Author: Adriana Larrea-Sarmiento; Anne M. Alvarez; James P. Stack; Mohammad Arif
Title: 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 Document date: 2019_3_11
ID: iniz1rjk_19
Snippet: Highly conserved regions within the major facilitator superfamily (MFS) transporter gene for C. michiganensis subsp. nebraskensis, ABC transporter ATP-binding protein CDS/ABC-transporter permease genes for C. michiganensis subspecies were used to design two sets of primers for both conventional end-point PCR and TaqMan real-time qPCR (Table 2; S2 Fig) . The primers and probes evaluated in silico showed high specificity for C. m. subsp. nebraskens.....
Document: Highly conserved regions within the major facilitator superfamily (MFS) transporter gene for C. michiganensis subsp. nebraskensis, ABC transporter ATP-binding protein CDS/ABC-transporter permease genes for C. michiganensis subspecies were used to design two sets of primers for both conventional end-point PCR and TaqMan real-time qPCR (Table 2; S2 Fig) . The primers and probes evaluated in silico showed high specificity for C. m. subsp. nebraskensis (Cmn) and C. michiganensis subspecies in general ( Table 2) . No nucleic acid similarity to orthologous genes or conserved domains were found for the major facilitator superfamily (MFS) supporting the unique presence of MFS gene only in C. michiganensis subsp. nebraskensis. On the other hand, highly conserved domains of sugar ABC transporter permease and ABC transporter ATP-binding genes within C. michiganensis subspecies were selected to design specific primers and probe, resulting in a broad but specific detection of all nine subspecies within C. michiganensis. Primer and probe sets were highly specific as revealed by NCBI BLASTn analysis.
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