This research tested 96 potato leaf samples for three viruses (PVS, PVY, PVX) using two detection methods: Sandwich ELISA and Direct ELISA. Sandwich ELISA found higher infection rates, with 79.2% of samples positive for PVS, 64.6% for PVY, and 2.1% for PVX. Direct ELISA showed lower rates, with 50% positive for PVS, 3.1% for PVY, and 0% for PVX. The results indicate Sandwich ELISA is more accurate than Direct ELISA for virus detection in potatoes due to its use of two sets of antibodies rather than one.

1. Potato Virus Detection and
Removal
Mai Nyia Vang, Lila Westreich, Dr. Sanjay Gupta, Dr. Christian Thill
Department of Horticulture Science, University of Minnesota
Potato Virus Detection and Removal

2. Mai Nyia Vang, Lila Westreich, Dr. Sanjay Gupta, Dr. Christian Thill
Department of Horticulture Science, University of Minnesota
Summary:
In this research, 96 potato copy samples were tested to detect Potato Virus S (PVS),
Potato Virus X (PVX), and Potato Virus Y (PVY). To detect the viruses, two methods were used,
Sandwich Enzyme Linked Immuno-Sorbent Assay (ELISA) and Direct ELISA. As expected, all
96 potato samples was infected with at least one virus. To detect the viruses, Sandwich ELISA
and Direct ELISA were used. In Sandwich ELISA, 79.2% of the samples were infected with
PVS, 64.6% PVY, and 2.1% PVX. For Direct ELISA, 50% of the samples shown were infected
with PVS, 3.1% PVY, and 0% PVX. The results turned out this way because in Sandwich
ELISA, two sets of antibodies are used and holds onto a specific virus, while in Direct ELISA,
all proteins and viruses are present and only one set of antibody is used. So in this case,
Sandwich ELISA would be most accurate compared to Direct ELISA.
Introduction:

3. Potatoes are the fourth largest crop production in the world. Every year, the potato crop
decreases because they are infected with viruses (Samsatly, 2014). Viruses are infections of the
cell in living organisms. There are many varieties of potato viruses, but three of the major potato
viruses are PVS, PVX, and PVY causes the downfall of potato production because they cause
defects on the potatoes, allowing them to grow smaller than the healthy potatoes. Symptoms
caused by the viruses show a mosaic pattern on the leaves. For the virus, Potato Leaf Roll Virus
(PLRV), the leaves are rolled, hence its name. For the potatoes, the viruses may cause ring spots
and strains. Potato viruses are mostly caused by aphids. Aphids are small insect that feed on
plant juice. Aphids are the virus vessels that transmit the virus into the plant. There are many
species of aphids and because of that, a potato
plant may be infected with more than one virus.
For potatoes infected with PVX, the virus is not
transmitted from aphids. Potato Virus X can only
be transmitted by man or machinery. Although
most potato viruses are spread by aphids. PVX is
spread through contact with other potatoes that
have the virus or by man and machinery. Other
than looking at the potato plant itself, it is possible
to detect viruses from the leaves. There are three
techniques that can be used to detect viruses,
Sandwich ELISA, Direct ELISA, and Dot Blot.
Once the virus has been detected, it is also possible
to remove the virus from the potato plant through tissue culture. The objective in this research is
to compare the alternative virus detection techniques on 96 potato leaf samples and how it can be
removed.
It was hypothesized that Direct ELISA can be used alternatively to detect the viruses
because it takes less time than the Sandwich ELISA and that it will give the same results as
Sandwich ELISA. Although, if Dot Blot was included in the experiment, Dot Blot would have
been the alternative technique because it takes less time than Direct ELISA. Dot Blot uses almost
the same process as Direct ELISA but have some advantages.
Material and Methods:
Sandwich Enzyme Linked Immuno-Sorbent Assay (ELISA):
Sandwich ELISA uses two sets of antibodies, capture antibodies and alkaline
phosphatase. A 96-well plate is used for both Sandwich and Direct ELISAs. In this experiment,
three viruses were tested, there should three well-plates. Make sure that the plates are labeled so
they don’t get mixed up with each other. Label the plates with the method used, your name, and
the date of performance. When doing Sandwich ELISA, capture antibodies, according to the
virus that is being detected, are used first to coat the plates so the infected proteins will attach to
the antibody. Ten microliters of the capture antibodies should be pipetted into each well using a

