The overall goal of this procedure is to accurately detect the citrus greening pathogen Candidatus liberibacter spp. in citrus leaf tissue. This method allows citrus farmers and researchers to quickly and accurately identify the presence of pathogen responsible for causing citrus greening.
Demonstrating procedure will be Ian Palmer, a graduate student from my laboratory. This method is efficient, cost effective, and adaptable for quantitative analysis. Additionally, it may be performed with a minimum level of laboratory experience and equipment.
First use clean scissors to cut a whole fresh leaf from a citrus tree, and place in a clean plastic sandwich bag. Then chill a mortar and pestle, by adding small quantity of liquid nitrogen. Next use the scissors to cut small pieces of the leaf tissue, approximately one square inch in size.
Place the leaf pieces in the mortar to freeze them immediately. Quickly start grinding the leaf tissue with the mortar, until the liquid nitrogen evaporates, and a fine green powder remains. Next chill a metal spatula in liquid nitrogen, for 10 to 15 seconds.
Then use the cold spatula to scoop the ground tissue from the mortar in a 1.5ML micro-centrifuge tube. Use a 1000ML pipet to add 600ML of nuclei lysis solution to the sample. Then vortex the sample for one to three seconds.
Incubate the tube at 65 degrees celsius, in a water bath for 15 minutes. Once the incubation period is over, use a 10ML pipet to add 3ML of RNA solution to the lycate to remove RNA. Then mix the contents of the tube by inverting it two to five times.
Incubate at 37 degrees celsius for 15 minutes in a cabinet incubator. After 15 minutes add 200ML of protein precipitation solution to the sample. Vortex the tube at high speed for 20 seconds.
Next centrifuge the sample at 13000G for three minutes. During the course of centrifugation add 600ML of isopropanol maintained at room temperature, to a new 1.5ML micro-centrifuge tube. After the centrifugation is over, carefully remove the supernatant from the centrifuge tube, to the tube containing isopropanol.
Discard the pellet. Invert the tube for the DNA to be visible like a thread like strand. Next centrifuge the sample at 13000G for a minute at room temperature.
Decant the supernatant after centrifugation. Then add 600ML of 70%ethanol to the pellet. Invert the tube several times to wash the pellet.
Centrifuge the tube at 13000G for one minute. After the centrifugation is over, carefully aspirate the supernatant. Then invert the tube with the lid open on an absorbent paper to air dry for 15 minutes.
Once dried add 100ML of DNA re-hydration solution to the pellet. Incubate the DNA suspension at 65 degrees celsius in a water bath for an hour. Tap the tube periodically while mixing.
Add 1ML of purified water in a micro-cuvette. Read the water as blank on the spectrophotometer. Then add 1ML of the sample in the micro-cuvette.
Place the micro-cuvette on the spectrophotometer, and obtain the DNA concentration. Constitute the PCR reaction by adding all the reagents, and 100 nanograms of genomic DNA in a 50ML PCR tube. Mix the reagents by gently flicking the tube, followed by a quick centrifugation.
Place the PCR tube in the thermo-cycler and run the program. To prepare the agarose gel, weigh 0.4 grams of agarose powder. Then add the agarose powder to a 200ML Erlenmeyer flask, adjusting the volume to 50ML with 1X TAE buffer.
Microwave the agarose mixture on high for about 1.5 minutes. Shaking every 30 seconds until the agarose dissolves completely. Next add 2.5ML of 10 milligram per milliliter of ethidium bromide to the liquid agarose.
Then shake the flask to mix the ethidium bromide, and quickly pour the liquid agarose gel in a level gel mold. Immediately insert a standard 12 tooth gel comb. Let the gel cool for about 20 minutes at room temperature, and then remove the gel comb.
Now place the solidified gel in the electrophoresis unit filled with 1X TAE buffer. Load the total amplified DNA sample and 5ML of DNA later in separate wells on the gel. Start the electrophoresis unit, at a constant 90 volts for 35 minutes.
Then remove the gel from the electrophoresis unit. Next place the gel on a UV transilluminator. Capture the photographs of the gel banding patterns in the transilluminator at 302 nanometers.
To identify the greening bacterium, the PCR amplified genomic DNAs obtained from both infected and uninfected samples are electrophoresed. In the gel these bands corresponding to 500 base pairs indicate a presence of a specific region of the Las16S ribosomal DNA in the CL asiaticas bacterium. These bands corresponding to 700 base pairs indicate the presence of LAA2/LAJ5, and insert present in the 16S ribosomal beta operon DNA sequence in the bacterium.
These distinct bands at 400 base pairs indicate the amplification of only the citrus F-box gene, which refers to the internal amplification control. To consider our results valid, presence of this band is required. On the contrary, presence of only a band at 400 base pairs, but lack of the other bands at 500 and 700 base pairs indicate a healthy citrus tissue.
Once mastered this technique can be done in about four hours if it's performed properly. This procedure may be adapted to allow for the performance of more quantitative methods, such as RTQPCR to detect the amount of pathogen present.
