Prática de Shadowing: 4 How to use PCR and qPCR - Aprenda a falar inglês com vídeo

Hi everyone, my name is Lisa Coluna.

Today I'm going to show you a little bit about PCR and qPCR,

which we use these genetic methods to be able to determine if acantinensis,

the rat lungworm parasite is in a given sample.

We use this technology on a variety of samples.

Most often we use it for detecting presence absence

or quantifying the rat longworm parasite in slugs or snails but we've also used it on blood samples,

human blood samples, human cerebral spinal fluid samples, CSF when you get a spinal tap and this is the technology

that is used by the department of health in order to determine

if you are sick with rat lung worm disease

so we have here today we have a very sterile environment over here we've got a general thermal cycler

that we have here that is used just to make many copies

of a particular region of DNA that you want to look at.

And then we have a clone zone, which is a sterile environment.

No DNA goes in here so that we make sure

that the reagents that we work with are not contaminated in any way.

So we make sure that the results that we see are actually from the sample and not from anything else.

And then down here we have what's called a qPCR, a real-time PCR, and that is able to detect a fluorescent probe

which allows us to either just do presence absence

or also we can quantify to know how many larvae are in a slug

or in a particular sample that we're looking at.

Where we're going to leave off is where in order to do this technology you have to first extract the total DNA.

And the movie on DNA extraction by Danez would allow you to know how to extract DNA.

And so we're going to take those samples and we are going to search for the rat lungworm DNA.

So what Danez did extracts all the DNA from everything that was in the sample, whether it's slug or horse or frog or human DNA,

plus all the bacteria and parasites, worms, whatever is in it.

So you have this tube that is a mixture of all the DNA from that sample that you placed in the tube.

And we are going to use genetic methods to search for the rat lungworm DNA.

This test is very sensitive and it's highly specific.

So there's with the test that was developed

from the CDC there's no other way and we've verified

that any signal that we see is definitely a cantonensis from rat lungworm.

The thermocycler is going to cycle temperatures from a higher temperature to a lower

and then a slightly higher and so these stages are called denaturing,

annealing and extension and so what that happens is Dines gives us double-stranded DNA

and so have two strands that are together

that are complementary to one another and

so the dna is made up of different chemical nucleotides called adenine guanine thymine and cytosine and

different base pairs will bond to each other and

so in the thermal cycling the first step is a warm period

that denatures the protein breaks the bonds and creates single-stranded dna

and then we have very specific primers that are just for the acantinensis, the rat lungworm DNA, that we're going to use

and we add those to our reaction mixture

and these primers sit on the single strain of DNA

and they bond directly to the DNA and then that's where our amplification

or where we're going to start making copies of the rat lungworm DNA.

And we need to make many copies of the DNA so that it can reach levels that are detectable by our equipment.

So the primer is going to sit down on the single-stranded DNA and then we use an enzyme,

an enzyme called TAC, that helps a chemical reaction move forward and it's going to build the complementary strand of DNA, the second strand, and that's called extension

when we build from a single strand to a double strand and we build the DNA into two.

And then we make that that cycles over

and over maybe 40 cycles of the thermal cycler

and eventually you're going to get millions

and millions of copies of the rat lungworm DNA of

that specific region of DNA that we know is rat lungworm in order for us to be able to detect.

With real-time PCR the difference between regular PCR and real-time PCR is

that that there's an extra probe that's involved that also has complementary base pairs and will sit down on the DNA.

And this probe has a quencher molecule and a reporter molecule.

And normally when the probe is intact, the quencher molecule quenches or absorbs the fluorescent signal that the reporter molecule will give off.

And so what happens is that as extension happens

and the enzyme starts to build the complementary strand in the second strand of DNA,

It's going to eat up the probe of the real-time PCR and release, and the quencher molecule and the reporter molecule will separate.

And so this disintegrated probe then allows the quencher or the reporter molecule to fluoresce, and that's what our equipment detects.

And so with every copy of DNA that's made, you have at least one reporter molecule,

and then the more and more copies that you make, the more and more reporter molecules, and the brighter the fluorescence signal gets.

There is some limiting reagent, maybe primers, maybe the individual nucleotides that you put in that stops the reaction from moving forward.

And so you can only make so many copies because you only have so many materials.

It's like if you were baking a cake and you ran out of eggs or something and the reaction would stop.

So when we work and when we start we're gonna take the DNA extraction from Dinez

and we're going to add that into a tiny tube with a bunch of different reagents

and it's like cooking

that you have to have all the right pieces in your tube in order to make copies of the DNA

and then those tubes will be put into the thermocycler.

Although today we're going be working with a real-time PCR unit.

This is the thermal cycler here, a normal, for normal polymerase chain reaction,

and this is an entry protocol that we could program for one step.

And so normally you start with a long denatry period to separate all your genomic DNA

because DNA is very long and stringy, and so you want to get it all into single-stranded DNA.

