TaqMan Real-Time PCR Amplification Procedure for P. ramorum   

TaqMan Real-Time PCR Amplification Procedure for P. ramorum
 
The following procedure is the TaqMan real-time PCR amplification procedure for P. ramorum as reported in Tooley et al. (2006).  The P. ramorum species-specific primer pair and the internal control plant primer pair are the same used for the conventional nested PCR amplification procedure.  Species specific primer pairs for other Phytophthora spp. have been developed (list of species, sequence alignment used for primer development - .msf, .nex, fastA)
 

Tooley, P.W., Martin, F.N., Carras, M.M. and Frederick, R.D. (2006) Real-time fluorescent PCR detection of Phytophthora ramorum and Phytophthora pseudosyringae using mitochondrial gene regions.  Phytopathology 96: 336-345 (pdf reprint)
 
 

 Modifications to amplification procedure

·         Potential alternative plant primers

·         Conventional PCR detection of P.  ramorum
 
Amplification Conditions
The following amplification procedures used TaqMan universal master mix, when BioRad master mix was used instead background amplification of 5 additional species was observed

  • Amplification conditions were
    • An additional 0.5 mM MgCl2 was added to the master mix
    • FMPr-1a, FMpr-7 used at 1,000 nM final concentration
    • TaqMan probe PrFAM used at 400 nM final concentration
    • FMPl2b, FMpl3b used at 100 nM final concentration
    • TaqMan probe Plant CALOrange used at 80 nM final concentration  
  • Primers

    Target

    Primer/probe

    Sequence (5’ to 3’ )

    Length

    Tma

    %GCb

    P. ramorum

    FMPr-1a

    GTATTTAAAATCATAGGTGTAATTTG

    26

    50.0

    23.1

    P. ramorum

    FMPr-7

    TGGTTTTTTTAATTTATATTATCAATG

    27

    51.9

    14.8

    P. ramorum

    PrFAM probe

    6-FAM d(CAGATATTAAACAAATTATATATAAAATCAAACAA) BHQ-1c

    35

    56.2

    14.3

    Plant

    FMPl-2b

    GCGTGGACCTGGAATGACTA

    20

    57.2

    55

    Plant

    FMPl-3b

    AGGTTGTATTAAAGTTTCGATCG

    23

    53.5

    34.8

    Plant

    Plant CALOrange probe

    CAL Orange d(CTTTTATTATCACTTCCGGTACTGGCAGG) BHQ-1

    29

    64.5

    44.8

    P. pseudosyringae

    FMPps1c

    AGTTTCATTAGAAGATTATTTAC

    23

    52.1

    21.7

    P. pseudosyringae

    FMPps2c

    AAAATTGTTTGATTTTATTAAGTATC

    26

    52.0

    15.4

    P. pseudosyringae

    PpsCALOrange probe

    CAL Orange d(TTAATAAAAAAATTATGATATTTAAACTAATTGGT) BHQ-1

    35

    56.3

    11.4



     

    Cycling conditions

        50 C for 2 minutes
        95 C for 10 minutes
        60 cycles of 95 C for 15 sec and 55 C for 1 minute

    Comments on amplification
  • The limit of detection when using purified culture DNA is 1 fg
  • The amplification can be 3 way multiplexed (plant control, P. ramorum and P. pseudosyringae)
    • Reaction volume was 50 µl
    • an additional 75 μM dNTPs was added to the master mix
    • the plant probe was used at 400 nM final concentration
  • DNA extracted from an artificially inoculated rhododendron leaf gave a positive result when diluted down as far as 10-5 (extrapolated to represent 1.7 fg pathogen DNA)
    • Positive results were also obtained with 10-6 dilutions, but with Ct­ values of 55.3 and were outside of the linear portion of the regression line
  • The real time PCR master mix can have an effect on the specificity of the marker
    • ABI TaqMan master mix worked well, but switching to BioRad caused 5 species to give a false positive