Contributed by Guillaume Bilodeau

G. J. Bilodeau, C. A. Lévesque, A. W. A. M. de Cock, C. Duchaine, S. Briére, P. Uribe, F. N. Martin, and R. C. Hamelin (2007) Phytopathology  97:632-642  (pdf of reprint)

MATERIALS AND  METHODS
Isolates.  
All isolates of Phytophthora used in this study are listed in Table 1. The P. ramorum collection comprised 38 isolates From European and North American origin isolated from different hosts. The Phytophthora collection comprised isolates of 65 different species and varieties, representing most of the recognized Phytophthora species currently available in pure culture. Mycelium was cultivated and DNA was extracted following the procedures described in De Cock et al. (1992) or Möller et al. (1992).

DNA sequencing.
The primers listed in Table 2 were used to amplify three gene regions of the nuclear DNA of P. ramorum and related Phytophthora species by PCR (Table 1). Primers for the β-tubulin and the elicitin genes were designed based on sequences of these genes obtained from P. parasitica (GenBank accession no. S67432) for elicitin, and P. cinnamomi (GenBank accession no. U22050) for β-tubulin. ITS primers used were from Bakkeren et al. (2000) and Mazzola et al. (2002), and the sequence AY038050 from GenBank for P. ramorum was used for alignment. These nuclear regions were selected because of the high level of sequence divergence among species that were observed in preliminary results and large number of sequence entries available in public databases. Genomic DNA from P. ramorum (CBS 101553, DAOM 230728) and other species was amplified using these genus specific primers. Most reactions yielded a single band. However, multiple bands were amplified for the elicitin gene of P. lateralis ATCC 201856, P. cactorum BR675, P. citricola BR 681, P. cinnamomi BR 680, and P. infestans CBS 366.51. A band of approximately 280 bp was cut and extracted from agarose gels with the QIAEX II agarose gel extraction kit (Qiagen, Valencia, CA). PCR products were then re-amplified. PCR products were purified with the QIAquick PCR purification kit (Qiagen) using the microfuge method, quantified, and sequenced using the same primers as for PCR. Sequencing reactions were performed with a Big Dye Terminator Sequencing kit on an ABI 310 automated sequencer (PE Applied Biosystems, Foster City, CA). Both strands were sequenced with the primers listed in Table 2. Sequences were aligned using Sequencher version 4.0.5 (Gene Codes Corporation, Ann Arbor, MI) and MegAlign version 5.08 (DNASTAR Inc., Madison, WI) using Clustal W. The sequences were deposited in GenBank and the accession numbers are listed in Table 3.

Design of primers.
The alignments of sequences listed in Table 3 were used to design PCR primers specific to P. ramorum (Table 2) using the software Primer Premier 5.00 (Premier Biosoft International, Palo Alto, CA). The selection criteria were the following: Tm (melting temperature) 55 to 65°C, primer length 18 to 22 bp, and absence of secondary structure whenever possible. Specific primers were designed so that the nucleotides unique to the target were at the 3′ end position of the primer. In primers Phy_ram_482U_LNA F and Prameli259L R (Table 2), positioning the discriminating site at the 3′ end resulted in secondary structures and the primers were moved toward the 5′ end. The primer pairs were designed such that PCR products were shorter than 200 bp, an important parameter for RT-PCR. In cases where only single nucleotide differences were present and unmodified primers did not allow specific amplifications, primers were synthesized with a lock nucleic acid (LNA) (Braasch and Corey, 2001) (Proligo LLC, Boulder, CO) to increase specificity. The ITS primers were different from other published primers (Garbelloto et al. 2003,Hayden et al., 2003, Hayden et al. 2006) and instead targeted positions 622 to 755 with mismatches at the 3′ site and the LNA modification.

