Wheat Streak Mosaic Virus Diagnosis using ELISA
The wheat curl mite vectors three viruses (wheat streak mosaic, Triticum mosaic, and High Plains wheat mosaic viruses) to winter wheat throughout the Great Plains. This video provides an overview of wheat streak mosaic virus symptoms and ELISA diagnosis procedure.
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[00:00:04.350]Wheat Streak Mosaic Virus Diagnosis
[00:00:08.400]Wheat streak mosaic virus affects wheat production
[00:00:11.050]across the globe.
[00:00:12.240]It is one of a complex of viruses,
[00:00:14.380]including Triticum mosaic virus
[00:00:16.330]and High Plains wheat mosaic virus.
[00:00:18.660]This complex causes significant yield losses
[00:00:21.210]in wheat production.
[00:00:25.750]Wheat streak mosaic virus
[00:00:27.320]is a single-stranded RNA-positive virus
[00:00:29.990]in the Potyviridae family in the genus Tritimovirus.
[00:00:33.700]It is vectored from plant to plant
[00:00:35.440]by the eriophyid mite Aceria tosichella.
[00:00:38.860]It was first observed in Nebraska in 1922
[00:00:42.490]and has continued to be an issue
[00:00:44.070]across much of the Great Plains region.
[00:00:48.290]Plants can be infected in the fall or the spring
[00:00:51.350]but symptoms typically begin developing
[00:00:53.400]in spring with warming temperatures.
[00:00:55.740]Young wheat is most susceptible to infection.
[00:00:59.010]Tight curling of the wheat leaf edge
[00:01:00.990]indicates the presence of the eriophyid mite.
[00:01:03.810]Viral infection symptoms develop progressively
[00:01:07.230]with light-yellow streaky mottling
[00:01:09.540]on the new growth occurring first.
[00:01:12.030]As the virus progresses,
[00:01:13.490]the symptoms become more pronounced.
[00:01:16.000]Eventually the mottling fades
[00:01:17.970]and becomes difficult to identify at later stages.
[00:01:21.540]Virus-infected plants are stunted
[00:01:23.690]and spraddle across the field.
[00:01:27.270]To test for the presence of wheat streak mosaic virus,
[00:01:30.520]diagnosticians will use a procedure called ELISA,
[00:01:33.810]enzyme linked immunosorbent assay.
[00:01:36.780]This assay is designed to confirm the presence
[00:01:39.700]of viral antigens in a sample and measure virus titer,
[00:01:43.820]the total amount of viral particles present.
[00:01:46.980]Antigens are the protein of the capsid coat
[00:01:49.570]surrounding the virus.
[00:01:51.410]The format used to diagnose wheat streak mosaic virus
[00:01:54.510]is a capture assay sandwich.
[00:01:56.690]The antigen is captured between two antibodies and detected.
[00:02:01.610]First the 96-well plates are coated
[00:02:04.400]with the primary antibody.
[00:02:06.450]This is the antibody that will bind to the wells
[00:02:08.810]and captures the antigen when it is applied.
[00:02:11.680]This antibody is allowed to rest for at least four hours
[00:02:15.060]at room temperature to facilitate binding to the wells.
[00:02:20.460]Samples gathered from the field are placed
[00:02:22.810]inside ELISA sample extraction bags.
[00:02:25.680]The heavy-duty plastic and plastic mesh inside
[00:02:28.490]facilitate the grinding.
[00:02:30.770]General extraction buffer is added
[00:02:32.730]to help stabilize the particles against degradation.
[00:02:36.210]Samples are ground with a tissue homogenizer
[00:02:38.730]to break up cells and release the cell contents,
[00:02:41.430]including the virus, into the solution.
[00:02:44.660]The sample solution is added to the wells
[00:02:47.140]and allowed to rest in 37 degrees Celsius
[00:02:50.150]to encourage binding to the antibody.
[00:02:54.700]After an hour, the samples are removed
[00:02:57.000]and rinsed off repeatedly
[00:02:58.300]with phosphate buffer saline with Tween, PBS-T for short.
[00:03:03.020]This removes extra material from the wells
[00:03:06.600]the present antibody-antigen binding reaction.
[00:03:11.500]The second antibody is added next,
[00:03:13.720]designed to bind the antigen, and contains a signal enzyme.
[00:03:17.510]Again the plate is placed in an oven at 37 degrees Celsius
[00:03:21.420]to facilitate binding between the antigen and the antibody.
[00:03:25.530]Again the wells are rinsed with PBS-T
[00:03:28.330]to remove any remaining detritus in the wells.
[00:03:33.460]The enzyme substrate reporter solution is added.
[00:03:36.420]Here PNP is the molecule being used.
[00:03:39.810]It provides the component that will be broken up apart
[00:03:42.370]by the enzyme present on the second antibody,
[00:03:45.040]producing a color result.
[00:03:47.140]Because this reaction is light sensitive,
[00:03:49.350]it is stored in darkness until readings are made.
[00:03:54.910]The plate is placed into a UV-Vis machine
[00:03:57.940]designed to take photometric readings
[00:03:59.910]of each individual well and report a value
[00:04:02.550]of how heavily colored it is.
[00:04:04.680]Yellow is positive for the virus
[00:04:06.430]and negative readings are clear.
[00:04:08.620]The brighter the color, the higher the virus titer
[00:04:11.450]and the more viral particles are present.
[00:04:13.960]Here you can see positive results in these wells
[00:04:17.010]and negative results here.
[00:04:19.540]From these readings a positive diagnosis
[00:04:21.820]for the virus presence can be made
[00:04:24.260]and followed by subsequent management decisions.
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