Catalog Number AP 107
Intended Use
The ELISA screens for the presence of soybean rust caused by Phakopsora
pachyrhizi. The test can detect the presence of the pathogen at the very
early stages of infection, from chlorotic lesions (before formation of a
pustule) to immature pustules (not releasing spores). During this period it is
often difficult and critical to differentiate the soybean rust symptoms from
other diseases caused by bacterial, viral or fungal infections and/or insect
damage. In addition, the test can also be used to detect advanced rust
symptoms with uredinospores and teliospores, complementing visual
inspections.
In controlled inoculation studies with levels as low as 100,000 spores/mL,
this kit has been shown to detect the presence of soybean rust infection
before the appearance of visual symptoms. Infection levels in the field may
vary depending on environmental conditions.
Materials Needed
• Tissue Extraction Kit, EnviroLogix Cat. # ACC 002 OR Multi-Wall
Mesh Pouch, EnviroLogix Cat. # ACC 021
• pipettes capable of delivering 100 µL
• marking pen (indelible)
• tape or Parafilm®
• timer
• distilled or deionized water for preparing Wash Buffer and for diluting 5x
Soy Leaf Extraction Buffer
• glass bottles or flasks with 250 mL capacity for storage of 1x Soy Leaf
Extraction Buffer and 1 liter capacity for Wash Buffer
• microtiter plate reader or strip reader
• wash bottle, or microtiter plate or strip washer
• multi-channel pipette that will measure 100 µL (optional)
• racked dilution tubes for loading samples into the plate with a multichannel
pipette (optional)
• orbital plate shaker (optional)
Preparation of Solutions
Wash Buffer:
Add the contents of the packet of Wash Buffer Salts (phosphate buffered
saline, pH 7.4 - Tween 20) to 1 liter of distilled or deionized water, and stir
to dissolve. Store refrigerated when not in use; warm to room temperature
prior to assay.
1x Soy Leaf Extraction Buffer
To prepare, warm 5x Soy Leaf Buffer supplied with the kit to room
temperature and mix well before diluting. Add one measure of 5x Buffer to
QualiPlate Kit for Soybean Rust
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Punch leaf sample
Use pestle to mash leaf tissue
Remove unneeded strips
Add sample extract
Bottle Wash method
four measures distilled or deionized water in a suitable container to create
1x Soy Leaf Buffer. Mix thoroughly to dissolve. Store refrigerated when
not in use; warm to room temperature prior to assay. 1X Soy Leaf Buffer is
stable for two weeks when stored refrigerated at 4-8°C and should be made
on an as-needed basis. Please call EnviroLogix Tech Service if you have
questions regarding calculating the appropriate amount of buffer to make
for the number of samples that will be processed.
Sample Preparation
Sampling Recommendation:
Take a leaf sample that includes a suspect spot or area. The ACC 002 punch
cap will result in a leaf sample of approximately 10mm diameter, weighing
about 0.01 grams. If using an alternate method, care must be taken to
ensure that the sample does not have an excess of non-affected tissue; this
may reduce sensitivity.
Sample Extraction:
1. Green leaf samples: Take a leaf punch sample by snapping the tube cap
of the Disposable Tissue Extractor down on the leaf, encompassing the
suspected rust spot. Insert the pestle into the tube and grind the tissue
by rotating the pestle against the sides of the tube with twisting motions.
Add 500 µl of Soy Leaf Extraction Buffer and continue grinding for 20-
30 seconds or until the leaf tissue is well ground. Use extreme caution
to prevent sample-to-sample cross-contamination with plant tissue or
exudate.
2. Similar grinding devices (e.g. Multi-Wall Mesh Bags) can be used with
a leaf to extraction buffer ratio (weight:volume) of 1:25 – 1:50 (eg. 3
mL to a 2.5 cm diameter leaf section). Using a hard object (e.g. coin)
rub across the surface of the mesh bag against a hard surface until the
leaf has transparent areas where the inside mesh has been forced
through the leaf. After adding Extraction Buffer, massage the exterior
of the mesh bag with fingers while holding the top of the bag closed to
prevent the buffer from spilling.
How to Run the Assay
• Read all of these instructions before running the kit.
• Allow all reagents to reach room temperature before beginning (at least
30 minutes with un-boxed plates and reagents at room temperature - do
not remove plates from bag with desiccant until they have warmed up).
• Organize all reagents, sample extracts, and pipettes so that step 1 can
be performed in 15 minutes or less. If more than three strips are to be
run at one time, the loading time will most likely exceed 15 minutes,
and the use of a multi-channel pipette is strongly recommended in steps
1, 5, 8 and 9.
• If three or fewer strips are to be run, use a disposable-tip, airdisplacement
pipette and a clean pipette tip to add each Standard and
sample extract to the wells. Conjugate, Substrate, and Stop Solution
may be added in the same manner; alternatively, use a repeating pipette
with a disposable tip for these three reagents.
• Once all components have reached room temperature, remove the plate
from the pouch. If fewer than all twelve strips are used, reseal the
remaining strips and the desiccant in the foil pouch, and refrigerate.