And then you have somewhere between 35 and 50 cycles depending on what you're working with and it will cycle between

denaturing annealing and extension cycles where you denature

so you go from double stranded to single stranded dna

and then your primers will anneal and your probe

and will sit down onto the single strand of dna

and extension is where you build the second strand of dna to make it double stranded again

and then that's one copy and you cycle again and you separate those copies, primer sit down and extend over and over until you get millions of copies.

Okay so we're ready to start our reaction and

so what we're going to do in here no DNA comes into this cabinet

so it's a very clean sterile environment so we can make sure we don't have DNA contamination.

So what I have here is I have this TACMAN environmental master mix and this has some general reagents,

the enzyme called TAC that helps build our double-stranded DNA.

It has what's called dNTPs, which are the individual nucleotides to help us build more copies of DNA.

And there's also some other reagents and buffers in here to make sure

that the pH is just right and that the enzyme is activated when we're ready.

And so that's a standard that many people can use in different reactions.

And then I have a tiny aliquot of the assay that is specific to us.

We make very small portions of it because freezing

and thawing this assay is not good and that can affect the sensitivity and how we read.

So we make very small aliquots, so we only use a little bit at a time in order for us.

And in this tube, that's where the primers for acantinensis

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and the probe for acantinensis exist and

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so this is very specific for our rat lungworm testing

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so i have a recipe and then i just have some water that is very clean sterile water

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so and then i have one cocktail tube so what i'm going to do is i'm going to make a master

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mix and then so that it's all homogenized i'll vortex it and spin it up mix it up really good

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and then I'll aliquot that into individual PCR tubes for each individual reaction.

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So I always start with some sort of run sheet so

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that I know exactly the proportions of everything

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that I need to work with and so I start usually adding with my water so I need 18.1 microliters of water.

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Now the master mix is very thick and viscous

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so whenever you pipette anything thick and viscous you have to be very slow

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and cautious so that it draws up into the pipette accurately.

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And then I need 8.9 microliters of the assay.

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and with q pcr we often do what's called mix by pipetting to flush out the tip

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so that we make sure everything that's in the tip gets in the tube

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and that helps with our accuracy especially between replicates

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so all of our samples all of the standards that we have

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and all the samples that we have we run in triplicate

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so that we can verify exactly if there is dna and how much dna that is in there.

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Okay so now I have the cocktail mix that's here with all the enzymes

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and the individual nucleotides and my primers and probes and I'm ready to make copies of DNA.

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So I'm going to mix this here with our little vortexer, give it a quick spin so that it's all homogeneous,

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and then I'm going to put 11 microliters into each of my tubes.

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Again, you want to pipette slowly and carefully to make sure that you have accurate volumes.

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qPCR is very sensitive and it will your replicates will come out different

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if your pipetting is off

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so you have to be very careful to make sure your pipetting is accurate okay

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and so now I've aliquoted into every individual tube the reagents

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that we're going to need and then I'm going to pull this tray out

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and add the DNA from Danez into each tube but before I do

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we have to add some water to the negative controls.

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And so every PCR reaction needs to have a negative control where you just add water to the reagents and no DNA.

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And what this control does is that it helps us look for contamination.

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So if there was contamination and rat lungworm DNA in this tube that we were measuring instead of our samples,

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then it would show up in our negative control

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and we would throw out the entire run of samples and have to redo them.

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We clean and get new reagents to make sure that the results

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that we see are actually from the samples that we collected.

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So I'm just going to add a little bit of water.

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So I'm going to add nine microliters of water to each of my negative controls.

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And then while I'm in the sterile cabinet,

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I'm going to close my negative controls to make sure that nothing else gets inside of them.

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Okay the rest of these tubes I'm just going to lightly

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kind of half close the lids just to help make sure there's no contamination in any other reaction

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and then I'm going to move this tray out onto the bench in order to add our DNA

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because we never bring DNA into this cabinet so all the pipettes and the tips and everything stay clean and stale.

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Now we're going to add DNA to our samples.

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We also have, in addition to the negative controls that we just added water to, we also have what's called positive controls that we have standards that we use.

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And so when we quantify how much rat lungworm DNA is in a sample, we have standards

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that serve like a ruler to say this is what 17 larvae per reaction looks like

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and this is what half a larva per reaction looks like.

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And we quantify based on how much DNA we put in the tube

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and then extrapolate to the organism or the tissue sample that we worked with.

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So the standards that we have here, we know this works.

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So the other part of a positive control

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that you always run a sample that you know amplifies with the primers

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and the reagents and the different temperatures from the thermal cycler

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so that if you look and you see no results

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and no amplification at all and no copies then something's wrong

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and maybe the enzyme maybe the master mix

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that you're using is is old or it was left out in warm temperatures and it's no good

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and so you'd have to replace that so positive control serves two purposes it makes sure that

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amplification can occur and then we also use it as standards reference standards to know how much

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and be able to quantify how many larvae are in a reaction. So

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when you add a sample you have to think every tube

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has a one quantity of rat lung worm in it of the DNA, acantinensis DNA.

