PLoS ONE (2017) 12 (9 - e0185427)

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Takao Ito and Koichi Suzaki (2017)
Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides
PLoS ONE 12 (9 - e0185427)
Abstract: Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.
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Research topic(s) for pests/diseases/weeds:

Pest and/or beneficial records:

Beneficial Pest/Disease/Weed Crop/Product Country Quarant.
Xylella fastidiosa
16SrI phytoplasma group
16SrII phytoplasma group
16SrX phytoplasma group
16SrXII phytoplasma group
16SrXII phytoplasma group
16SrVII phytoplasma group
16SrV phytoplasma group
16SrVI phytoplasma group
16SrIV phytoplasma group
16SrIII phytoplasma group
16SrIX phytoplasma group
16SrXI phytoplasma group
16SrXV phytoplasma group
16SrXXXI phytoplasma group
16SrXIX phytoplasma group