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<p>Phytoplasmas are phloem-restricted, insect-transmitted plant pathogens responsible for destructive diseases in numerous crops. Their obligate parasitism, inability to be cultured in vitro, and typically low titers in infected tissues create a critical need for highly sensitive and reliable diagnostic tools. Here, we developed and validated a droplet digital PCR (ddPCR) assay targeting a conserved region of the 16&#xa0;S rRNA gene for broad-range detection and absolute quantification of phytoplasmas. Using the UniRNA primer/probe set, the assay’s specificity was confirmed through in silico analyses and multiple sequence alignments against various phytoplasma ribosomal groups and non-target pathogens. Linearity was observed across a broad dynamic range, detecting 55-1576 copies µl<sup>−</sup>¹ from serial dilutions of naturally infected tomato DNA. Probit regression analysis demonstrated that ddPCR outperformed qPCR in sensitivity- exhibiting a 5.82-fold lower detection limit (LOD95 of 38.41 copies/reaction)- and provided more robust quantification for low-titer samples. Field-collected symptomatic tomato plants were identified as ‘<i>Candidatus</i> Phytoplasma trifolii’ through nested PCR, sequencing, and virtual RFLP analyses. The developed ddPCR system also successfully detected multiple reference phytoplasma groups, validating its broad applicability. This broad range ddPCR assay enhances early disease detection, supports accurate surveillance, and offers a robust diagnostic tool for pathogen monitoring and phytosanitary programs.</p>

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A 16 S rRNA- targeted droplet digital PCR (ddPCR) assay for sensitive detection and absolute quantification of phytoplasmas

  • 期刊论文

  • 发表日期：

    2026年5月23日

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  • Yigit Sabri Unlu,
  • Isil Tulum

摘要

Phytoplasmas are phloem-restricted, insect-transmitted plant pathogens responsible for destructive diseases in numerous crops. Their obligate parasitism, inability to be cultured in vitro, and typically low titers in infected tissues create a critical need for highly sensitive and reliable diagnostic tools. Here, we developed and validated a droplet digital PCR (ddPCR) assay targeting a conserved region of the 16 S rRNA gene for broad-range detection and absolute quantification of phytoplasmas. Using the UniRNA primer/probe set, the assay’s specificity was confirmed through in silico analyses and multiple sequence alignments against various phytoplasma ribosomal groups and non-target pathogens. Linearity was observed across a broad dynamic range, detecting 55-1576 copies µl−¹ from serial dilutions of naturally infected tomato DNA. Probit regression analysis demonstrated that ddPCR outperformed qPCR in sensitivity- exhibiting a 5.82-fold lower detection limit (LOD95 of 38.41 copies/reaction)- and provided more robust quantification for low-titer samples. Field-collected symptomatic tomato plants were identified as ‘Candidatus Phytoplasma trifolii’ through nested PCR, sequencing, and virtual RFLP analyses. The developed ddPCR system also successfully detected multiple reference phytoplasma groups, validating its broad applicability. This broad range ddPCR assay enhances early disease detection, supports accurate surveillance, and offers a robust diagnostic tool for pathogen monitoring and phytosanitary programs.