Rapid and Sensitive Detection of Streptococcus iniae in Trachinotus ovatus Based on Multienzyme Isothermal Rapid Amplification

doi:10.3390/ijms24097733

. 2023 Apr 23;24(9):7733.

doi: 10.3390/ijms24097733.

Rapid and Sensitive Detection of Streptococcus iniae in Trachinotus ovatus Based on Multienzyme Isothermal Rapid Amplification

Yifen Wang^{1

2}, Jingjing Niu¹, Minmin Sun^{1

2}, Ziyi Li^{1

2}, Xiangyuan Wang^{1

2}, Yan He^{1

2}, Jie Qi^{1

2}

Affiliations

¹ Key Laboratory of Tropical Aquatic Germplasm of Hainan Province, Sanya Oceanographic Institute, Ocean University of China, Sanya 572025, China.
² MOE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, 5 Yushan Road, Qingdao 266003, China.

PMID: 37175440
PMCID: PMC10178759
DOI: 10.3390/ijms24097733

Rapid and Sensitive Detection of Streptococcus iniae in Trachinotus ovatus Based on Multienzyme Isothermal Rapid Amplification

Yifen Wang et al. Int J Mol Sci. 2023.

. 2023 Apr 23;24(9):7733.

doi: 10.3390/ijms24097733.

Authors

Yifen Wang^{1

2}, Jingjing Niu¹, Minmin Sun^{1

2}, Ziyi Li^{1

2}, Xiangyuan Wang^{1

2}, Yan He^{1

2}, Jie Qi^{1

2}

Affiliations

¹ Key Laboratory of Tropical Aquatic Germplasm of Hainan Province, Sanya Oceanographic Institute, Ocean University of China, Sanya 572025, China.
² MOE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, 5 Yushan Road, Qingdao 266003, China.

PMID: 37175440
PMCID: PMC10178759
DOI: 10.3390/ijms24097733

Abstract

Infectious diseases caused by Streptococcus iniae lead to massive death of fish, compose a serious threat to the global aquaculture industry, and constitute a risk to humans who deal with raw fish. In order to realize the early diagnosis of S. iniae, and control the outbreak and spread of disease, it is of great significance to establish fast, sensitive, and convenient detection methods for S. iniae. In the present study, two methods of real-time MIRA (multienzyme isothermal rapid amplification, MIRA) and MIRA-LFD (combining MIRA with lateral flow dipsticks (LFD)) for the simA gene of S. iniae were established, which could complete amplification at a constant temperature of 42 °C within 20 min. Real-time MIRA and MIRA-LFD assays showed high sensitivity (97 fg/μL or 7.6 × 10² CFU/mL), which were consistent with the sensitivity of real-time PCR and 10 times higher than that of PCR with strong specificity, repeatability simplicity, and rapidity for S. iniae originating from Trachinotus ovatus. In summary, real-time MIRA and MIRA-LFD provide effective ways for early diagnosis of S. iniae in aquaculture, especially for units in poor conditions.

Keywords: Streptococcus iniae; lateral flow dipsticks; multienzyme isothermal rapid amplification; rapid detection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Figure 1**
Amplicons of twenty-five primer combinations using the basic MIRA assay. F: forward primer; R: reverse primer; M: 100 bp DNA Ladder. (A) The primer combinations of F1R1–F4R2; (B) The primer combinations of F4R3–F5R5.

**Figure 2**
Optimum reaction temperature for real-time MIRA assays. NC: negative control.

**Figure 3**
Specificity of real-time MIRA and MIRA-LFD assays. NC: negative control. (A) Specificity of real-time MIRA assays; (B) Specificity of MIRA-LFD assays.

**Figure 4**
Comparison of DNA sensitivity. NC: negative control. DNA detection limits were determined using DNA diluted at 9.7 ng/μL–9.7 fg/μL as template for PCR (A), real-time PCR (B), real-time MIRA (C), and MIRA-LFD (D) assays.

**Figure 5**
Comparison of the sensitivity of bacterial suspensions. NC: negative control. The detection limits of bacterial suspensions were determined using DNA extracted from a dilution series of gradient bacterial suspension samples containing 7.6 × 10⁷−10⁰ CFU/mL of *S. iniae* as templates for PCR (A), real-time PCR (B), real-time MIRA (C), and MIRA-LFD (D) assays.

**Figure 6**
Reproducibility of real-time MIRA and MIRA-LFD assays. NC: negative control. (A,B) DNA detection limit (97 fg/μL) and bacterial suspensions detection limit (7.6 × 10² CFU/mL) of the real-time MIRA reproducibility assays, respectively. (C,D) DNA minimum detection limit (97 fg/μL) and bacterial fluid minimum detection limit (7.6 × 10² CFU/mL) of the MIRA-LFD reproducibility assays, respectively.

**Figure 7**
Evaluation of the practicability of real-time MIRA and MIRA-LFD based on real-time PCR. 1–12:12 samples of suspected *S. iniae* infection; NC: negative control. (A–C) The detection results of real-time PCR, real-time MIRA, and MIRA-LFD respectively.

