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

A new primer set P16s-F1 (5'-AGACTCCTACGGGAGGCAGCA-3') and P16s-R1 (5'-TTGACGTCATCCCCACCTTCC-3') targeting the 16S ribosomal RNA region (16S rRNA) of plant bacteria was designed and used to identify eleven isolates from corn leaves that showed characteristic symptoms of Goss's disease; the strains were provided by DuPont Pioneer Plant Disease Diagnostic Laboratory during the 2015 growing season ("DP" number; S1 Table) . Also, a new set of primers CM-dnaA-F1 (5'-ACGAAGTACGGCTTCGACAC-3') and CM-dnaA-R1 (5'-GCGGTGTGGTTGATGATGTC-3') was designed to amplify a partial sequence of the chromosomal replication initiator gene, dnaA, of C. michiganensis. PCR assays for 16S rRNA and dnaA were performed with the following conditions: initial denaturation at 94°C for 5 min, 35 cycles of denaturation at 94°C for 20 sec, annealing at 58°C for 30 sec, extension at 72°C for 1 min, and a final extension at 72°C for 3 min. Five μ l of PCR product was cleaned enzymatically using 2 μ L of ExoSAP-IT™ following the manufacturer's protocol (Affymetrix Inc, Santa Clara, CA). Sanger sequencing of both sense and anti-sense strands was performed at GENEWIZ facility (Genewiz, La Jolla, CA). Obtained sense and anti-sense sequences were aligned and manually edited for accuracy using Geneious version 10.1.3. Consensus sequences were compared against the NCBI GenBank nucleotide and genome databases using BLASTn tool [20] .