Molecular Diagnostics Part 1

Don Lee Author

06/10/2016 Added

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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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[00:00:06.199]Hello, Don Lee from UNL,
[00:00:08.619]and this is gonna be part four,
[00:00:11.758]Molecular Diagnosis in Oomycete Disease Management.
[00:00:16.470]And again, I'm gonna be emphasizing
[00:00:18.230]on telling the story of work that's coming out of
[00:00:21.629]the Chilvers Lab at
[00:00:24.001]Michigan State University,
[00:00:26.758]and the emphasis is putting agronomists
[00:00:30.706]in a position where they are working with the farmer
[00:00:35.184]who's field we see here,
[00:00:36.972]obviously great potential for soybean crop production,
[00:00:42.068]but some problem areas.
[00:00:43.765]And so better understanding the biology
[00:00:45.903]that's the cause behind this poor stand
[00:00:51.590]and obvious seedling disease issues,
[00:00:56.648]and understanding then where technology comes in
[00:01:01.019]to give the farmer some options
[00:01:03.613]to get a better result.
[00:01:05.477]And the technology can come in a number of forms,
[00:01:08.424]but an agronomist of course should be aware
[00:01:10.899]of varieties and resistance they might have
[00:01:13.677]to specific seedling diseases,
[00:01:16.019]and then seed treatments that can be applied
[00:01:19.835]as a preventative to managing these seedling diseases.
[00:01:24.091]But you gotta know the cause of the seedling disease
[00:01:28.088]and in our case, we want to try to better understand
[00:01:31.016]the oomycete community that might be
[00:01:33.509]living in this part of the field,
[00:01:35.759]so what we'll see here with this presentation,
[00:01:38.327]and I'll break it into two parts, is that
[00:01:43.528]pathologists are developing DNA-based detection methods
[00:01:49.933]that could deliver answers to agronomists
[00:01:54.544]in a couple of different ways, and then they can
[00:01:56.782]work with their farmers to come up with the best
[00:01:58.890]technology approach to minimize
[00:02:02.536]or avoid these problems in the future.
[00:02:04.507]So we're gonna look at DNA based testing,
[00:02:07.320]and scenarios where you might want to sample
[00:02:12.180]in the field as we've been describing,
[00:02:14.428]and then send those samples off to the lab,
[00:02:17.836]but also scenarios where you would sample
[00:02:21.263]and conduct a test in the field.
[00:02:24.162]And the key to the choices here
[00:02:27.454]really are the DNA analysis technology
[00:02:30.446]that's developing in all areas of the life sciences,
[00:02:34.862]where we can detect specific DNA sequences
[00:02:40.027]and also quantify the relative amounts of those.
[00:02:44.268]You can get actually very incredibly detailed information
[00:02:48.452]about the DNA sequence make-up of entire communities
[00:02:52.242]if you use advanced high through-put DNA sequencing.
[00:02:56.286]We won't talk about that in this presentation,
[00:03:00.759]but now there are new emerging ways
[00:03:03.576]of conducting DNA testing with this process
[00:03:07.014]called PCR that can be done
[00:03:11.061]without the need of a laboratory expert.
[00:03:13.543]It can be done in the field,
[00:03:15.083]and so we'll try to show you how those work.
[00:03:18.755]I wanna emphasize that if you
[00:03:21.262]are familiar with DNA testing technology,
[00:03:24.915]understand how DNA is tested,
[00:03:28.415]detected with he PCR reaction,
[00:03:32.447]then you're ready to go with understanding
[00:03:34.400]what I'll present here.
[00:03:35.774]If you don't have that understanding,
[00:03:37.857]then we encourage you to
[00:03:40.452]watch the companion presentations in this part
[00:03:44.120]of our lesson.
[00:03:46.501]We have a presentation on how PCR works,
[00:03:49.548]the polymerase chain reaction,
[00:03:51.243]and then we also have a presentation
[00:03:53.268]that shows how a version of PCR
[00:03:55.771]called quantitative PCR
[00:03:57.574]that's got a slight difference works.
[00:04:00.199]So I won't take the time here to present that.
[00:04:04.161]It would be highly recommended to watch these
[00:04:07.446]two companion presentations
[00:04:09.058]as a part of your learning.
[00:04:10.871]Okay, so we're gonna take a look at
