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<p>Apple necrotic mosaic virus (ApNMV) (species: <i>Ilarvirus ApNMV</i>), the predominant causal agent of apple mosaic disease in Asia, poses a major threat to apple cultivation, leading to significant yield losses. Considering the graft-transmissibility of ApNMV and the lack of resistant cultivars, reliable detection methods are crucial for managing disease spread through planting materials. In this study, we developed a rapid, sensitive, and field-deployable reverse transcription-recombinase polymerase amplification (RT-RPA) assay for ApNMV detection. Three primer sets targeting the RNA-dependent RNA polymerase (RdRp) and coat protein genes were designed, of which the RdRp-specific primer pair (282&#xa0;bp) showed high specificity. Crude leaf extracts prepared in NaOH:EDTA (1:1) buffer provided the most suitable template, eliminating the need for RNA purification. The optimized assay performed best at 42&#xa0;°C for 35&#xa0;min, detecting ApNMV up to 10<sup>–5</sup> dilution in RNA and 10<sup>–4</sup> dilution in crude extract template, thereby exhibiting tenfold higher sensitivity than conventional RT-PCR. No cross-reactivity was observed with other common apple viruses (ASPV, ASGV, ACLSV, ApMV). ApNMV was detected by RT-RPA in 75% (55 of 73) of field samples compared to 71% (52 of 73) by RT-PCR. For on-site application, the assay was coupled with the hydroxy naphthol blue (HNB) dye, producing distinct colour changes for positive and negative reactions, fully concordant with gel electrophoresis. In parallel, a SYBR Green-based RT-qPCR assay targeting RdRp gene was developed for absolute quantification, yielding a robust standard curve (R<sup>2</sup> = 0.99, efficiency = 98.6%) and detecting viral loads ranging from 12.9 to 86.9 copies in field samples. Collectively, RT-RPA offers a rapid, simple, and field-adaptable diagnostic tool, while RT-qPCR enables precise quantification, together supporting enhanced surveillance and management of ApNMV in apple production systems.</p>

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Colorimetric RT-RPA and quantitative RT-qPCR assays for rapid and field-deployable detection of apple necrotic mosaic virus

  • 期刊论文

  • 发表日期：

    2026年2月12日

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  • Nehala Shaji,
  • Rakesh Kumar,
  • Damini Diksha,
  • Pooja Bhardwaj,
  • Jayesh Gehlot,
  • Aarzoo Qamar,
  • Nitika Gupta,
  • Pankhuri Singhal,
  • Sajad Un Nabi,
  • Virendra Kumar Baranwal,
  • Susheel Kumar Sharma

摘要

Apple necrotic mosaic virus (ApNMV) (species: Ilarvirus ApNMV), the predominant causal agent of apple mosaic disease in Asia, poses a major threat to apple cultivation, leading to significant yield losses. Considering the graft-transmissibility of ApNMV and the lack of resistant cultivars, reliable detection methods are crucial for managing disease spread through planting materials. In this study, we developed a rapid, sensitive, and field-deployable reverse transcription-recombinase polymerase amplification (RT-RPA) assay for ApNMV detection. Three primer sets targeting the RNA-dependent RNA polymerase (RdRp) and coat protein genes were designed, of which the RdRp-specific primer pair (282 bp) showed high specificity. Crude leaf extracts prepared in NaOH:EDTA (1:1) buffer provided the most suitable template, eliminating the need for RNA purification. The optimized assay performed best at 42 °C for 35 min, detecting ApNMV up to 10–5 dilution in RNA and 10–4 dilution in crude extract template, thereby exhibiting tenfold higher sensitivity than conventional RT-PCR. No cross-reactivity was observed with other common apple viruses (ASPV, ASGV, ACLSV, ApMV). ApNMV was detected by RT-RPA in 75% (55 of 73) of field samples compared to 71% (52 of 73) by RT-PCR. For on-site application, the assay was coupled with the hydroxy naphthol blue (HNB) dye, producing distinct colour changes for positive and negative reactions, fully concordant with gel electrophoresis. In parallel, a SYBR Green-based RT-qPCR assay targeting RdRp gene was developed for absolute quantification, yielding a robust standard curve (R2 = 0.99, efficiency = 98.6%) and detecting viral loads ranging from 12.9 to 86.9 copies in field samples. Collectively, RT-RPA offers a rapid, simple, and field-adaptable diagnostic tool, while RT-qPCR enables precise quantification, together supporting enhanced surveillance and management of ApNMV in apple production systems.