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References

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1. Feng Y., Bai M., Geng Y., Chen D., Huang X., Ouyang P., Guo H., Zuo Z., Huang C., Lai W. The potential risk of antibiotic resistance of Streptococcus iniae in sturgeon cultivation in Sichuan, China. Environ. Sci. Pollut. Res. Int. 2021;28:69171–69180. doi: 10.1007/s11356-021-15501-1. - DOI - PubMed
1. Hayat M., Yusoff M., Samad M., Razak I.A., Yasin I., Thompson K., Hasni K. Efficacy of Feed-Based Formalin-Killed Vaccine of Streptococcus iniae Stimulates the Gut-Associated Lymphoid Tissues and Immune Response of Red Hybrid Tilapia. Vaccines. 2021;9:51. doi: 10.3390/vaccines9010051. - DOI - PMC - PubMed
1. Guo S., Mo Z., Wang Z., Xu J., Li Y., Dan X., Li A. Isolation and pathogenicity of Streptococcus iniae in offshore cage-cultured Trachinotus ovatus in China. Aquaculture. 2018;492:247–252. doi: 10.1016/j.aquaculture.2018.04.015. - DOI
1. Zhou Y., Xiao J., Ma X., Wang Q., Zhang Y. An effective established biosensor of bifunctional probes-labeled AuNPs combined with LAMP for detection of fish pathogen Streptococcus iniae. Appl. Microbiol. Biotechnol. 2018;102:5299–5308. doi: 10.1007/s00253-018-9016-3. - DOI - PubMed

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[1] Ma Y.-M., Yang M.-J., Wang S., Li H., Peng X.-X. Liver functional metabolomics discloses an action of L-leucine against Streptococcus iniae infection in tilapias. Fish Shellfish Immunol. 2015;45:414–421. doi: 10.1016/j.fsi.2015.04.037. - DOI - PubMed

[2] Ma Y.-M., Yang M.-J., Wang S., Li H., Peng X.-X. Liver functional metabolomics discloses an action of L-leucine against Streptococcus iniae infection in tilapias. Fish Shellfish Immunol. 2015;45:414–421. doi: 10.1016/j.fsi.2015.04.037. - DOI - PubMed

[3] Feng Y., Bai M., Geng Y., Chen D., Huang X., Ouyang P., Guo H., Zuo Z., Huang C., Lai W. The potential risk of antibiotic resistance of Streptococcus iniae in sturgeon cultivation in Sichuan, China. Environ. Sci. Pollut. Res. Int. 2021;28:69171–69180. doi: 10.1007/s11356-021-15501-1. - DOI - PubMed

[4] Feng Y., Bai M., Geng Y., Chen D., Huang X., Ouyang P., Guo H., Zuo Z., Huang C., Lai W. The potential risk of antibiotic resistance of Streptococcus iniae in sturgeon cultivation in Sichuan, China. Environ. Sci. Pollut. Res. Int. 2021;28:69171–69180. doi: 10.1007/s11356-021-15501-1. - DOI - PubMed

[5] Hayat M., Yusoff M., Samad M., Razak I.A., Yasin I., Thompson K., Hasni K. Efficacy of Feed-Based Formalin-Killed Vaccine of Streptococcus iniae Stimulates the Gut-Associated Lymphoid Tissues and Immune Response of Red Hybrid Tilapia. Vaccines. 2021;9:51. doi: 10.3390/vaccines9010051. - DOI - PMC - PubMed

[6] Hayat M., Yusoff M., Samad M., Razak I.A., Yasin I., Thompson K., Hasni K. Efficacy of Feed-Based Formalin-Killed Vaccine of Streptococcus iniae Stimulates the Gut-Associated Lymphoid Tissues and Immune Response of Red Hybrid Tilapia. Vaccines. 2021;9:51. doi: 10.3390/vaccines9010051. - DOI - PMC - PubMed

[7] Guo S., Mo Z., Wang Z., Xu J., Li Y., Dan X., Li A. Isolation and pathogenicity of Streptococcus iniae in offshore cage-cultured Trachinotus ovatus in China. Aquaculture. 2018;492:247–252. doi: 10.1016/j.aquaculture.2018.04.015. - DOI

[8] Guo S., Mo Z., Wang Z., Xu J., Li Y., Dan X., Li A. Isolation and pathogenicity of Streptococcus iniae in offshore cage-cultured Trachinotus ovatus in China. Aquaculture. 2018;492:247–252. doi: 10.1016/j.aquaculture.2018.04.015. - DOI

[9] Zhou Y., Xiao J., Ma X., Wang Q., Zhang Y. An effective established biosensor of bifunctional probes-labeled AuNPs combined with LAMP for detection of fish pathogen Streptococcus iniae. Appl. Microbiol. Biotechnol. 2018;102:5299–5308. doi: 10.1007/s00253-018-9016-3. - DOI - PubMed

[10] Zhou Y., Xiao J., Ma X., Wang Q., Zhang Y. An effective established biosensor of bifunctional probes-labeled AuNPs combined with LAMP for detection of fish pathogen Streptococcus iniae. Appl. Microbiol. Biotechnol. 2018;102:5299–5308. doi: 10.1007/s00253-018-9016-3. - DOI - PubMed

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Rapid and Sensitive Detection of Streptococcus iniae in Trachinotus ovatus Based on Multienzyme Isothermal Rapid Amplification

Affiliations

Rapid and Sensitive Detection of Streptococcus iniae in Trachinotus ovatus Based on Multienzyme Isothermal Rapid Amplification

Authors

Affiliations

Abstract

Conflict of interest statement

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