Parameters for specific detection of Clavibacter michiganensis subsp. sepedonicus in potato stems and tubers by multiplexed PCR-ELISA

American Journal of Potato Research, Jul/Aug 2003 by Mills, Dallice, Russell, Brian W

Development of an Internal Control to Specifically Monitor Cms72 Primer Function and PCR Inhibition

In three separate reactions, sea anemone DNA (a generous gift from Virginia Weiss, Oregon State University) was used as the template to generate randomly amplified fragments by PCR from the forward and reverse primers that amplify Cms50, Cms72, and Cms85. To ensure that the primers would anneal to random fragments, less stringent conditions were initially used. The conditions were 50 cycles 30 s at 92 C, 30 s at 37 C, and 60s at 72 C. Fragments were separated by gel electrophoresis and those of approximately 100-250 bp in size were excised from the gel by a toothpick and transferred to water to dissolve the DNA. The fragments were amplified again using the standard PCR conditions for amplification of Cms50, Cms72, and Cms85 fragments. Specific fragments were gel-purified and cloned into the Xcml-derived vector (Borovkov and Rivkin 1997) and one designated SA72 was used in this study to specifically monitor Cms72 primer function with tuber-derived samples.

Immunodetection of Cms Strains

Pure cultures of bacteria and putative infected plant material were analyzed by an indirect fluorescent antibody staining (IFAS) procedure (De Boer and Wieczorek 1984) using a kit (Agdia, Inc., Elkart, IN, USA) that employs the 9A1 anti-Cms monoclonal antibody.

RESULTS

Development of Conditions for Multiplex PCR

The forward and reverse primer sets used for PCR amplification of Cms50, Cms72, and Cms85 sequences in this study produce single PCR products of 192 bp, 161 bp, and 205 bp, respectively, from Cms genomic DNA. The reaction conditions were established for multiplex PCR that included optimizing the annealing temperature for the three primer sets, establishing primer concentrations, and designing a new reverse primer for the amplification of Cms50 (see Materials and Methods) to obtain primers with similar annealing temperatures for near uniform amplification of all three fragments. Using these new PCR conditions, all three PCR products were synthesized in approximately equal amounts when Cms genomic DNA was not limiting (Figure 1). When the sample was diluted to 3 CFU per 10 [mu]L in the potato core fluid approximately equal amounts of the Cms50 and Cms72 products were observed in the gel, but the Cms85 product was barely visible (Figure 1). No additional PCR products were visualized indicating that members of different primer sets were not priming the amplification of DNA from other sites within the genome.

The specificity of this multiplex PCR system was then examined with representative strains of Cms, as well as closely related Clavibacter subspecies and other species of bacteria (Figure 2 and Table 1). The multiplex PCR conditions primed the amplification of the three fragments from all 45 Cms strains analyzed regardless of their colony morphology and origin, and the typical gel pattern for the three fragments is shown in Figure 2 lane 11. Non-specific amplicons were occasionally detected from DNA templates of the eight heterologous strains examined (Figure 2, lanes 3-10), and a fragment of approximately 148 bp was amplified from genomic DNA of C. m. subsp. insidiosum (Figure 2A, lane 3). However, probes made of Cms50, Cms72 and Cms85 did not hybridize to Southern blots with PCR products made from the 8 heterologous strains (data not shown). Fragments of 50 to 75 bp that were observed in all lanes, including the tube that had no template DNA (lane 2), were assumed to be primer dimers. To demonstrate that the DNA templates of the eight species were suitable for amplification, a reaction was carried out using forward and reverse primers that amplify a conserved 100 bp sequence from the 16S ribosomal subunit rDNA repeat. For each DNA sample, a robust PCR product of the appropriate size was obtained (data not shown).