4. P100 pipette with 12 pipette tips. Then the plates is incubated for two hours on a shaker. While
the plates are incubating, the sample potato leaves should be extracted. To extract proteins the
leaves, the potato leaves must be grind into small pieces. Membrane extraction buffer (MEB)
should be made to mix with the grind leaves and as they mix, the buffer will pull out proteins
from the leaves. MEB is made with Phosphate Buffered Saline with Tween-20 (PBST) wash
buffer, tween-20, and nonfat dried milk (for the exact amount of ingredients, please look at the
PVY-N Reagent Set or the Reagent Set). When the incubation is over, the plates are washed with
PBST Wash Buffer thoroughly. When the samples are grind and have been extracted to liquid,
the liquid is pipetted into the wells. There should be a layout of how the samples are organized
on the plates. There three wells for each sample. Since 24 samples were tested in each plate, the
template is a little different because there is also the positive and negative controls. After
incubating for two hours, the plates are washed again. After
washing, the alkaline phosphatase is made and pipetted into the
wells according to the virus, and is incubated for another two hours
and washed again. For PVY, because the detection is a little
different from PVS and PVX, along with the alkaline phosphatase,
it also uses a detection antibody, which makes it a triple antibody
virus. Before washing the plates, the substrate is made with p-
Nitrophenyl Phosphate (PNP) and put into the wells. Make sure
that the plates are washed thoroughly because any leftover
antibodies with enzymes will affect the outcome. It takes an hour
for the color to develop. It is best if the plates are covered from
intense light because the light will affect the PNP by making the
substrate have color in the negative controls and make the results
turn out differently. When the color has been developed, the plates
are then read on a microplate reader. Optical density of the samples
were recorded at 405 nm wavelength. All the samples with two
folds or more optical density than the negative control were considered positive for the virus. In
order for all 96 potato samples to be finished testing, this process must be done three more times.
Direct ELISA:
Direct ELISA has almost the same procedure as Sandwich ELISA, but Direct ELISA
does not use capture antibodies. At the same time as the samples are put into the Sandwich
ELISA plate, it is best to start the Direct ELISA because this ELISA starts with putting in the
samples and incubating. Starting from the samples, the rest of the procedure will be the same as
Sandwich ELISA.
Dot Blot:
In Dot Blot, instead of using a 96-well plate,
a nitrocellulose membrane is used. Squares are drawn
on the membrane to separate the samples. Then one
dot of the samples are placed in a square. Once the

5. squares are filled, the samples are set to dry and put into a milk solution for an hour to keep the
proteins in place. After one hour has past, the membrane is then washed with Tris Buffer Saline
(TBS) twice for three to five minutes each. When the washing is done, the antibodies linked with
enzyme alkaline phosphatase are added onto the membrane and incubated for one hour and
washed again. When making the substrate, instead of using the PNP, Nitro Blue Tetrazolium
(NBT) is used. NBT is used because when the samples are placed on the membrane, the
chlorophyll is shown as a yellow-green dot, and if PNP was used, it would be difficult to
differentiate between the two, while NBT shows a purple color if infected. Instead of taking
about five hours to detect viruses, Dot Blot only takes two and a half hours to detect the virus,
which makes Dot Blot an efficient way to detect viruses.
Tissue Culture:
Tissue culture is used to remove the virus. Tissue culture is a non-contaminated process
which allows a plant to grow without soil. In this experiment, potato plants grown in culture
tubes were used to grow new potato plants. Culture tubes are used
because the potato plant will grow vertically and be easier to cut into
smaller pieces. In the culture tubes are media. Media is a plant nutrition
gel that gives the plants what they need. In tissue culture, any plant can
be grown and any container may be used as long as they are sanitized.
To remove the virus from the plants, tissue culture takes six to eight
months, so in this particular experiment, the tissue culture was not
completed. Tissue culture is step in removing a virus.
Media Preparation:
To make one liter of media for tissue culture, 1 liter of water,
4.43 grams of MS Media plus Vitamins (M519), 30 grams of sucrose,
and 7.5 grams of agar are needed. While making the media, it is best to
start separate by having agar in its own beaker. Start out with half a liter
in both beakers, one will be stirring, while the other is heating and stirring. Add the agar into the
heating beaker and let it stir and saturate. Then add the MS Media plus Vitamins (M519) and
sucrose into the other beaker and let it stir and saturate. Once the sucrose and media solution
have been saturated, melt the agar in the microwave. Although the agar has been heating, there
are still some pieces of the agar visible. When the agar is fully melted, it is added to the sucrose
and media solution. Once everything has been mixed, it is poured into a big jar to dispense the
media into the tubes. Twelve milliliters of the media are poured into each tube while the media is
still warm because once it cools down, it will turn into a solid and it will be unable to be
dispensed. When all the media is done and the tubes are filled, the culture tube cap are put on.
After that, the tubes are going to go into the autoclave to be sanitized, but before that, the
autoclave tape is put on across the tubes to make sure that they have been sanitized. When going
into the autoclave room, make sure that no one is using it. If no one is using it, open the door and
put in the culture tubes. To be safe, wear gloves. Make sure the tubes are in a basket so it will be