Design of molecular beacon and TaqMan probes.
Molecular beacons were designed using Mfold version 3.1 (DNA mfold server: 1996 to 2003, Michael Zuker, Rensselaer Polytechnic Institute) and Beacon designer 3 software (Premier Biosoft International, Palo Alto, CA) to calculate the Tm and the structure of the molecule. The molecular beacon was labeled with fluorescein (6-FAM^tm) at the 5′ end and with the quencher Dabcyl at the 3′ end (Bonnet et al., 1999, Tyagi and Krammer, 1996). TaqMan probes (Heid et al., 1996) were designed with Primer Premier 5.00. We used the following parameters for the design: Tm 10°C higher than the primers, 15 to 30 bp in length and the total number of G's or C's in the last five nucleotides at the 3′ end of the primer not exceeding two. The mismatching nucleotide was positioned as close as possible to the middle of the probe rather than at the ends while avoiding positions with secondary structures. The TaqMan probes were labeled with fluorescein (6-FAM^tm) at the 5′ end and with the quencher Black Hole Quencher^tm-1 (BHQ^tm-1) at the 3′ end (Integrated DNA Technologies Inc., Coralville, IA).

PCR amplification.
Real-time PCR was performed with a DNA Engine Opticon 2 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). Fluorescent molecules (SYBR Green, dual-labeled probe [TaqMan or molecular beacons]) were included in the PCR master mix (QuantiTect, Qiagen). All reactions were performed in 25 µl volumes. The DNA concentration used in the reaction was determined with a Nanodrop spectrophotometer ND-1000 (Nanodrop Technologies, Wilmington, DE) and ranged from 0.05 to 60 ng/µl for P. ramorum and from 5 and 110 ng/µl for other    Phytophthora sp. Molecular beacon, TaqMan and SYBR Green real-time PCR assays were used in a preliminary experiment with the three gene regions using a subset of isolates. The real-time PCR on P. ramorum tested on the three gene regions were made in triplicates, and the mean and standard error were calculated for each. Subsequently, TaqMan assays were used for further studies using 48 plant tissue samples from seven hosts. P. ramorum CBS 101553 was used as positive control, and P. lateralis CBS 168.42 and no template DNA were used as negative controls. The analysis software Opticon Monitor version 2.01.10 (Bio-Rad Laboratories) was used to analyze the data (cycle range set at 1 to 21). Data were exported as cycle threshold (Ct) values and analyzed for comparisons among samples in Excel spreadsheets. Statistical analyses were performed with Excel (Microsoft Excel version 9.0.3821 SR-1, Redmond, WA). The specific reaction conditions for each of the three detection technologies tested were set up as follows.

SYBR Green.
The PCR assay contained 0.4 µM of each LNA primer or regular primer (Table 2), 1× QuantiTect SYBR Green PCR Kit (Qiagen), and template DNA. PCR cycling conditions were set at 95°C for 15 min, 40 cycles at 94°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Fluorescence was read during the extension at 72°C.

TaqMan probe.
The PCR assay contained 0.4 µM of each LNA primer or regular primer depending on the region used, 0.2 µM TaqMan (dual-labeled probe) (Table 2), 1× QuantiTect Probe PCR Master Mix (Qiagen), and template DNA. PCR cycling conditions were set at 95°C for 15 min, 36 cycles at 94°C for 15 s, and 65°C for 60 s (68°C for 60 s for elicitin). Cycle number was extended to 40 to 45 for β-tubulin and ITS on environmental samples to increase sensitivity. Fluorescence was read during the extension at 65 to 68°C.

Molecular beacon.
The PCR assay contained 0.4 µM of each LNA primer for β-tubulin (Table 2), 0.2 µM molecular beacon probe (Table 2), 1µ QuantiTect Probe PCR Master Mix, and template DNA. PCR cycling conditions were set at 95°C for 15 min, 36 cycles at 94°C for 15 s, 65°C for 30 s, and 72°C for 30 s. Fluorescence was read after the annealing at 65°C. This allows the molecular beacon to open and anneal with the target sequence.

Alignments of β-tubulin used to design primers

Alignments of elicitin used to design primers

References

Guus Bakkeren, James W. Kronstad, C. Andre Levesque 2000 Comparison of AFLP Fingerprints and ITS Sequences as Phylogenetic Markers in Ustilaginomycetes Mycologia 92:510-521