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<p><i>Candidatus</i> Midichloria mitochondrii (<i>Ca.</i> M. mitochondrii), an endosymbiont intracellular bacterium living in the mitochondria of several tick species, especially ixodid species, has been proposed as a potential marker for monitoring tick-bite exposure. Therefore, the present study aimed to develop two different diagnostic methods, TaqMan-based real-time PCR (rt-PCR) and Droplet Digital PCR (dd-PCR), targeting the 16&#xa0;S rRNA gene and <i>gyrB</i> gene for the detection of <i>Ca</i>. M. mitochondrii in different wildlife species from several areas of southern Italy. Both techniques were validated using 10-fold serial dilutions of a sequenced positive control up to reach 10<sup>−6</sup> final dilution. Among wildlife field samples, both the techniques identified <i>Ca</i>. M. mitochondrii DNA, although dd-PCR showed higher sensitivity, being able to detect the target DNA in a higher dilution and in several spleen samples scored negative by rt-PCR. Noteworthy, these molecular methods revealed for the first time the presence of <i>Ca</i>. M. mitochondrii DNA in red foxes (<i>Vulpes vulpes</i>), Eurasian badgers (<i>Meles meles</i>), otters (<i>Lutra lutra</i>), porcupines (<i>Hystrix cristata</i>), European hares (<i>Lepus europaeus</i>), and alpacas (<i>Vicugna pacos</i>), suggesting a tick-bite exposure of these animals in the study area.</p>

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Development of TaqMan real-time PCR and droplet digital PCR protocols for the detection of Candidatus Midichloria mitochondrii and evaluation of exposure among wildlife

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年7月1日

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  • Alessia Pucciarelli,
  • Lorena Cardillo,
  • Maurizio Viscardi,
  • Gerardo Picazio,
  • Nicola D’Alessio,
  • Giovanni Sgroi,
  • Antonio Rinaldi,
  • Vincenzo Veneziano,
  • Giovanna Fusco,
  • Claudio de Martinis

摘要

Candidatus Midichloria mitochondrii (Ca. M. mitochondrii), an endosymbiont intracellular bacterium living in the mitochondria of several tick species, especially ixodid species, has been proposed as a potential marker for monitoring tick-bite exposure. Therefore, the present study aimed to develop two different diagnostic methods, TaqMan-based real-time PCR (rt-PCR) and Droplet Digital PCR (dd-PCR), targeting the 16 S rRNA gene and gyrB gene for the detection of Ca. M. mitochondrii in different wildlife species from several areas of southern Italy. Both techniques were validated using 10-fold serial dilutions of a sequenced positive control up to reach 10−6 final dilution. Among wildlife field samples, both the techniques identified Ca. M. mitochondrii DNA, although dd-PCR showed higher sensitivity, being able to detect the target DNA in a higher dilution and in several spleen samples scored negative by rt-PCR. Noteworthy, these molecular methods revealed for the first time the presence of Ca. M. mitochondrii DNA in red foxes (Vulpes vulpes), Eurasian badgers (Meles meles), otters (Lutra lutra), porcupines (Hystrix cristata), European hares (Lepus europaeus), and alpacas (Vicugna pacos), suggesting a tick-bite exposure of these animals in the study area.