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Development of Two Recombinase Polymerase Amplification EXO (RPA-EXO) and Lateral Flow Dipstick (RPA-LFD) Techniques for the Rapid Visual Detection of Aeromonas salmonicida

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Abstract

Aeromonas salmonicida is the pathogen underlying furunculosis, causing a septicemic infection that influences both salmonid and non-salmonid fish. Early diagnosis of these contagions is essential for disease surveillance and prevention, so a rapid and sensitive approach is needed. Herein, a recombinase polymerase amplification EXO (RPA-EXO) assay and RPA with a lateral flow dipstick (RPA-LFD) were produced for testing A. salmonicida. The RPA-EXO and RPA-LFD primer sets were devised based on the conserved fragment sequence of the vapA gene. Then, RPA-EXO and RPA-LFD reaction systems were established, and the reaction temperature and time were optimized. After optimization, the RPA-EXO method was capable of testing A. salmonicida within 10 min, and the RPA-LFD method could detect A. salmonicida in only 5 min. The RPA-EXO and RPA-LFD methods exhibited high specificity with no cross-reaction with other strains. To assess sensitivity, a partial vapA gene was cloned, and serial plasmid dilutions were created ranging from 1 × 106 to 1 × 10−1 copies/μL. The detection limit of RPA-EXO was 1 × 102 copies/μL, and the detection limit of RPA-LFD was 1 copy/μL. For spiked turbot tissue samples, the sensitivity detection of A. salmonicida was 1.2 × 101 CFU/mL and 1.2 CFU/mL by RPA-EXO and RPA-LFD, respectively. In comparative analyses of clinical samples, the diagnostic results of RPA-EXO and RPA-LFD were compared with those of the standard conventional PCR test and showed nearly 100% consistency. Therefore, our RPA-EXO and RPA-LFD assays exhibited excellent specificity and sensitivity, which provided two simple, fast and dependable methods to conduct large-scale field investigations of A. salmonicida in resource-limited settings.

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Data Availability

All data generated or analyzed during this study are included in this published article.

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Funding

This work was supported by National Natural Science Foundation of China (No. 32002421), the Advanced Talents Foundation of QAU (Grant No. 6651118016), Natural Science Foundation of Shandong Province (Grant No. ZR2019BC009), Fish Innovation Team of Shandong Agriculture Research System (SDAIT-12–06), “First class fishery discipline” program in Shandong Province, special top talent plan “One Thing One Decision (Yishi Yiyi)”, Breeding Plan of Shandong Provincial Qingchuang Research Team (2019).

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Author notes

  1. Shun Zhou and Xujia Zheng contributed equally to this work.

Authors and Affiliations

  1. School of Marine Science and Engineering, Qingdao Agricultural University, Qingdao, 266109, China

    Shun Zhou, Xujia Zheng, Zongrui Yang, Qing Huang, Jingyuan Yi, Lin Su, Baoshan Guo & Yunji Xiu

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  1. Shun Zhou
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  2. Xujia Zheng
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  3. Zongrui Yang
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  4. Qing Huang
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  6. Lin Su
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Contributions

SZ proposed concepts and methods. XJZ analyzed the data and wrote the paper. ZYY designed the experiment and performed the experiments. QH and JYY participated in the RPA-EXO detection, and LS and BSG participated in the RPA-LFD detection experiments. YJX edited the manuscript and supervised the project. All the authors read and approved the final manuscript.

Corresponding author

Correspondence to Yunji Xiu.

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Ethical Approval

This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, Qingdao Agricultural University. The protocol was approved by the Committee on the Ethics of Animal Experiments of Qingdao Agricultural University IACUC (Institutional Animal Care and Use Committee).

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The authors declare no competing interests.

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Zhou, S., Zheng, X., Yang, Z. et al. Development of Two Recombinase Polymerase Amplification EXO (RPA-EXO) and Lateral Flow Dipstick (RPA-LFD) Techniques for the Rapid Visual Detection of Aeromonas salmonicida. Mar Biotechnol 24, 1094–1109 (2022). https://doi.org/10.1007/s10126-022-10170-8

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