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Rapid Visual Detection of Spiroplasma eriocheiris by Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue Dye

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Abstract

In recent years, a new type of Spiroplasma has been found that can cause “tremor disease” of the Chinese mitten crab Eriocheir sinensis. The outbreak of epidemic tremor disease has caused a serious setback in the Chinese mitten crab farming industry, with an incidence rate of more than 30% and mortality rates of 80–100%. Therefore, finding a sensitive method to detect tremor disease in E. sinensis has become a current research focus. In this research, a loop-mediated isothermal amplification detection method coupled with hydroxynaphthol blue dye (LAMP-HNB) was developed and used to rapidly detect Spiroplasma eriocheiris. First, we designed and synthesized specific outer primers, inner primers and loop primers based on the 16S ribosomal RNA gene of S. eriocheiris. Second, the LAMP-HNB detection method for S. eriocheiris was successfully established by screening the primers, adjusting the temperature and time of the reaction, and optimizing the concentrations of Mg2+ and dNTPs. In the specific tests, only samples infected with S. eriocheiris showed positive results, and other infections caused by bacteria and parasites tested negative, proving that the test has high specificity. Moreover, the detection limit was 2.5 × 10–6 ng/µL, indicating high sensitivity. This method for detecting S. eriocheiris provides rapid visual output based on LAMP-HNB detection and is a simple, fast, sensitive, and inexpensive method that can be applied to a wide range of field investigations.

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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

My Research Project was partially (or fully) sponsored by [Qingdao Agricultural University Doctoral Start-Up Fund (6631122030), Fish Innovation Team of Shandong Agriculture Research System (SDAIT-12–06), National Natural Science Foundation of China (32002421), the Advanced Talents Foundation of QAU (6651118016), Shandong Engineering Research Center for Prevention and Control of Aquatic Animal Disease, "First Class Fishery Discipline" Programme [(2020)3] in Shandong Province].

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Author notes

  1. Shun Zhou and Zongrui Yang contributed equally to this work.

Authors and Affiliations

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

    Shun Zhou, Zongrui Yang, Baoshan Guo, Jingyuan Yi, Yunfei Pang, Ruixin Feng, Jiaxue Song & Yunji Xiu

Authors
  1. Shun Zhou
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  2. Zongrui Yang
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  3. Baoshan Guo
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  7. Jiaxue Song
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  8. Yunji Xiu
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Contributions

SZ proposed concepts and methods. ZRY analyzed the data and wrote the paper. BSG designed the experiment and performed the experiments. JYY and YFP participated in the RPA-EXO detection, and RXF and JXS 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.

Ethics declarations

I am enclosing herewith a manuscript entitled “[Rapid visual detection of Spiroplasma eriocheiris by loop-mediated isothermal amplification with hydroxynaphthol blue dye]” submitted to AQUACULTURE for possible evaluation. With the submission of this manuscript, I would like to confirm that the above-mentioned manuscript has not been published elsewhere, accepted for publication elsewhere or under editorial review for publication elsewhere; and that my Institute’s [School of Marine Science and Engineering, Qingdao Agricultural University] representative is fully aware of this submission.

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).

Competing Interests

The authors declare no competing interests.

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Zhou, S., Yang, Z., Guo, B. et al. Rapid Visual Detection of Spiroplasma eriocheiris by Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue Dye. Mar Biotechnol 26, 116–124 (2024). https://doi.org/10.1007/s10126-023-10282-9

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