6. easier to take them out of the autoclave. Once everything is set, close the door and seal it so
nothing will leak out. Press one of the presets to start the autoclave, then wait until the autoclave
is over. For this research, it took 30 minutes for the autoclave to be done, but it took a little
longer for the temperature to go down since it gets really hot, so it is best to wait for 50 minutes.
When taking out the culture tubes, make sure to wear the gloves to protect yourself because the
autoclave, the tubes, rack, and the bucket are still very hot to touch. If touched, the autoclave can
burn your skin.
Subculture:
Before starting tissue culture, make sure everything needed is there; potato plants in
culture tubes, new culture tubes, knife, tong, etc. Before turning on the hood in the culture room,
make sure the spray the entire inside of the hood with 7% ethanol alcohol to make sure the hood
is sanitized, then turn on the light and fan to dry the ethanol alcohol. Also, always sanitize your
hands before touching any thing and placing things on the hood. Place the new tubes, tubes with
potato plants in a rack, petri dishes, knife, tong, 95% ethanol alcohol, and the flame bottle on the
hood. Make sure the flames bottle and the ethanol alcohol are not too close to each other. When
cutting the potato plant, the plant must be taken out of the tube and be cut on a petri dish. The
knife should be dipped into 95% ethanol alcohol before each use to ensure that there will be no
contamination. When the cutting is finished, the tongs are sanitized with the ethanol alcohol and
flamed to dry. It is most important that the tong and knife are sanitized before each use to make
sure that the plant is not contaminated. Each piece of the potato plant are placed into the culture
tubes and a small section of the plant goes into the media. After all plant pieces are in the tubes,
the tubes are labeled with what was written on the original tube. For example, if the original
label was MN07612WB-01, then that is what will be written on the new tubes. Along with that
the date is written on the tube. After everything is finished, the new tubes are taken into the
potato breeding room to grow. The plant should take about one month to grow tall enough to be
cut again and is repeated again for at least six months.
Results and Discussion:
Potato Virus Detected
+ Positive of Virus
- Negative of Virus
PVS: PVY: PVX:
Sample: Sandwich Direct Sandwich Direct Sandwich Direct
124 MN09054BW-01Rus + + + - - -
125 W9200-13 - - + + - -
126 MN10026WW-02Rus - - + - - -

7. 127 AF4124-7 + + + - + -
128 MN02467 + + + - - -
129 AC00206-2W - - + - - -
130 AF4725-14 + + + - - -
131 MonDak Gold (MN15620) + + + - - -
132 AF44130-7 + + + - - -
133 AF4281-3 + + + - - -
134 MN07312BB-01 + + - - - -
135 T186-1Y + + + - - -
181 W9252-7 + - + - - -
182 NorValley - - + - - -
183 W9322-2 + - + - - -
184 MN07179GFB-01 + - + - - -
185 MN10016WW-03 + + + - - -
186 W10676-1Rus + - + - - -
187 J112-8 - - + - - -
188 NYH15-7 - - + - - -
189 W9604-1Rus + - - - - -
202 MN10056WB-05Rus + + - - - -
203 MSR128-4Y + + - - - -
204 MN10010WW-05Rus + + - - - -
205 MN10056WW-05Rus + + - - - -
206 MN08213BW-01Rus + + - - - -
207 MN10006WW-03Rus + + + - - -
208 MN10006WW-03Rus + + - - - -
209 MN08083BW-01 - - - - - -
210 AF4965-2 + + - - - -
211 W9313-2 + + - - - -
212 MN09045BW-01Rus + + - - - -