[00:04:12.896]situations where you'd want to sample
[00:04:15.874]in the field, and you have some time
[00:04:19.132]to make a decision, so you can send
[00:04:21.412]your sample off for DNA analysis.
[00:04:24.029]So I found kind of a parallel application.
[00:04:28.188]What if you're a wildlife biologist,
[00:04:30.940]a conservation officer, and
[00:04:35.042]you want to know if samples that you've confiscated
[00:04:41.017]are a particular species,
[00:04:43.156]so the wildlife crimes unit's
[00:04:46.018]analysis laboratories are developing
[00:04:49.880]the same kind of DNA based identification methods
[00:04:54.850]that I'll describe here,
[00:04:56.443]that have been developed by scientists
[00:04:58.949]such as the authors for this publication,
[00:05:02.763]where you can, if you know the specific DNA sequences
[00:05:07.365]of the group of organisms you're interested in,
[00:05:10.296]you can use that information to design PCR
[00:05:14.181]in a detection assay.
[00:05:17.029]So I'll go through a story shared
[00:05:19.456]by the Chilvers Lab, where an agronomist
[00:05:22.994]may be working with a farmer
[00:05:24.809]that could have Phytophthora species
[00:05:28.140]in their oomycete community,
[00:05:30.146]and actually might have a situation
[00:05:33.696]where they could be dealing with
[00:05:35.598]Phytophthora sansomeana, or Phytophthora sojae,
[00:05:39.878]and the reason, knowing the difference is important,
[00:05:43.679]is because of the biological differences
[00:05:47.152]between these two Phytophthora.
[00:05:49.124]Sansomeana has a wide host range,
[00:05:51.444]so there could be a number of crops
[00:05:53.452]that could be affected by this particular Phytophthora,
[00:05:58.513]whereas Phytophthora sojae
[00:06:01.042]has a narrower host range,
[00:06:03.374]so knowing the species difference
[00:06:06.111]could be an important bit of information
[00:06:09.467]that the farmer can make decisions with,
[00:06:14.466]and working with an agronomist that understands that
[00:06:17.027]can be very important.
[00:06:18.488]So let's take a look at the genetic information
[00:06:21.848]that's laid out here in this slide.
[00:06:24.167]So Phytophthora and pythium is oomycetes,
[00:06:29.146]they're eukaryotes, they have compartments
[00:06:31.408]in their cells that are mitochondria,
[00:06:34.077]and mitochondria have their own set of genes,
[00:06:37.173]their own little circular genome.
[00:06:39.826]There might be about a hundred genes
[00:06:42.428]that are a part of the mitochondrial genome,
[00:06:46.144]and so by sequencing the DNA from the mitochondrial genome
[00:06:51.705]of various Phytophthora species,
[00:06:54.080]you can focus in on regions where the sequence difference
[00:06:59.299]is different between Phytophthora sojae
[00:07:02.432]and Phythphthora sansomeana.
[00:07:04.519]You can also zero in on regions where that
[00:07:08.068]sequence is highly conserved,
[00:07:09.814]so if that DNA is from a species
[00:07:13.352]that's a Phytophthora, these sequences will
[00:07:16.911]be retained, will match up.
[00:07:18.756]And so you can take advantage of those
[00:07:20.982]and make those conserved regions into your primers,
[00:07:24.592]and you can therefore guarantee
[00:07:26.395]that you're gonna make copies of the DNA
[00:07:30.645]if it's in your sample and it's from a
[00:07:36.109]Phytophthora, okay.
[00:07:37.854]Now the region that we're looking at here
[00:07:39.337]is just this small region of genes
[00:07:43.039]that are a part of the mitochondrial DNA
[00:07:46.595]in these oomycetes.
[00:07:48.290]So in addition to the primers that you need
[00:07:50.488]to initiate and target your PCR,
[00:07:54.548]where is the DNA gonna get copied?
[00:07:56.915]You also have to have probes.
[00:07:58.774]Probes get incorporated into the DNA copies
[00:08:02.172]as they get made, and here is where
[00:08:05.034]there is a genus-specific probe,
[00:08:08.172]and then there is sequence differences
[00:08:10.409]between the two species within the genus
[00:08:12.998]that can work as a species-specific probe.
[00:08:17.575]So when you conduct the PCR reaction,
[00:08:20.707]when this is carried out in a laboratory
[00:08:24.106]that's conducting quantitative PCR,
[00:08:26.985]the Phytophthora probe would get incorporated
[00:08:29.332]as long as these copies are being made
