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Background <p>Lactococcosis is a bacterial <!--Query ID="Q1" Text="Please check if article title was captured and presented correctly. " Resolved="yes"-->disease in fish caused by several <i>Lactococcus</i> species, including <i>L. garvieae</i> and <i>L. petauri</i>, with clinical signs frequently overlapping with those caused by other streptococcal species. This diagnostic overlap necessitates a prompt and precise diagnostic tool for guiding accurate treatment and control. To address this diagnostic challenge, we developed basic recombinase polymerase amplification (basic-RPA) and RPA combined with lateral flow dipstick (RPA-LFD) assays for the detection of lactococcosis<i>-</i>associated <i>Lactococcus</i> species.</p> Results <p>Both methods exhibited a detection<!--Query ID="Q2" Text="Please confirm if the author names are presented accurately and in the correct sequence. Otherwise amend if necessary. " Resolved="yes"--> limit of 10<sup>3</sup> copies/µL for the recombinant plasmid pMD19-<i>adhE</i>-<i>ywdF</i> and 10 pg/µL for <i>L. garvieae</i> genomic DNA. Specificity testing using DNA from <i>L. garvieae</i>, <i>Streptococcus agalactiae</i>, <i>Aeromonas hydrophila</i>, <i>Edwardsiella piscicida</i>, and <i>Streptococcus iniae</i> confirmed that amplification occurred exclusively with <i>L. garvieae</i>, showing specificity against the tested panel. The assay also detected <i>L. petauri</i>, indicating that it recognizes both major lactococcosis-associated <i>Lactococcus</i> species. When tested with the plasmid standard, the RPA assays were 100-fold more sensitive than PCR; with genomic DNA, all three methods showed the same detection limit. When applied to 20 samples from experimentally infected tilapia, the RPA-LFD results were fully consistent with those of conventional PCR.</p> Conclusions <p>The established methods are straightforward,<!--Query ID="Q3" Text="Please check if affiliations were captured and presented correctly. " Resolved="yes"--> sensitive, and specific, offering a promising candidate tool for the rapid on-site diagnosis of piscine lactococcosis.</p>

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Establishment of methods for visual and rapid detection of piscine lactococcosis based on isothermal recombinase polymerase amplification

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月28日

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  • Hanyu Luo,
  • Zhihao Wu,
  • Weiqi Fan,
  • Hui Zeng,
  • Jie Li,
  • Yong-An Zhang,
  • Yang Zhou

摘要

Background

Lactococcosis is a bacterial disease in fish caused by several Lactococcus species, including L. garvieae and L. petauri, with clinical signs frequently overlapping with those caused by other streptococcal species. This diagnostic overlap necessitates a prompt and precise diagnostic tool for guiding accurate treatment and control. To address this diagnostic challenge, we developed basic recombinase polymerase amplification (basic-RPA) and RPA combined with lateral flow dipstick (RPA-LFD) assays for the detection of lactococcosis-associated Lactococcus species.

Results

Both methods exhibited a detection limit of 103 copies/µL for the recombinant plasmid pMD19-adhE-ywdF and 10 pg/µL for L. garvieae genomic DNA. Specificity testing using DNA from L. garvieae, Streptococcus agalactiae, Aeromonas hydrophila, Edwardsiella piscicida, and Streptococcus iniae confirmed that amplification occurred exclusively with L. garvieae, showing specificity against the tested panel. The assay also detected L. petauri, indicating that it recognizes both major lactococcosis-associated Lactococcus species. When tested with the plasmid standard, the RPA assays were 100-fold more sensitive than PCR; with genomic DNA, all three methods showed the same detection limit. When applied to 20 samples from experimentally infected tilapia, the RPA-LFD results were fully consistent with those of conventional PCR.

Conclusions

The established methods are straightforward, sensitive, and specific, offering a promising candidate tool for the rapid on-site diagnosis of piscine lactococcosis.