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<p>Although lab-based protocols are available for detection, a simple and less time-consuming on-site kit development would help in the early detection of pathogens for their timely management. In the present study, a recombinase polymerase amplification-based lateral flow assay (RPA-LFA) was developed for the simultaneous detection of <i>Phytophthora</i> and <i>Pythium</i> species in black pepper. Primers were designed from conserved regions of ITS and the conditions were optimized for duplex RPA and RPA-LFA assays. Based on the results, 20&#xa0;mM MgoAc concentration, 39&#xa0;°C, and 30&#xa0;min incubation time were chosen as the optimum conditions for duplex RPA-LFA. The addition of 1.2&#xa0;M betaine further improved the specificity of the assay. The sensitivity of duplex RPA-LFA was 0.01&#xa0;ng of <i>Phytophthora</i> and 1&#xa0;ng of <i>Pythium</i> DNA in 50&#xa0;ng of healthy black pepper leaf DNA. The assay was highly specific and did not show any cross-reaction with other pathogens of black pepper and also pathogens of other spice crops grown as component crops. The assay could successfully detect different species of <i>Phytophthora</i> and <i>Pythium</i> infecting black pepper. In a validation test, duplex RPA-LFA detected the pathogens using crude extract from the field samples and also from artificially inoculated samples.</p>

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Betaine-assisted recombinase polymerase amplification-lateral flow assay (RPA-LFA) for simultaneous detection of oomycete pathogens infecting black pepper

  • 期刊论文

  • 发表日期：

    2026年7月27日

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  • A. Jeevalatha,
  • Malavika Pradeep,
  • A. I. Bhat

摘要

Although lab-based protocols are available for detection, a simple and less time-consuming on-site kit development would help in the early detection of pathogens for their timely management. In the present study, a recombinase polymerase amplification-based lateral flow assay (RPA-LFA) was developed for the simultaneous detection of Phytophthora and Pythium species in black pepper. Primers were designed from conserved regions of ITS and the conditions were optimized for duplex RPA and RPA-LFA assays. Based on the results, 20 mM MgoAc concentration, 39 °C, and 30 min incubation time were chosen as the optimum conditions for duplex RPA-LFA. The addition of 1.2 M betaine further improved the specificity of the assay. The sensitivity of duplex RPA-LFA was 0.01 ng of Phytophthora and 1 ng of Pythium DNA in 50 ng of healthy black pepper leaf DNA. The assay was highly specific and did not show any cross-reaction with other pathogens of black pepper and also pathogens of other spice crops grown as component crops. The assay could successfully detect different species of Phytophthora and Pythium infecting black pepper. In a validation test, duplex RPA-LFA detected the pathogens using crude extract from the field samples and also from artificially inoculated samples.