Voiceover: Don Lee, here again,
from the University of Nebraska at Lincoln.
And so, Molecular Diagnosis, this will be part two.
So we've looked at sampling in the field
and then sending your sample to a laboratory
to get the best possible DNA-based testing information.
Now we're going to look at a situation
where the agronomist might be under a more rapid
information turnaround demand.
They may be out in the field.
They're looking at the situation.
The farmer has to make a choice,
and they have to make it within a timeframe
that's shorter than testing
in a laboratory will allow.
Is there a way that an agronomist
can get DNA-based information about the oomycetes
that are living in this part of the field
and use that information with a certain level
of confidence to make decisions?
That's what we're going to take a look at.
So the key tool to making this a reality
for agronomists is an instrument called
an isothermal DNA system.
RPA is one of the kinds
of isothermal
or constant temperature PCR that can happen,
and we'll take you through how this instrument
and how the setup of this analysis can work.
And so, again, I was looking for an example
of research that we might be able to relate to.
This was an article looking
at periodontal-type of bacteria.
So imagine you're at the dentist's office.
They see a problem might be caused
by a certain type of microorganism
living in your mouth.
Can they get information right away
that then can tell them the best possible treatment
that they can impose where hours matter?
And so, the same kind of situation would be
what the Chilvers Lab is looking at
as they're developing this DNA detection technology
and then sharing the results with their
scientific peers.
So let's take a look at how they would
be doing this assay.
So again, you're out in the field.
You have to have equipment that you can run
out in the field.
And so, the first thing is to take your sample
as they're showing here.
And the goal here is just to macerate your sample,
break up the cells so that the DNA comes
out of solution.
And the DNA will be DNA from the organisms
that are living in that tissue sample
as well as DNA from the plant.
But we're going to do PCR so that we make
specific copies of the DNA from the organisms
that we're interested in.
So we can separate the solution
that would have DNA in it away from all
the other debris material in our samples.
And then we can actually conduct PCR
under a constant temperature
and then use this device to detect the DNA copies
as they are getting made.
So let's take a look at this cartoon picture
and get an understanding of the very basics
of this RPA cycle.
The key is, you have to have your primers,
your DNA sequences that will specifically target
the DNA from the, in this case,
the oomycete genus
or species that we're interested in.
But then, instead of using temperature
to denature the double-stranded DNA,
they use special recombinase and single strand
DNA binding proteins that are added to that test tube.
And those proteins, in collaboration
with the single-stranded primers will bind
to the targeted DNA, denature it for them
with what they call a D loop.
And then the third enzyme you put in
is a DNA polymerase, this little purple depicted molecule,
will read the original DNA,
make a double-stranded copy.
So you can start with one double-stranded DNA,
get two, and then you can repeat cycles of these:
two to four, four to eight.
And as the copies are getting integrated,
we can, with the use of those probe molecules,
detect DNA as it's being incorporated.
And that's the detection capabilities,
what that little device does.
So again, you have to know the DNA sequence
of the species that you're interested in
and have a probe that will specifically bind
to that DNA.
And in this experiment that the Chilvers Lab shared,
they set up their PCR two different ways.
You could use a primer that's found
in targeting a sequence found in all phytophthora.
And then you can add a second primer
that's specific for the sansomeana sequence
or a primer specific for the sojae sequence.
And so, if you get a PCR product
that's where this probe is integrated using this primer,
that means you have sansomeana there.
If you get this PCR product incorporated
when you use this primer,
that means your phytophthora sojae's there.
If the PCR doesn't work, this probe can't incorporate it,
you don't get any signal.
And you can get the signal produced in real-time
so you can get your answer faster.
And so, the output is the same.
It takes a few cycles, and then you start
to make copies and then copies of the copies.
And that device detects those copies
as they get made.
And the use of your specific primers
will tell you if the targeted species
that you're interested is present
and you're detecting its DNA in your sample.
You can also do a similar kind of assay
where they use these
sequence detection strips,
and you either get a plus or a minus answer
with your sample.
It's called end-point diagnostic data.
Either way, you can conduct this sampling
and testing in the field,
and from your answer,
decide on a management strategy.
So the question that the Chilvers Lab proposed then is,
"How reliable is this in the field testing going to be?
"Can we come up with ways and make this
"a procedure that rivals
"the reliability
"and detection sensitivity of the quantitative PCR,
"the Taqman PCR?"
So let's take a look at their results
of this experiment.
They had 74 samples
that came from soil sampled
in 21 fields from nine different states.
And so, if we take a look at their numbers,
if they ran those samples on both Taqman PCR
and then this isothermal RPA PCR.
And the greens tell you when the two methods
were in agreement.
The reds tell you when the two methods
were not in agreement.
So for example, you could have a false positive.
That's where you get a sample in the field
that says, "Hey, I'm detecting
"the phytophthora here,"
but when you ran that analysis
of the same sample in the laboratory method,
it would be a negative sample.
That would be a false positive.
Okay, so you can get an idea
of the overall reliability
and the overall sensitivity of the test.
So there might be situations
where you cannot detect the presence of the DNA
with the isothermal in the field assay
but you can detect it with the Taqman.
It has a sensitivity that's not as high
as the laboratory test.
So both of those issues are important.
If you're going to make a management decision
based on the field test, you have to have an idea
of how confident you should be in it by relating it
to the "in the lab" test.
So based on these results,
obviously, the Chilvers Lab is very optimistic
about the potential to develop these DNA testing tools
as a resource that agronomists can have
to give farmers the best possible information.
You could develop them for a number
of different seedling disease-causing pathogens.
And if agronomists combined
with the skills of diagnosticians
in different regional testing labs
come together, collectively this team
should be able to do a better job
of helping farmers manage seedling diseases.