Synergetic effect of non-complementary 5’ AT-rich sequences on the development of a multiplex TaqMan real-time PCR for specific and robust detection of Clavibacter michiganensis and C. michiganensis subsp. nebraskensis

Adriana Larrea-Sarmiento; Anne M. Alvarez; James P. Stack; Mohammad Arif

Potential gene targets for broad range and specific detection of C. michiganensis subspecies and C. m. subsp. nebraskensis, in particular, were identified by comparative genomic analysis using Mauve [25] and Geneious. To calculate the core genome in the C. michiganensis subspecies and the variable genomes for each subspecies, the annotated genomes of gram-positive bacteria, C. m. subsp. nebraskensis NCPPC 2581 (NC_020891), C. m. subsp. michiganensis NCPPB 382 (NC_009480), C. m. subsp. sepedonicus ATCC 33113 (NC_010407), C. m. subsp. insidiosus strain R1-1 (NZ_CP011043), and C. m. subsp. capsici strain PF008 (NZ_CP012573) were used. Moreover, Rathayibacter tritici (NZ_CP015515), R. toxicus (NZ_CP013292), Rhodococcus fascians (CP015235), Corynebacterium efficiens (CP004369), and Curtobacterium sp. (CP009755) genomes were included to confirm primer specificity developed in this study. Manually filtered orthologous genes present in the core genome of the five subspecies were identified as potential targets for detection of all known nine C. michiganensis subspecies. Putative and characterized genes in the accessory genome of C. m. subsp. nebraskensis, no homology with genes from other C. michiganensis subspecies, were selected as possible targets to detect C. m. subsp. nebraskensis. Homology and specificity of the C. michiganensis-specific (ABC transporter ATP-binding protein CDS/ABC-transporter permease CDS) and C. m. subsp. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/566281 doi: bioRxiv preprint nebraskensis-specific (Major facilitator superfamily (MFS) transporter CDS) candidate genes were validated in-silico using NCBI GenBank BLASTn analysis tool. The probe CM-P and Cmn11-P were labeled with Cy5 and FAM reporter dyes, respectively. All primers and probes were designed using Primer3; thermodynamic parameters, internal structures, and self-dimer formation were evaluated in silico Primer3 and mFold web server (Table 2) . Primers and probes were synthesized by Integrated DNA technologies Inc. (Coralville, IA). To evaluate the potential impact of 5' AT-rich flap sequences, additional primer sets were synthesized after incorporating a customized 5' AT-rich flap as described by Arif and Ochoa-Corona [17] (Table 2 ; Fig 2) . The customized flaps were designed to finely tune the tm, primer length, and GC content to optimize the reaction efficiency. Eleven and ten nucleotide long flaps were customized and added to the primer sets for C. michiganensis and C. m. subsp. nebraskensis, respectively. No 5' AT-rich flap sequences were added to the probes. μ l of nuclease-free water. Thermal cycling parameters were the same as for the individual single reactions, but the annealing time was increased to 60 secs. PCR assays were conducted in a T100™ BioRad thermal cycler (Bio-Rad, Hercules, CA). Non-template controls were included in each PCR amplification -1 μ l of nuclease-free water was used instead of genomic DNA. A volume of 13 μ l of amplified PCR product was used for electrophoresis in a 3% agarose gel; 1X Tris-acetate-EDTA (TAE) buffer at 80 V for 90 min was used for electrophoresis. Amplicon sizes were estimated using 100 bp DNA ladder (New England Biolabs, Ipswich, MA). Genomic DNA of C. m. subsp. nebraskensis strain A6206 was used to perform the PCR sensitivity assays with primer sets CM-F/R and Cmn11-F/R. The copyright holder for this preprint (which was not peer-revie