8. 213 Red Pontiac + + + + - -
280 A03440-2C + + - - - -
281 Yukon Gold + + - - - -
282 MN09059BB-01 - - - - - -
283 CO00270-7W - - - - - -
284 J2-2 - - - - - -
285 A01143-3C + + - - - -
286 MN09082BW-01Rus + + - - - -
287 MN10049BB-01Rus + + - - - -
288 CO05061-6W + + - - - -
289 ND071336-1 + + - - - -
290 ND8305-1 + + - - - -
291 Af4518-1 - - - - - -
292 MN07289BB-01 + - - - - -
293 AF4975-3 + - - - - -
294 MN07289BB-01 + - + - - -
295 W8822-2 - - + - - -
296 W6609-3 + + + - - -
297 MN09010BW-01R - - + - - -
298 CO03243-3W + - - - - -
299 R093-3 - - + - - -
300 W450-1 + - + - - -
301 MN09102BW-01Rus - - + - - -
302 MSQ086-3 + - - - - -
303 AF4648-2 + - - - - -
325 MN07330BB-01 + - + - - -
326 MSL007-B + + + - - -
327 W9281-14 + - + - - -
328 MN09032BB-01 + + + - - -

9. 329 AF4113-2 + + + - + -
330 ND071369b-6 + + + - - -
331 W5015-12 + - + - - -
332 W8867-5 + - + - - -
333 MN10011WW-03Rus + + + - - -
334 T184-3 + + + - - -
335 ND8316-2 + - + - - -
336 AC034352-2W + - + - - -
472 AC034352-2W + - + - - -
473 MN09054BW-01Rus + + + - - -
474 MN10056WW-04Rus + + + + - -
484 MN09045BW-01Rus + - + - - -
485 MSS297-3 + - + - - -
486 MN10017WB-01Rus + + + - - -
487 MN09079BB-01Rus + + + - - -
488 NYH25-4 - - + - - -
489 MN09152BW-01Rus + - - - - -
490 MN02467 + - + - - -
491 MN10023BB-01Rus + - + - - -
492 W8405-1R + - + - - -
493 CO05061-2P + - + - - -
494 MN08155BW-01Rus - - + - - -
495 MN08085BW-01 + - + - - -
496 AND00618-2RussY - - - - - -
497 AF0338-17 - - + - - -
498 MN04844-07 - - + - - -
499 W9960-1R/Y + + + - - -
500 CO05061-7W + + - - - -
501 MSQ086-3 + + + - - -

10. 502 CO03187-1RU + + + - - -
503 MN08213BW-01Rus + + + - - -
504 W6234-4Rus + + + - - -
529 MN08122BW-01Rus + + + - - -
530 CO02033-1W + + - - - -
531 MN08207BW-01Rs + + - - - -
In 2012, tubers were first planted in an inoculation trial of PVY. These tuber (highlighted
samples) were harvested and planted in 2013 to evaluate expression of PVY infections.
Potato Virus Detection Percentage
PVS PVY PVX
Sandwich ELISA 79.2% 64.6% 2.1%
Direct ELISA 50.0% 3.1% 0.0%
While conducting this experiment, two methods were used, Sandwich ELISA and Direct
ELISA. Although they may be similar, Direct ELISA does not use capture antibodies. Aside
from that, it was expected that both methods would have the same results. As it turn out, most of
the outcome in PVS for Sandwich and Direct ELISA were very similar. For PVX, the outcomes
were also very similar. But as for PVY, the outcomes were only similar with some samples. The
virus would show most in the Sandwich ELISA because it uses two antibodies with a certain
protein, while Direct ELISA only uses one antibody and has all the proteins available. The
reason why the results had some difference was probably because 24 samples were at use and
was being tested for three different viruses at the same time.
As the three viruses were being detected, PVS, PVY, and PVX, it has come to conclude
that PVS is most commonly found in the potato samples that were tested. It was also concluded
that PVX appears the least in these samples. Most of these samples appear to have more than one
virus. Since the potato viruses are transmitted through aphids, and as a result of many species of
aphids, more than one species may have transmitted the viruses into the same plant.
Sandwich ELISA is most useful when indicating how much the potato is infected. Direct
ELISA is useful when indicating which potato plant is infected, although Sandwich ELISA is a
good use for that, it takes a much longer process than Direct ELISA. Either way, they should

