Notes on amplification

• The plant primers are present at 1/10^th the concentration of the Phytophthora genus specific primers due to the greater amount of plant DNA that is present in the sample.  If a greater amount of the plant primers are used they compete with the genus specific primers for amplification and will provide a fainter Phytophthora genus specific band (the plant primers amplify a smaller fragment and are therefore more efficient in amplification).
• In each amplification I include a known sample (P. ramorum DNA) as well as a blank tube with no DNA
• The presence of glycerol enhances the specificity of the amplification, without it a band may be amplified by some, but not all, plant species
• Adding BSA to enhance the level of template amplification has been tried
• BSA: from New England BioLabs, dilute a 100 mg/ml stock solution 1:20 and add at a rate of 1µl per tube (make in 100 µl aliquots and store in the freezer; after repeated freeze-thaw cycles or if it is more than a few weeks old discard and remake)
• BSA is not essential for the amplification, it just tends to give more amplified product but you may encounter nonspecific background bands

• Samples from the first round amplification are diluted 1:100
• There are several reasons for this:
  •  It significantly dilutes the plant DNA so the second round of amplification with the genus specific primers (which is done in the absence of glycerol and at a lower annealing temp so if one is used, the species specific fragments amplify) will not amplify plant sequences
  • The plant primers currently in use generate a fragment that is close in size to the P. ramorum diagnostic fragment, so diluting the samples this much will eliminate this as a potential source of false positives
  • Dilutions of 1:25. 1:50 work as well, but I like to use the 1:100 just to make sure.  There does not appear to be any difference in the final level of the amplified diagnostic fragment between any of the dilutions
  • New plant primers have been constructed that will not interfere with this assay.  I have not finished testing them on all plant species yet so I have kept the prior primers (please see below for details)
  • As noted above, the pathogen DNA is often present in very low concentrations relative to the plant DNA, which means that often times it will not amplify a genus specific fragment with just a single round.  Unfortunately adding more DNA to the reaction mix may inhibit amplification due to the presence of PCR inhibitors, so the approach I have chosen is do a second round of amplification with the genus-specific primers
  • The primer concentration for this amplification is lower than the first round so it does not generate nonspecific background problems

Second Amplification Master Mix

       2.5 µl    Buffer
       3.0 µl    MgCl₂ (3 mM)
       0.5 µl   dNTP mix
       0.2 µl   FMPhy-8b (12.5 µM)
       0.2 µl   FMPhy-10b (12.5 µM)

• Bring vol to 19 µl/reaction with water, add 1µl of diluted DNA and place on ice.
• Add 5 µl of diluted AmpliTaq (0.2 µl AmpliTaq + 4.8 µl H₂O per amplification.)
• Start the amplification program on the thermalcycler and put on “pause”
• Once the lid temperature and block temperature are reached, the tubes are placed in the wells, the lid closed and the program restarted.
• After amplification, 12 µl of the reaction mix is run on a 3% NuSieve 3:1 gel to visualize the bands.
• If the water blank has a band or the known tube does not amplify, the entire amplification is discarded and the samples rerun.

       1.   95 C for 3 minutes
       2.   95C for 30 sec
       3.   64 C for 30 sec
       4.   go to 2 and repeat 34 more times
       5.   72 C for 5 minutes
             10 C, hold

•        Use of primer pair Phy-8b and Phy-10b
  •        Under the conditions indicated above Phy-8b + 10b primer pair amplifies all of the Phytophthora species with the exception of low amplification of P. lateralis and P. sojae. 
  •        Both these species are amplified by the earlier version of this primer pair, Phy 8 + 10.
    •        Phy 8 + 10 will amplify all Phytophthora spp. and none of the plant material tested, it also amplifies Pythium spp. as well.  For this reason it cannot be used for assaying any plant part that comes into contact with the soil.
    •        This primer pair is more robust than Phy-8b and Phy-10b and will generate a higher concentration of template.
  •        The Phy-8b and Phy-10b primer pair did not amplify any of the Pythium spp. tested.

•        One of the first things that will need to be tested when this procedure is use done on a different PCR machine is to evaluate the effect of annealing temp on amplification.  The annealing temperature is very important to for the specificity of the genus–specific primers and differences have been encountered when using different thermalcyclers.
  •        At 65 annealing temp with glycerol the primers do not amplify plant DNA from the species that have been tested using the Eppendorf thermalcycler (most of the hosts from which P. ramorum has been isolated, lettuce, citrus, pine, watermelon, cucumber, tomato, lettuce, cherry, juniper, redwood, sugar beet, strawberry, ..).  However, using an Applied Biosystems 9700 thermalcycler in Paul Tooley’s lab in Ft. Detrick low background amplification was observed Kalmia latifolia ‘Madeline’ (approximately 0.3 kb), U. californica (approximately 0.8 kb), Glycine max (approximately 0.4 kb) and Solanum tuberosum (approximately 0.3 kb). 
  •        Removing the glycerol form the amplification mixture and using a 65 C annealing temp will allow background amplification of cherry, citrus, live oak, Juniper, and strawberry. 
  •        With a 66 C annealing temp without addition of glycerol no amplification of plant DNA is observed.

§ The annealing temperatures that we used with our thermalcycler (Eppendorf) for the first and second round amplification was 66 C and 64 C, respectively. However, when we used a MJ unit the temperatures had to be reduced by 1 C for the marker system to work. I believe this is due to differences in block calibration between machines (possibly differences in ramp rates as well) and highlights the importance of validating your machine before getting started with using the diagnostic assay.  Several suggestions:

§ use purified DNA of the pathogen that may be present in your infected tissue and healthy plant DNA from the hosts you will be evaluating to check the annealing temps for specificity. If the annealing temp is too low for the first round amplification some plant DNA (citrus) may give a band the same size as the Phytophthora genus‑specific amplicon.

·   After the optimum annealing temperatures have been determined the plant and pathogen DNA are combined in the master mix and 12 tubes are placed in a uniform pattern across the thermalcycler block to test for block uniformity.  Both the first and second amplification cycles are tested this way and the separate tubes are compared for intensity of amplification and lack of background bands.  To ensure our thermalcycler is functioning properly we repeat this test on a regular basis.

§ If the annealing temp for the first round amplification with the Phytophthora genus‑specific primer pair is to high there will be low level of genus‑specific amplicon generated n the first round amplification, which will reduce the sensitivity of detection using the species specific primer pair.

 TROUBLE SHOOTING

• Bands in the 450-500 bp range when plant DNA only is in the amplification mixture – the stringency needs to be increased.  Make sure the glycerol is included in the master mix, increase the annealing temperature by 1 C,

• Inconsistent amplifications – if there is inconsistent amplification of positive controls or amplification of negative controls the components of the master mix are discarded and fresh tubes opened.  The uniformity of temperature control of the block is also tested.