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

The detection limits of TaqMan real-time qPCR single reactions for both primer and probe sets were 100 fg using purified bacterial genomic DNA (Fig 4A and B) . However, low reaction efficiencies of 82% and 91% were observed for C. michiganensis species and C. m. subsp. nebraskensis targets, respectively, suggesting inhibition or sub-optimal conditions for amplification ( Fig 4A-B Table 3 ). To optimize the amplification conditions, customized 5'ATrich sequences were added to each primer; the addition of AT-rich sequences at the 5' position remarkably enhanced the reaction efficiency to 99-104% (Fig 4C-H) . The most pronounced positive effect was observed with the C. michiganensis specific primer and probe set; the sensitivity was enhanced 10-fold and detected the target sequence in 10 fg of genomic DNA ( Fig 4A and B ; Table 3 ). No adverse effect was observed when 1 µl host plant DNA was added in each sensitivity reaction (spiked test, Fig 4E) . Similarly, no effect was observed with the C. m. subsp. nebraskensis-specific primers and probe designed using the MFS gene region. The ATrich sequence effect was also evaluated with endpoint PCR, the C. michiganensis assay sensitivity was enhanced from 100 fg to 10 fg (S3 Fig). Additionally, analysis of the amplification curves indicated that the use of 5'AT-rich sequences did not affect the sigmoidal shape with the concentration of target in sensitivity assays. In contrast, use of non-tailed primers negatively impacted the amplifications as well as the efficiencies (Fig 4) . The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 a Flap primers with AT 5'-rich non-complementary nucleotides; b spiked test was performed only in multiplex reactions; each reaction was performed three replicates and the average C T values are provided with their standard deviation; d reaction efficiency were calculated using the Rotor-Gene Q Series Software.