Molecular Diagnostics Part 2

Don Lee Author

06/10/2016 Added

160 Plays

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For more information please visit the Oomycete Disease Diagnostics website. https://ge.unl.edu/oomycete-disease-diagnostics/

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[00:00:06.131]Don Lee, here again,
[00:00:07.292]from the University of Nebraska at Lincoln.
[00:00:09.730]And so, Molecular Diagnosis, this will be part two.
[00:00:13.631]So we've looked at sampling in the field
[00:00:17.741]and then sending your sample to a laboratory
[00:00:20.342]to get the best possible DNA-based testing information.
[00:00:25.218]Now we're going to look at a situation
[00:00:26.843]where the agronomist might be under a more rapid
[00:00:31.394]information turnaround demand.
[00:00:33.879]They may be out in the field.
[00:00:35.458]They're looking at the situation.
[00:00:37.292]The farmer has to make a choice,
[00:00:39.127]and they have to make it within a timeframe
[00:00:42.656]that's shorter than testing
[00:00:46.092]in a laboratory will allow.
[00:00:47.996]Is there a way that an agronomist
[00:00:50.736]can get DNA-based information about the oomycetes
[00:00:55.160]that are living in this part of the field
[00:00:58.399]and use that information with a certain level
[00:01:00.907]of confidence to make decisions?
[00:01:02.741]That's what we're going to take a look at.
[00:01:04.320]So the key tool to making this a reality
[00:01:08.616]for agronomists is an instrument called
[00:01:11.240]an isothermal DNA system.
[00:01:17.018]RPA is one of the kinds
[00:01:20.851]of isothermal
[00:01:22.537]or constant temperature PCR that can happen,
[00:01:26.740]and we'll take you through how this instrument
[00:01:29.027]and how the setup of this analysis can work.
[00:01:32.278]And so, again, I was looking for an example
[00:01:34.786]of research that we might be able to relate to.
[00:01:38.873]This was an article looking
[00:01:41.123]at periodontal-type of bacteria.
[00:01:43.819]So imagine you're at the dentist's office.
[00:01:46.466]They see a problem might be caused
[00:01:48.579]by a certain type of microorganism
[00:01:51.403]living in your mouth.
[00:01:53.873]Can they get information right away
[00:01:56.520]that then can tell them the best possible treatment
[00:02:00.328]that they can impose where hours matter?
[00:02:05.460]And so, the same kind of situation would be
[00:02:08.687]what the Chilvers Lab is looking at
[00:02:11.659]as they're developing this DNA detection technology
[00:02:15.119]and then sharing the results with their
[00:02:17.673]scientific peers.
[00:02:19.879]So let's take a look at how they would
[00:02:22.735]be doing this assay.
[00:02:24.036]So again, you're out in the field.
[00:02:26.381]You have to have equipment that you can run
[00:02:28.842]out in the field.
[00:02:30.468]And so, the first thing is to take your sample
[00:02:33.347]as they're showing here.
[00:02:46.815]And the goal here is just to macerate your sample,
[00:02:50.112]break up the cells so that the DNA comes
[00:02:53.548]out of solution.
[00:02:55.383]And the DNA will be DNA from the organisms
[00:02:59.043]that are living in that tissue sample
[00:03:01.513]as well as DNA from the plant.
[00:03:03.463]But we're going to do PCR so that we make
[00:03:05.808]specific copies of the DNA from the organisms
[00:03:09.314]that we're interested in.
[00:03:10.684]So we can separate the solution
[00:03:13.192]that would have DNA in it away from all
[00:03:15.793]the other debris material in our samples.
[00:03:18.719]And then we can actually conduct PCR
[00:03:21.458]under a constant temperature
[00:03:23.270]and then use this device to detect the DNA copies
[00:03:26.521]as they are getting made.
[00:03:28.935]So let's take a look at this cartoon picture
[00:03:31.397]and get an understanding of the very basics
[00:03:34.137]of this RPA cycle.
[00:03:37.411]The key is, you have to have your primers,
[00:03:39.686]your DNA sequences that will specifically target
[00:03:43.146]the DNA from the, in this case,
[00:03:45.737]the oomycete genus
[00:03:47.767]or species that we're interested in.
[00:03:50.739]But then, instead of using temperature
[00:03:52.782]to denature the double-stranded DNA,
[00:03:55.151]they use special recombinase and single strand
[00:03:58.727]DNA binding proteins that are added to that test tube.
[00:04:03.366]And those proteins, in collaboration
[00:04:06.411]with the single-stranded primers will bind
[00:04:09.710]to the targeted DNA, denature it for them
[00:04:12.310]with what they call a D loop.
[00:04:13.773]And then the third enzyme you put in
[00:04:15.794]is a DNA polymerase, this little purple depicted molecule,
[00:04:19.961]will read the original DNA,
[00:04:22.342]make a double-stranded copy.
[00:04:23.874]So you can start with one double-stranded DNA,
[00:04:26.498]get two, and then you can repeat cycles of these:
[00:04:29.656]two to four, four to eight.
[00:04:31.490]And as the copies are getting integrated,
[00:04:36.413]we can, with the use of those probe molecules,