QualiPlate Kit for Soybean Rust
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Rev. 04-19-07
Incubate
Test can be read visually, or…
…complete protocol and add
Stop Solution
Read plate in a Plate Reader
within 30 minutes of the addition of
Stop Solution
• Use the well identification markings on the plate edge as a guide when
adding the samples and reagents. It is recommended that at least two
wells each of Blank (Extraction Buffer) and a known-negative soy leaf
extract be run on each plate. Additional quality control samples may be
added at the discretion of the user. The kit Positive Control is provided
to show an example of a strong positive result. Sample extracts may be
run in either single or duplicate wells.
1. Add 100 µL of Extraction Buffer Blank, 100 µL of the Soybean Rust
Kit Positive Control, 100 µL of any user-prepared negative control
leaf extract, and 100 µL of each sample extract to their respective
wells. Follow the same order of addition for all reagents.
NOTE: It is strongly recommended that a multi-channel pipette be
used in steps 1, 5, 8 and 9.
2 Thoroughly mix the contents of the wells by moving the plate in a rapid
circular motion on the bench top for a full 20-30 seconds. Be careful
not to spill the contents!
3. Cover the wells with tape or Parafilm to prevent evaporation and
incubate at ambient temperature for 1 hour. If an orbital plate
shaker is available shake plate at 200 rpm.
4. After incubation, carefully remove the covering and vigorously shake
the contents of the wells into a sink or other suitable container. Flood
the wells completely with Wash Buffer, then shake to empty. Repeat
this wash step three times.
5. Add 100 µL of Soybean Rust Enzyme Conjugate to each well.
6. Thoroughly mix the contents of the wells, as in step 2. Cover the wells
with new tape or Parafilm and incubate for 1 hour at ambient
temperature. Use orbital shaker if available.
7. Wash the wells again as described in step 4. Alternatively, perform four
washes (300 µL/well) with a microtiter plate or strip washer. Slap the
plate on a paper towel to remove as much water as possible.
8. Add 100 µL of Substrate to each well. Mix thoroughly as in step 2.
Cover the wells with new tape or Parafilm and incubate for 20 minutes
at ambient temperature. Use orbital shaker if available.
NOTE: At this point, results may be scored visually. Test wells that are
blue are positive for soybean rust. Very weakly positive (ie very light blue)
results may require the use of a plate reader for confirmation. If the plate
reader is to be used, continue with step 9.
Caution: Stop Solution is 1N Hydrochloric acid. Handle carefully.
9. Add 100 µL of Stop Solution to each well and mix thoroughly. This
will turn any positive well contents yellow.
NOTE: Read the plate within 30 minutes of the addition of Stop Solution.
How to Interpret the Results
Visual Inspection
After step 8 above, the well containing the Positive Control should show a
distinct blue color. If not, that may indicate an invalid assay due to possible
improper protocol, and the test should be repeated.
QualiPlate Kit for Soybean Rust
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If after step 8 above, a test well is blue, this is interpreted as positive for
Phakopsora pachyrhizi. Very weakly positive results may require the use
of a plate reader for confirmation.
Spectrophotometric Measurement
Set the wavelength of the microtiter plate reader to 450 nanometers (nm).
(If it has dual wavelength capability, use 600, 630 or 650 nm as the
reference wavelength.)
Interpreting Results
Compare the Optical Density (OD) of the sample extracts to those of the
mean Extraction Buffer Blank wells, or preferably, to known-negative leaf
extract wells, to determine presence or absence of soybean rust in your
sample extract. Samples with absorbances significantly greater than those
of the Blank and/or negative leaf extract wells are presumed to be positive
for soybean rust.
Cross-Reactivity
The kit does not cross react with several other rust infections caused by
Uromyces, Puccinia and Melampsora species. No cross reactivity was
observed with other common fungal genera including Aspergillus,
Cercospora kikuchii or C. sojina, Fusarium, Penicillium, Peronospora
mansurica, Pseudomonas savastanoi pv. Glycinea, Septoria, Rhizoctonia,
Rhizopus, and Xanthomonas campestris pv. Glycinea. No cross reactivity
has been observed with similar-looking diseases such as frogeye leafspot,
powdery mildew, downy mildew, brown spot, bacterial blight, bacterial
pustule.
Precautions and Notes
• Observe any applicable regulations, federal or state guidelines, or inhouse
lab safety protocols when disposing of samples and kit reagents.
• Store all QualiPlate components at 4°C to 8°C (39°F to 46°F) when not
in use.
• Do not expose QualiPlate components to temperatures greater than
37°C (99°F) or less than 2°C (36°F).
• Allow all reagents to reach ambient temperature (18°C to 27°C or 64°F
to 81°F) before use.
• Do not use kit components after the expiration date.
• Do not use reagents or test plates from one QualiPlate with reagents or
test plates from a different QualiPlate.
• Do not expose Substrate to sunlight during pipetting or while
incubating in the test wells.
• Do not dilute or adulterate test reagents or use samples not called for in
the test procedure.
• As with all tests, it is recommended that results be confirmed by an
alternate method when necessary.
QualiPlate Kit for Soybean Rust
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Rev. 04-19-07
For Technical Support
Contact Us At:
EnviroLogix
500 Riverside Industrial
Parkway
Portland, ME 04103-1486
USA
Tel: (207) 797-0300
Toll Free: 866-408-4597
Fax: (207) 797-7533
e-mail:
info@envirologix.com
website:
www.envirologix.com
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