybody's really keen to get up to date before the end of the year. And yeah, all that's left for me to say is just thank you so much everybody for joining, thanks for your questions, and we hope you enjoyed today's session and hope to see you again very soon.
Thanks Melanie. Thanks everyone. Thanks.
