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

. CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 Multiplex TaqMan qPCR assays for detection of C. michiganensis species and C. m. subsp. nebraskensis are robust, accurate, sensitive and reliable. The developed assays can be used to accelerate phytosanitary diagnostics and target pathogen detection. Sensitive detection can help in early detection of latently-infected plant materials and support further studies in pathogen dissemination during interstate or international commerce. The addition of 5' AT-rich sequences lead to the development of thermodynamically competent primers for sensitive detection. This concept also will enable development of improved protocols for different pathogens that can work under the same PCR conditions. Development of mix and match diagnostic protocolswhere the primers can be multiplexed based on individual needs will be possible since each primer's thermodynamics, GC content, annealing temperature and primer length can be customized to fit existing PCR conditions. 1 6 Supporting information captions S1 Fig. dnaA phylogeny within the nine C. michiganensis subspecies inferred using the maximum likelihood (ML) method in MEGA 7.0.25. The evolutionary distances were computed using a General Time Reversible model with gamma distribution (GTR + G). Forty-three partial sequences based on dnaA gene of Clavibacter strains were rooted with 2 closest taxa Leifsonia xyli and Rathayibacter triciti, served as an outgroup. A 1,000 replicates were performed to calculate the bootstrap value which is shown over the branches; only bootstrap values greater than 70% are presented. Fig. Target selection. A. Primers and probe (Cmn11-F/R/P) targeting the MFS transporter gene specific for C. m. subsp. nebraskensis. B. Primers and probe (CM-F/R/P) targeting the sugar ABC transporter permease and ABC transporter ATP-binding genes for specific detection of C. michiganensis species. In both cases, the probe is located in the inner part of the amplicon, between the sense and anti-sense primers. 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 the C. michiganensis and C. m. subsp. nebraskensis with/without 5' AT-rich sequences, respectively; (C). Single reactions targeting MSF gene of C. m. subsp. nebraskensis; (D) Single reactions targeting the sugar ABC transporter permease and ABC transporter ATP-binding genes for specific detection of C. michiganensis subspecies; (E) 1 µl host corn DNA was added in each reaction of ten-fold serially diluted sensitivity assay for simultaneous detection of C. m. subsp. nebraskensis and other C. michiganensis subspecies. All the experiments were conducted the same day using genomic DNA from C. m. subsp. nebraskensis. A molecular-weight size marker of 100 bp from BioLabs was used as a standard reference to determine the product size. All PCR products were electrophoresed in a 3% agarose gel.