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<p>Morphological similarity with related whitefly species and limited access to rapid diagnostics hinder timely detection and management of <i>Aleurotrachelus trachoides </i><i>(Back)</i>. To address this gap, a geographically validated, rapid, and potentially field-deployable colorimetric recombinase polymerase amplification (RPA) was developed for decentralized detection of <i>A. trachoides</i>. Specimens collected from seven agroclimatic zones in India were confirmed through morphological identification and mitochondrial cytochrome oxidase I (<i>mtCOI</i>) sequencing, with phylogenetic analysis confirming <i>A. trachoides</i> as a distinct monophyletic clade. A species-specific 150&#xa0;bp <i>mtCOI</i> region was identified through in silico and validated by cloning and Sanger sequencing to design species-specific primers for PCR and RPA assays. The RPA assay detected as little as 10 pg/µL, outperforming conventional PCR (1 ng/µL), and generated results within 20&#xa0;min at 38&#xa0;°C without a thermal cycler. No cross-reactivity was observed with the nine nontarget whitefly species. Colorimetric detection using SYBR Safe and hydroxynaphthol blue (HNB) dyes enabled direct visual detection. Large-scale geographical validation (160 target and 90 nontarget samples) showed 98.81% sensitivity and 99.26% specificity, and detection from crude DNA extracts via HNB dye confirmed its field applicability. This study presents a species-specific colorimetric RPA assay with potential as a rapid and reliable molecular tool to support early surveillance, quarantine inspection, and sustainable management of <i>A. trachoides</i> in affected agroecosystems.</p>

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A rapid, potentially field-deployable colorimetric recombinase polymerase amplification assay for molecular surveillance of the invasive whitefly, Aleurotrachelus trachoides (Back), vector of begomovirus

  • 期刊论文

  • 发表日期：

    2026年7月15日

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  • Darshana Brahma,
  • Manjunatha Channappa,
  • Selvaraj Krishnan,
  • Suman Talya Chandrashekara,
  • Nalimala Veeramanikanta Reddy,
  • Selva Babu Selvamani,
  • Aditya Kukreti,
  • Doddachowdappa Sagar,
  • Venkatesan Thiruvengadam

摘要

Morphological similarity with related whitefly species and limited access to rapid diagnostics hinder timely detection and management of Aleurotrachelus trachoides (Back). To address this gap, a geographically validated, rapid, and potentially field-deployable colorimetric recombinase polymerase amplification (RPA) was developed for decentralized detection of A. trachoides. Specimens collected from seven agroclimatic zones in India were confirmed through morphological identification and mitochondrial cytochrome oxidase I (mtCOI) sequencing, with phylogenetic analysis confirming A. trachoides as a distinct monophyletic clade. A species-specific 150 bp mtCOI region was identified through in silico and validated by cloning and Sanger sequencing to design species-specific primers for PCR and RPA assays. The RPA assay detected as little as 10 pg/µL, outperforming conventional PCR (1 ng/µL), and generated results within 20 min at 38 °C without a thermal cycler. No cross-reactivity was observed with the nine nontarget whitefly species. Colorimetric detection using SYBR Safe and hydroxynaphthol blue (HNB) dyes enabled direct visual detection. Large-scale geographical validation (160 target and 90 nontarget samples) showed 98.81% sensitivity and 99.26% specificity, and detection from crude DNA extracts via HNB dye confirmed its field applicability. This study presents a species-specific colorimetric RPA assay with potential as a rapid and reliable molecular tool to support early surveillance, quarantine inspection, and sustainable management of A. trachoides in affected agroecosystems.