[00:04:40.778]detect DNA as it's being incorporated.
[00:04:45.376]And that's the detection capabilities,
[00:04:48.952]what that little device does.
[00:04:51.065]So again, you have to know the DNA sequence
[00:04:53.526]of the species that you're interested in
[00:04:56.823]and have a probe that will specifically bind
[00:05:00.097]to that DNA.
[00:05:01.723]And in this experiment that the Chilvers Lab shared,
[00:05:05.484]they set up their PCR two different ways.
[00:05:07.992]You could use a primer that's found
[00:05:10.570]in targeting a sequence found in all phytophthora.
[00:05:14.285]And then you can add a second primer
[00:05:16.653]that's specific for the sansomeana sequence
[00:05:19.672]or a primer specific for the sojae sequence.
[00:05:23.782]And so, if you get a PCR product
[00:05:26.220]that's where this probe is integrated using this primer,
[00:05:30.608]that means you have sansomeana there.
[00:05:32.814]If you get this PCR product incorporated
[00:05:35.391]when you use this primer,
[00:05:36.994]that means your phytophthora sojae's there.
[00:05:39.687]If the PCR doesn't work, this probe can't incorporate it,
[00:05:42.845]you don't get any signal.
[00:05:44.424]And you can get the signal produced in real-time
[00:05:48.000]so you can get your answer faster.
[00:05:50.252]And so, the output is the same.
[00:05:52.180]It takes a few cycles, and then you start
[00:05:54.200]to make copies and then copies of the copies.
[00:05:56.707]And that device detects those copies
[00:05:58.751]as they get made.
[00:06:00.724]And the use of your specific primers
[00:06:04.785]will tell you if the targeted species
[00:06:08.317]that you're interested is present
[00:06:10.686]and you're detecting its DNA in your sample.
[00:06:14.053]You can also do a similar kind of assay
[00:06:17.745]where they use these
[00:06:20.508]sequence detection strips,
[00:06:22.714]and you either get a plus or a minus answer
[00:06:26.708]with your sample.
[00:06:29.146]It's called end-point diagnostic data.
[00:06:31.259]Either way, you can conduct this sampling
[00:06:33.883]and testing in the field,
[00:06:36.112]and from your answer,
[00:06:38.387]decide on a management strategy.
[00:06:41.894]So the question that the Chilvers Lab proposed then is,
[00:06:45.423]"How reliable is this in the field testing going to be?
[00:06:49.440]"Can we come up with ways and make this
[00:06:53.457]"a procedure that rivals
[00:06:56.832]"the reliability
[00:06:58.728]"and detection sensitivity of the quantitative PCR,
[00:07:02.768]"the Taqman PCR?"
[00:07:04.510]So let's take a look at their results
[00:07:06.739]of this experiment.
[00:07:07.807]They had 74 samples
[00:07:09.223]that came from soil sampled
[00:07:12.474]in 21 fields from nine different states.
[00:07:18.163]And so, if we take a look at their numbers,
[00:07:20.508]if they ran those samples on both Taqman PCR
[00:07:24.107]and then this isothermal RPA PCR.
[00:07:28.566]And the greens tell you when the two methods
[00:07:30.911]were in agreement.
[00:07:32.049]The reds tell you when the two methods
[00:07:34.765]were not in agreement.
[00:07:36.507]So for example, you could have a false positive.
[00:07:41.569]That's where you get a sample in the field
[00:07:45.098]that says, "Hey, I'm detecting
[00:07:47.513]"the phytophthora here,"
[00:07:49.766]but when you ran that analysis
[00:07:52.355]of the same sample in the laboratory method,
[00:07:55.675]it would be a negative sample.
[00:07:57.185]That would be a false positive.
[00:07:58.973]Okay, so you can get an idea
[00:08:01.503]of the overall reliability
[00:08:04.987]and the overall sensitivity of the test.
[00:08:08.864]So there might be situations
[00:08:10.536]where you cannot detect the presence of the DNA
[00:08:15.250]with the isothermal in the field assay
[00:08:18.129]but you can detect it with the Taqman.
[00:08:21.937]It has a sensitivity that's not as high
[00:08:24.956]as the laboratory test.
[00:08:27.162]So both of those issues are important.
[00:08:29.553]If you're going to make a management decision
[00:08:31.480]based on the field test, you have to have an idea
[00:08:34.871]of how confident you should be in it by relating it
[00:08:38.168]to the "in the lab" test.
[00:08:40.977]So based on these results,
[00:08:42.440]obviously, the Chilvers Lab is very optimistic
[00:08:45.459]about the potential to develop these DNA testing tools
[00:08:48.942]as a resource that agronomists can have
[00:08:52.796]to give farmers the best possible information.
[00:08:55.978]You could develop them for a number
[00:08:58.090]of different seedling disease-causing pathogens.
[00:09:01.643]And if agronomists combined
[00:09:04.326]with the skills of diagnosticians
[00:09:07.067]in different regional testing labs
[00:09:09.863]come together, collectively this team
[00:09:12.904]should be able to do a better job
[00:09:14.529]of helping farmers manage seedling diseases.

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Molecular Diagnostics Part 2

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