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<p><i>Glugea plecoglossi</i>, a microsporidia of the <i>Glugea</i> genus, can cause an infamous disease <i>Plecoglossus altivelis</i> in East Asia, resulting in heavy economic losses. At present, the main diagnostic methods for this disease include microscopy examination, quantitative real-time PCR, and loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD). In this study, a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) method, targeting the <i>beta-tubulin</i> gene, was developed to detect <i>G. plecoglossi</i>, three sets of primers and probes were designed and screened, after which the initial reaction system was established. The RPA-LFD method for <i>G. plecoglossi</i> could complete nucleic acid amplification at 39 °C for 10 min, after which the amplification product was dropped on the LFD strip, and the results could then be observed within 5 min. A specificity assay revealed that there was no cross-reactivity with other protozoa except <i>G. plecoglossi</i>. A sensitivity assay revealed that the detection limit was 9.38×10<sup>−6</sup> ng/µL, which was more sensitive than that of conventional PCR. Compared with conventional detection methods, the novel RPA-LFD method has the advantages of simple operation, short operation time, high sensitivity, and high specificity for <i>G. plecoglossi</i> detection, indicating its potential use in rapid field detection of <i>G. plecoglossi</i>.</p>

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Construction and application of the RPA-LFD rapid detection method for Glugea plecoglossi

  • 期刊论文

  • 发表日期：

    2025年4月10日

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  • Jiaxue Song,
  • Ruixin Feng,
  • Yunfei Pang,
  • Qingyue Xu,
  • Dong Zheng,
  • Yunji Xiu,
  • Shun Zhou

摘要

Glugea plecoglossi, a microsporidia of the Glugea genus, can cause an infamous disease Plecoglossus altivelis in East Asia, resulting in heavy economic losses. At present, the main diagnostic methods for this disease include microscopy examination, quantitative real-time PCR, and loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD). In this study, a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) method, targeting the beta-tubulin gene, was developed to detect G. plecoglossi, three sets of primers and probes were designed and screened, after which the initial reaction system was established. The RPA-LFD method for G. plecoglossi could complete nucleic acid amplification at 39 °C for 10 min, after which the amplification product was dropped on the LFD strip, and the results could then be observed within 5 min. A specificity assay revealed that there was no cross-reactivity with other protozoa except G. plecoglossi. A sensitivity assay revealed that the detection limit was 9.38×10−6 ng/µL, which was more sensitive than that of conventional PCR. Compared with conventional detection methods, the novel RPA-LFD method has the advantages of simple operation, short operation time, high sensitivity, and high specificity for G. plecoglossi detection, indicating its potential use in rapid field detection of G. plecoglossi.