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<p>Environmental DNA (eDNA) analysis is an important tool for assessing the population status and distribution of the threatened species, whose habitats have been declining or becoming fragmented. In this study, we developed a visual loop-mediated isothermal amplification (LAMP) method for the on-site detection of the eDNA of the giant water bug <i>Kirkaldyia deyrolli</i> in freshwater habitats. Species-specific primers were designed based on the mitochondrial DNA sequence of <i>K. deyrolli</i>, specifically targeting the cytochrome C oxidation enzyme subunit II (<i>COII</i>) gene. The effectiveness of these primers was confirmed using turbidity and visual LAMP tests, which demonstrated their high specificity and sensitivity. Field tests were conducted using commercial portable equipment, allowing eDNA extraction and visual LAMP reactions to be performed directly at various pond sites. The results revealed rapid and accurate detection within 30&#xa0;min, demonstrating the potential of this method for the quick assessment of the presence of <i>K. deyrolli</i> in natural habitats. This technique offers a cost-effective and rapid alternative to real-time PCR, thereby directly enabling effective conservation efforts in the field.</p>

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Development of a visual loop-mediated isothermal amplification method to detect environmental DNA of the giant water bug Kirkaldyia deyrolli in the field

  • 期刊论文

  • 发表日期：

    2025年4月28日

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  • Jun-ichi Takahashi,
  • Shin-ya Ohba

摘要

Environmental DNA (eDNA) analysis is an important tool for assessing the population status and distribution of the threatened species, whose habitats have been declining or becoming fragmented. In this study, we developed a visual loop-mediated isothermal amplification (LAMP) method for the on-site detection of the eDNA of the giant water bug Kirkaldyia deyrolli in freshwater habitats. Species-specific primers were designed based on the mitochondrial DNA sequence of K. deyrolli, specifically targeting the cytochrome C oxidation enzyme subunit II (COII) gene. The effectiveness of these primers was confirmed using turbidity and visual LAMP tests, which demonstrated their high specificity and sensitivity. Field tests were conducted using commercial portable equipment, allowing eDNA extraction and visual LAMP reactions to be performed directly at various pond sites. The results revealed rapid and accurate detection within 30 min, demonstrating the potential of this method for the quick assessment of the presence of K. deyrolli in natural habitats. This technique offers a cost-effective and rapid alternative to real-time PCR, thereby directly enabling effective conservation efforts in the field.