Gotaq flexi

DNA extractions were carried out either following Randall et al.^{50 (link)} after samples were ground under liquid nitrogen, or using the Qiagen DNeasy plant kit (Qiagen, Manchester, UK). The complete second internal transcribed spacer of nuclear ribosomal DNA (ITS2) and partial 5.8S and 26S sequences were amplified using primer pair S2F and S3R^{35 (link)}. Where amplification with this primer pair failed, a second ITS2 primer pair were tried, ITS-p3 and ITS-p4^{38 (link)}. PCRs were carried out in 10 µL reaction volumes containing 2 µL DNA template, 1x PCR buffer, 2.0 mM MgCl₂, 0.2 µM of each primer (at 10 mM), 0.2 mM of each dNTP and 1 U Go Taq Flexi (Promega, Southampton, UK). For problematic samples, a multiplex PCR mix (Qiagen, Manchester, UK) was used, with primers and DNA at the same concentration and volume described above. Reaction conditions were an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 56 °C for 30 s and 72 °C for 1 min, and a final extension of 72 °C for 10 min. PCR products were sequenced in both directions by Eurofins Genomics (Wolverhampton, UK). Contigs were constructed and consensus sequences created in Sequencher version 5.4.6⁵¹ or MEGA6^{52 (link)} after manually editing sequences. Consensus sequences were aligned using automated ClustalW alignment in BioEdit⁵³ or ClustalX^{54 (link)}, for in silico analysis (see below).

Singleplex PCR condition was initially optimized according to GoTaq Flexi manual instruction (Promega, USA). Multiplex PCR was optimized to ensure appropriate amplification. Extension temperatures of 68°C and 72°C were tested in this experiment. Magnesium chloride concentration, ranging from 1.5–4.0 mM, was adjusted to give the most intensity for all PCR products. Annealing temperature, ranging from 55°C-61°C, was optimized using a gradient thermal cycler. PCR reaction was carried out in a total volume of 20μl per reaction. Multiplex PCR amplification was performed on Thermalcycler (Applied Biosystems, USA) under the following conditions; 94°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 55–61°C for 1 min, 68/72°C for 1 min, and final extension at 68°C for 10 min.

Bacterial suspensions of each strain were prepared at 1 × 10⁷ CFU ml^-1 and were boiled at 95°C for 10 min before PCR. Aliquots of boiled cells were kept at -20°C. Primers for amplification of 14 VNTR loci from X. citri pv. citri (Bui Thi Ngoc et al., 2009 (link)) were tested with five strains of pathovar viticola and strain 306 of pathovar citri. Reaction mix contained 1X GoTaq Flexi buffer (Promega), 1.5 or 3.0 mM MgCl₂, 62.5 μM each dNTP, 0.125 μM of each primer, 0.25 U of GoTaq Flexi DNA polymerase and 1 μl of bacterial cells suspension. Conditions for amplification were as follows: 95°C for 5 min followed by 32 cycles of 95°C for 30 s; 60, 64 or 68°C, depending on the primer set, for 30 s and 72°C for 30 s and a final extension at 72°C for 10 min. PCR products were separated on 2.5% agarose gels in 1X Tris acetate EDTA buffer (TAE) and visualized after ethidium-bromide staining. When poor amplification occurred, PCR was optimized by testing different MgCl₂ concentrations (1.5 and 3.0 mM) and annealing temperatures (60, 64, 68°C). VNTR loci were selected based on reproducibility (amplicons of same size produced in different PCR runs) and polymorphism detection among pathovar viticola strains, verified by gel electrophoresis.

To perform the first PCR, 5 μl of genomic DNA was used as a template in a final volume of 30 μl containing 0.33 mM dNTPs (Eurobio, France), 1X PCR buffer, 2.5 mM MgCl₂, 1 μM of each primer, 0.2 μl (1 U) of GoTaq Flexi (Promega) and deionized water. PCR steps included 95 °C for 5 min - 40 cycles of 30 s at 95 °C, 30 s at the selected annealing temperature (Table 1), 30 s at 72 °C - and a final elongation step of 5 min at 72 °C. Four nested PCRs were run (one for each cluster II, III and IV, and one with cluster I-IV primers), using 10 μl of the first amplicon (diluted 1:100) as template. Presence and sizes of the amplicons were controlled under UV on an ethidium bromide-stained 1.5% agarose gel.
To control the specificity of the cluster-specific nested PCR, amplicons were purified using ExoSAP-IT (Ozyme, Saint Quentin en Yvelines, France) and sent for Sanger conventional sequencing in both directions (GATC, Germany). Conserved internal primers (Ana-Int-F2/Ana-Int-R3), framing a highly discriminatory region between clusters (position from nt 854 to 1182), were specifically designed to allow cluster assignment.