11. have the same, if not similar, results. In total of testing 96 different samples, it took two weeks to
complete because there were some mistakes made in the processes.
Advantages of Alternative Techniques
If it is to only find which potato samples are infected with a virus, Dot Blot would be
most useful because it takes less time than Sandwich ELISA and Direct ELISA. Dot Blot also
uses less resources. But if it is to find which sample has more infection than the other, Sandwich
ELISA is the best method because it uses two sets of antibodies, so it is more accurate.
Conclusion:
Direct ELISA resulted to be not as efficient than Sandwich ELISA, even though it took
less time. When the virus detection was over and the results were out, it was seen that Direct
ELISA did not have the same results as Sandwich ELISA, although they did have some
similarities. Sandwich ELISA was shown as the most efficient and had a greater result compared
to Direct ELISA. As for the tissue culture, the experiment was not complete because the time
frame was too short. But as far as the tissue culture went, there were no contamination found in
the tubes. Each plant was growing healthy, but some did not grow as fast as the other because
while cutting and putting the plant into new tubes, the plant tissues may have been disrupted,
which makes the plant have difficulties growing.
References:
● Agdia Inc. PVY-N Reagent Set, Catalog Number: SRA 26001, Elkhart Indiana.
● Reagent Set, Agdia Inc., DAS ELISA, Elkhart Indiana
● Salazar, L.F. 2003. Potato Viruses After the XXth Century: Effects, Dissemination and
Their Control. Crop Protection Department, CIP, Lima, Peru.
● Samsatly, J., Jawhari, M., Najjar, C., Sobh, H., & Abou-Jawdah, Y. 2014. Modification
of Serological Techniques and Their Evaluation for Detection of Potato Viruses in Seed
Certification Related Activities. Crop Protection 16: 51-57.
● Selvaraj, S., & Ganeshamoorthi, P. (n.d.). Insect Pests of Potato and its
Management.Insect Pests of Potato and its Management. Retrieved July 14, 2014, from
http://www.krishisewa.com/cms/disease-management/137-potato-insect-pests.html
Acknowledgements:
This project was supported by American Chemical Society Project SEED, Minnesota
Local Section of the American Chemical Society and 3M Foundation. Special thanks to Dr.
Sanjay Gupta, Dr. Christian Thill and Lila Westreich for teaching me about potato virus
detection, tissue culture and for letting me use their lab to do my project. I would also like to
thank the University of Minnesota, Twin Cities for the location of Project SEED.

12. Research Reflection:
When I first started,researching potato virus was kind of unusual because it was not what I was
expecting to doing. But as I learn techniques about virus detection and tissue culture and watching the
graduate student doing her own research,I started to get fascinated, and I was getting comfortable doing
things on my own. On my first week, I was unable to understand what they were telling me to do because
this was all new to me and I didn’t know how to do it. The graduate student, Lila, gave me a protocol on
the virus detection. On the first day, she tried to teach me how to do the virus detection. At first I was
kind of intimidated because I was worried that I was doing to do something wrong, but then she was
telling me what to do, so I thought I couldn’t have made any mistake. She helped me here and there the
first week. In fact, she was the one who taught me most of things I did for my research project. One thing
she didn’t teach was the tissue culture because she didn’t have he things with her to show me, and it was
kind of confusing when it is explained verbally.
For my second week,I was able to start my research project,virus detection. Virus detection was
a simple project, at least that was what I had thought. I found out that I had to test 96 potato samples with
three different viruses, PVX, PVS,and PVY. It did same difficult at first because it was a lot of sample
testing. But then Dr. Gupta suggested that I do 24 samples at a time, so then I would be doing the same
procedure four times to complete the 96 samples. I thought it was simple enough, but when it came to
actually the experiment, it was very tiring. When I had to extract the samples, it took about half an hour
because samples had to be picked, the extraction buffer had to be made, and they all had to be put into
small tubes. And when I was pipetting the samples into the wells, it took about an hour and a half because
each sample had to be pipetted into three wells and there were six plates that had to be filled. So it was
very tiring going back and forth from tube to well. Other than that, it was a lot of waiting because after
something has been put into the wells, they had to be incubated for two hours or overnight. So that wasn’t
the most fun, but in the end when the all the result were out and the 96 samples were finished, it was a
great experiment.
When the virus detection was finished, I was able to start on the second part of my research
project, tissue culture. I thought It was really fun because I was able to cut things and lit something on
fire. There was something about it that made me fascinated. One of the fun things when doing tissue
culture was making the media. Making the media was probably one of the most simplest thing that could
be done in this lab. It was very simple and it was fun dispensing them into the culture tubes. But the scary

13. part was when I had to put culture tubes with media into the autoclave to sterilize the tubes because when
the autoclave was finished, it was really hot that it can burn your skin, so I was really cautious around the
autoclave. The tissue culture didn’t last long because I had to wait for weeks for the potato plants to grow
tall enough to be able to cut them again, but it was still very fun to do tissue culture for a little while.
My research project didn’t really take me long to finish. It probably only took 3 weeks total,
though it could have taken less time if I didn’t had to redo some of the virus detections. Overall, it was
really great to have this opportunity to do these kind of things when I’m just in high school.