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<p>Okra (<i>Abelmoschus esculentus</i> L.) is an important vegetable crop valued for its nutritional and medicinal properties. Enation leaf curl disease, caused by okra enation leaf curl virus (OELCuV; <i>Begomovirus abelmoschusenation</i>), severely affects okra production in India, leading to leaf curling, petiole bending, stunted growth and yield losses. Rapid, sensitive and field-deployable diagnostics are critical for timely management of virus disease. Here, we developed and validated a colorimetric recombinase polymerase amplification (RPA) assay for specific detection of OELCuV from crude okra leaf extracts. Fifteen symptomatic samples from ICAR-IARI, New Delhi, were initially detected for OELCuV by PCR using universal begomovirus and enation leaf curl virus-specific primers, while confirmatory identification was achieved through rolling circle amplification (RCA), cloning, sequencing, and sequence analysis. RPA is a rapid, highly sensitive and user-friendly isothermal technique that detects low RNA or DNA targets at a constant temperature, even in crude samples, offering advantages such as minimal equipment requirement, rapid turnaround time, multiplexing capability, and suitability as a portable and cost-effective platform for on-site virus detection and adaptation to other plant viruses. Two primer sets targeting the coat protein (AV1) and pre-coat protein (AV2) were designed, with AV1 primers showing high specificity and no cross-reactivity with other okra virus. Nine extraction buffers were evaluated, with phosphate buffer providing optimal templates. The RPA assay was optimized at 37°C for 30&#xa0;min, detecting OELCuV up to 10<sup>–9</sup> dilution and demonstrating higher analytical sensitivity than conventional PCR. The validation using of 90 okra field samples collected from six states of India demonstrated comparable detection efficiency of the developed RPA assay and PCR, with consistent results across both methods. Inclusion of hydroxynaphthol blue (HNB) enabled visual endpoint detection, eliminating agarose gel-based analysis or other specialized equipment. Sequence analysis of the RCA-amplified complete DNA-A genome segment revealed 91.5%–99.5% nucleotide identity with global OELCuV isolates (NCBI accession PP236444). This cost-effective assay offers a portable platform for on-site OELCuV detection and can be adapted for other viruses, promoting sustainable okra cultivation.</p>

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Field-compatible detection of okra enation leaf curl virus using colorimetric RPA from crude leaf extract

  • 期刊论文

  • 发表日期：

    2026年3月23日

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  • Nishant,
  • Ramesh Kumar Yadav,
  • Damini Diksha,
  • Rakesh Kumar,
  • Malyaj R. Prajapati,
  • Reshav Naik,
  • Nitika Gupta,
  • Virendra K. Baranwal,
  • Susheel Kumar Sharma

摘要

Okra (Abelmoschus esculentus L.) is an important vegetable crop valued for its nutritional and medicinal properties. Enation leaf curl disease, caused by okra enation leaf curl virus (OELCuV; Begomovirus abelmoschusenation), severely affects okra production in India, leading to leaf curling, petiole bending, stunted growth and yield losses. Rapid, sensitive and field-deployable diagnostics are critical for timely management of virus disease. Here, we developed and validated a colorimetric recombinase polymerase amplification (RPA) assay for specific detection of OELCuV from crude okra leaf extracts. Fifteen symptomatic samples from ICAR-IARI, New Delhi, were initially detected for OELCuV by PCR using universal begomovirus and enation leaf curl virus-specific primers, while confirmatory identification was achieved through rolling circle amplification (RCA), cloning, sequencing, and sequence analysis. RPA is a rapid, highly sensitive and user-friendly isothermal technique that detects low RNA or DNA targets at a constant temperature, even in crude samples, offering advantages such as minimal equipment requirement, rapid turnaround time, multiplexing capability, and suitability as a portable and cost-effective platform for on-site virus detection and adaptation to other plant viruses. Two primer sets targeting the coat protein (AV1) and pre-coat protein (AV2) were designed, with AV1 primers showing high specificity and no cross-reactivity with other okra virus. Nine extraction buffers were evaluated, with phosphate buffer providing optimal templates. The RPA assay was optimized at 37°C for 30 min, detecting OELCuV up to 10–9 dilution and demonstrating higher analytical sensitivity than conventional PCR. The validation using of 90 okra field samples collected from six states of India demonstrated comparable detection efficiency of the developed RPA assay and PCR, with consistent results across both methods. Inclusion of hydroxynaphthol blue (HNB) enabled visual endpoint detection, eliminating agarose gel-based analysis or other specialized equipment. Sequence analysis of the RCA-amplified complete DNA-A genome segment revealed 91.5%–99.5% nucleotide identity with global OELCuV isolates (NCBI accession PP236444). This cost-effective assay offers a portable platform for on-site OELCuV detection and can be adapted for other viruses, promoting sustainable okra cultivation.