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<p>The South American tomato pinworm, <i>Tuta absoluta</i> (Meyrick) (Lepidoptera: Gelechiidae) is an economically important pest worldwide. <i>Tuta absoluta</i> is invasive in India where it causes severe damage to tomato and other solanaceous crops. On-site rapid detection of <i>T. absoluta</i> is essential to adopt appropriate management strategies. Taxonomy-based identification is a promising tool, but it is time-consuming and needs adequate taxonomic expertise. Here, we developed an on-site and field deployable recombinase polymerase amplification (RPA) assay for the detection of <i>T. absoluta.</i> RPA primers were designed to target the cytochrome c oxidase subunit 1 (COI) gene of <i>T. absoluta</i>. RPA assay was performed using a simple crude extract of this insect in 0.02&#xa0;M Ethylenediaminetetraacetic acid (EDTA), followed by incubation at 37 ºC for 25&#xa0;min. The assay was further simplified by adding colorimetric dye (HNB), which eliminates the gel-electrophoresis step. The presence of <i>T. absoluta</i> was detected by visual color change from dark blue to sky blue. The entire assay from crude extraction to calorimetric reading was rapid, user-friendly, and lasted 45&#xa0;min. The assay was validated by setting a pheromone trap in the field, followed by crude extraction of the trapped moths in 0.02&#xa0;M EDTA and their detection with RPA assay. This on-site, rapid detection method for <i>T. absoluta</i> can be utilized by non-expert personnel for quarantine and practical management purposes.</p>

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Development of colorimetric recombinase polymerase amplification (RPA) assay for rapid detection of Tuta absoluta

  • 期刊论文

  • 发表日期：

    2025年4月25日

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  • Arindam Kumar,
  • Damini Diksha,
  • Susheel Kumar Sharma,
  • Suman Barman,
  • Mukesh Kumar Dhillon,
  • Pathour R. Shashank

摘要

The South American tomato pinworm, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is an economically important pest worldwide. Tuta absoluta is invasive in India where it causes severe damage to tomato and other solanaceous crops. On-site rapid detection of T. absoluta is essential to adopt appropriate management strategies. Taxonomy-based identification is a promising tool, but it is time-consuming and needs adequate taxonomic expertise. Here, we developed an on-site and field deployable recombinase polymerase amplification (RPA) assay for the detection of T. absoluta. RPA primers were designed to target the cytochrome c oxidase subunit 1 (COI) gene of T. absoluta. RPA assay was performed using a simple crude extract of this insect in 0.02 M Ethylenediaminetetraacetic acid (EDTA), followed by incubation at 37 ºC for 25 min. The assay was further simplified by adding colorimetric dye (HNB), which eliminates the gel-electrophoresis step. The presence of T. absoluta was detected by visual color change from dark blue to sky blue. The entire assay from crude extraction to calorimetric reading was rapid, user-friendly, and lasted 45 min. The assay was validated by setting a pheromone trap in the field, followed by crude extraction of the trapped moths in 0.02 M EDTA and their detection with RPA assay. This on-site, rapid detection method for T. absoluta can be utilized by non-expert personnel for quarantine and practical management purposes.