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And so this is very important to make sure that we're very accurate.

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So I'm going to mix the sample briefly on a vortex to make sure

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that it's homogeneous and then when I pull the sample out I want to make sure

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that I don't cross contaminate with my gloves.

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All right I'm going to take a sample out

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and add it to the specific tube

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and then I'm going to mix by pipetting to make sure

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that every last bit of DNA in this pipette tip comes out into the tube

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and I'll do that over and over to help make sure I have tight replicates between samples.

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Part of the reason with PCR and qPCR that I'm using gloves

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and the purpose of using gloves is not necessarily to protect myself.

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DNA is not hazardous and the reagents that I'm working with are not hazardous to me or my health.

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I'm actually wearing gloves and PPE to help protect my samples.

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So I want to protect my samples against cross-contamination

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and there's also enzymes on our hands that help break down

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and destroy DNA and

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so I don't want any of those enzymes from my own skin to get into the sample

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and inhibit the PCR reaction that's going to move forward.

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With PCR and qPCR it's very important that you stay very organized

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and you know exactly which sample you're putting in which tube.

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If you get results at the end that look weird, you don't want to ever question exactly which sample got put into which tube.

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And so that is in part why I've organized myself in this manner.

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And so that I always take the sample that I need to load is here on my right.

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And then as I load it, and when I'm finished with it I place it on my left

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so that there's no question if somebody interrupts me

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while I'm working I know exactly where I'm at and I know exactly which sample needs to get loaded

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make sure that there's no bubbles Okay, now we've got our samples all ready to go.

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We've got the reagents in there

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that will help us make many copies of the DNA as well as the DNA that Dinez extracted from us, for us.

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I forgot to mention that we use special tubes for the real-time PCR

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that are different than normal PCR that have an optically clear cap

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so that the laser and the equipment can measure the fluorescent the fluorescence level of that reporter molecule that's in our assay.

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So we're going to go ahead and open and place our tubes into the qPCR, into the real-time PCR.

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I always use a little chem wipe to make sure

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that the tops are clean and free of any dust or fingerprints or anything like that.

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We've centrifuged the samples to make sure that there's no bubbles in the mixture.

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That's important for if a bubble pops in the middle of a reading that can affect the fluorescent level.

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So it's important to have no bubbles.

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So we put these in the system and then we've programmed,

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pre-programmed the software so that we know exactly which samples are in which well,

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which are standards, the positive controls and which are negative controls, which are unknown.

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And so we control the real-time PCR through the computer and we just press run

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and then the system is going to talk to our real-time PCR and connect to it and program our run.

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Okay so now our run is off and running and we've got roughly an hour and a half until

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our run is done.

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If you wanted to you could sit and watch the fluorescence

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and the data will come directly from the real-time PCR to the computer

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and you'll start to watch the amplification of our region of DNA that we're looking for.

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For the real-time PCR when we

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when you see the amplification you tend to see an exponential curve

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and that's because DNA is doubling every single time you have a cycle on every copy it makes.

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So you go from

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if we had one copy of the gene of region we

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would go from one copy to two to four to eight to 16 to 32 to 64 128

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and so on and so on and so

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that builds exponentially until the reaction stops because you run out of a certain reagent.

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That's the plateau phase.

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So while this is going, we could watch that, but I'll show you another file.

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Here you can see the finished version, and here we have a variety of our standards and other samples that we've run here,

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and so we know that these have amplified.

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A negative sample that does not have rat lungworm DNA would have a signal below with no amplification curve in it.

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Okay so this method that we have here for real-time PCR is the best method to be able to determine

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if what you're working with is acantinensis or not.

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Even when we look under the microscope and we see a worm that's there, we can't say definitively that it is acantinensis just by looking at it

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and so currently the only way that we know for sure

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that it's acantinensis is through this genetic testing there are in some cases we've made um hypotheses

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that we're likely working with acantinensis

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because of swimming behavior that's very characteristic uh to acantinensis

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and particular structures within the nematode itself that's very characteristic of acantinensis

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but for the moment this is the only test we have.

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And if you get sick with rat lungworm, this is a test when they're asking for a spinal tap, this is a test that the state lab,

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we're not able to do this on human samples for clinical diagnosis.

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We do, we test human samples for research purposes, but clinical diagnosis, the state lab uses the same technology,

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the same primers and probe assay in order to determine if you have rat lungworm DNA or not.

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Prática de Shadowing: 4 How to use PCR and qPCR - Aprenda a falar inglês com vídeo

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O que é a Técnica de Shadowing?