[00:08:31.068]from any of the Phytophthora species
[00:08:34.761]within the Phytophthora genus,
[00:08:36.941]and then the sojae probe would get incorporated
[00:08:41.046]if it's sojae DNA,
[00:08:43.429]and the sansomeana probe would get incorporated
[00:08:46.783]if it's sansomeana DNA.
[00:08:49.361]So as the DNA gets copied, we can detect
[00:08:52.645]those copies being made in a quantitative PCR assay.
[00:08:57.404]So remember, there's a lot of different
[00:09:00.080]Phytophthora and sojae,
[00:09:01.535]a lot of different oomycete species,
[00:09:04.087]and you could have many of these
[00:09:05.381]living in the same soil ecosystem,
[00:09:08.879]so then as you're quantitative PCR reaction
[00:09:13.710]takes place, we're gonna specifically
[00:09:16.460]think about if I have DNA from this Phytophthora species
[00:09:20.443]or this Phytophthora species,
[00:09:22.335]they sort out into different genetic groups
[00:09:25.688]based on some specific genetic sequence differences
[00:09:29.515]in their mitochondrial DNA,
[00:09:31.734]and the PCR can detect those.
[00:09:34.564]So the initial cycles of the PCR,
[00:09:36.828]we just start to make copies
[00:09:38.827]and then copies of the copies,
[00:09:40.470]and the quicker these peaks arise,
[00:09:43.687]that means the more of that particular
[00:09:47.452]DNA sequence is present.
[00:09:50.053]And so what we're seeing here is
[00:09:52.130]there's lots of members of the Phytophthora genus,
[00:09:55.878]but there was just one DNA sample
[00:09:58.549]that contained the Phytophthora sojae,
[00:10:02.628]so you can have genus level detection,
[00:10:06.604]and that's what we're seeing with the blue lines,
[00:10:08.778]and then species level detection,
[00:10:11.419]that's what we're seeing with the green line.
[00:10:14.601]If there were lots of Phytophthora sojae
[00:10:17.426]containing samples, we'd have more of these present,
[00:10:21.320]if there were none, we wouldn't see this particular
[00:10:24.477]line appear.
[00:10:25.875]So this kind of PCR gives you a yes-no answer,
[00:10:30.624]is that particular type of DNA sample present,
[00:10:35.019]but it also gives you quantitative data.
[00:10:37.249]The more of that DNA that's present,
[00:10:40.087]the more rapidly your detection curve
[00:10:45.212]starts to appear, and you can use this
[00:10:47.953]to get an idea of the relative abundance
[00:10:50.676]of a particular type of DNA that's detected in this assay.
[00:10:55.804]So the ability to differentiate
[00:11:00.887]between samples that contain one species of Phytophthora
[00:11:04.716]from those that don't contain that species
[00:11:07.490]is the goal of doing molecular diagnostics.
[00:11:10.716]So we can take a look at an example then
[00:11:14.055]of a experiment conducted with two years
[00:11:16.849]worth of samples,
[00:11:18.683]in the lab at Michigan State University.
[00:11:23.454]In the first year, they had 42 samples,
[00:11:25.704]in the second year, 74,
[00:11:28.011]and so we can see that these were the numbers
[00:11:31.848]of Phytophthora-positive samples that they detected
[00:11:37.171]in both years, and then you can break that into
[00:11:41.422]the two species.
[00:11:43.146]All of them were Phytophthora sojae.
[00:11:45.759]In 2013 none of them were sansomeana.
[00:11:49.033]They detected one sansomeana
[00:11:51.434]out of the 25 species,
[00:11:57.574]one that had sansomeana, one that had Phytophthora sojae,
[00:12:01.957]several that had other Phytophthora
[00:12:05.448]that didn't fall into either of these species.
[00:12:07.720]Okay, so you can get genus level
[00:12:10.262]or species-specific information
[00:12:13.485]from this quantitative PCR.
[00:12:16.982]And there would be situations where
[00:12:19.499]sampling that field, knowing if you have
[00:12:22.286]this type of Phytophthora present,
[00:12:24.447]or this type of Phytophthora present
[00:12:26.624]can empower the farmer to make the proper decision
[00:12:30.845]if they're going to do replanting
[00:12:33.342]or a different kind of seed treatment,
[00:12:36.046]or go with a different crop choice
[00:12:40.693]in subsequent growing seasons.
[00:12:44.209]All right, so this will be the end of part one.

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Molecular Diagnostics Part 1

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