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Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification
Chapters
Summary June 25th, 2020
We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.
Transcript
Phytopathogen detection is a crucial step in plant disease management. This protocol provides a rapid detection method for irrigation water contamination due to a common waterborne fighter pathogen. Using this technique, specific pathogen, such as Phytophthora capcici, can be processed and rapidly detected from our large volume of irrigation water while remaining on site.
Demonstrating the procedure will be Owen Hudson, a Masters student from my laboratory, and Sumyya Waliullah, a PostDoctoral researcher from Dr.Pingxi G's laboratory to set up a pump and filter for on site Phytophthora capcici detection in irrigation water. Attach a filtering flask to a tube connected to a hand pump and fitted buchner funnel into the rubber stopper in the mouth of the filtering flask. Then fit an appropriately sized piece of filter paper with a 15 micron retention size into the funnel.
To obtain water samples collect irrigation water from the targeted source. The water may have small amounts of debris, but not significant sediment or soil. Slowly pour between 50 to 1000 milliliters of test water over the filter paper inside the funnel while using the hand pump to create a suction to pull the water through the funnel.
When all of the water has been filtered, use forceps to remove the filter paper from the funnel. And use sterile scissors to cut the paper into small pieces. Submerge eight to 12 pieces of paper in 400 microliters of extraction buffer in a 1.5 milliliter tube for a five minute incubation at room temperature.
Vortex or otherwise agitate the paper for 10 seconds once every minute. At the end of the incubation, use forceps to remove the paper with as little buffer as possible. And repeat the license with the next set of filter paper pieces.
For DNA extraction from the filter paper samples added 20 microliters of proteinase K and 10 microliters of 10 nanograms per microliter RNAs to the sample too and incubate the solution at room temperature for 15 minutes with vortexing or shaking every three minutes. At the end of the incubation, add 500 microliters of magnetic beads in binding buffer to the sample and mix well by shaking. After five minutes at room temperature, place the tube and the magnetic separator rack for two minutes before removing and discarding the supernatant.
Remove the tube from the magnetic separator and vigorously re-suspend the beads in 500 microliters of wash buffer. After 30 seconds return the tube to the magnet and wait two minutes before discarding the supernatant. Repeat the wash with 500 microliters of wash buffer too and 500 microliters of 80%ethanol as just demonstrated.
And air drive the magnetic bead pellet for 15 minutes at room temperature. At the end of the incubation re-suspend the beads in 50 microliters of elution buffer with pipetting for one minute before placing the tube back onto the magnet for two minutes to allow collection of the supernatant. After preparing the LAMP primer mix as indicated in the table, add at 2.5 microliters of primer mix 12.5 microliters of a lava LAMP mastermix one microliter of extracted DNA and nine microliters of double distilled water to a PCR tube and set up one positive and one negative control.
incubate all of the samples in a 64 degrees Celsius heat blog for 45 minutes or the genie three amplification instrument at the end of the incubation, a load of five microliters of each amplified sample onto a 1%agarose gel and image the resulting bands on an ultraviolet imaging machine. If a calorimetric dye was used, view the color change to determine the results as positive or negative. If a portable amplification instrument was used, view the amplification graph on the screen to determine the results.
The optical time for running the LAMP assay at 64 degrees is 45 minutes, as the lowest concentrations that were positive for detection are not amplified until 40 minutes, while higher concentrations are amplified at 20 minutes. Amplification can be confirmed on a 1%agarose gel labeled with a Nucleic acid stain. As illustrated conventional PCR is 40 times less sensitive than LAMP.
Detecting 4.8 times 10 to the third zoospores per milliliter concentrations at the lowest. In this analysis, water samples were collected from seven ponds used for commercial vegetable production in Tift County, Georgia. Three of which demonstrated positive LAMP results.
When the samples were tested by conventional PCR however, zoospores were detected in only one of the three positive samples. In this table, the differences between detection methods using such variables as sensitivity, time, required preparation, materials and cost are summarized. LAMP is the least expensive method among the tested methods, and is also the fastest, requiring only 30 to 60 minutes for amplification compared to conventional PCR amplification.
Using this protocol DNA extracted from closely related UMI seed pathogens results in no detection for any of the samples, confirming the specificity of the primers. Make sure not to pour the sample water too fast and be careful when adding extracted DNA to the LAMP tubes to limit contamination. Once other waterborne pathogens have had their specific primer sets developed, the filtration and lamp acid methods can be adapted for other pathogens.
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