Veterinary Research Forum

Cell SELEX-based DNA aptamer selection targeting Streptococcus agalactiae using high-throughput sequencing: toward multifunctional applications in aquaculture

Document Type : Original Article

Authors

Dung Ho My Nguyen 1
Kamolwan Khianchaikhan 2
Apinya Longya 2
Nararat Laopichienpong 2
Thuan Duc Lao 3
Pimwarang Sukkarun 4

1 Center for Life Science Research – CELI, Ho Chi Minh City Open University, Ho Chi Minh City, Vietnam

2 Department of Research and Development, Queen Saovabha Memorial Institute, Bangkok, Thailand

3 Department of Pharmaceutical and Medical Biotechnology, Faculty of Biotechnology, Ho Chi Minh City Open University, Ho Chi Minh City, Vietnam

4 Faculty of Veterinary Science, Rajamangala University of Technology Srivijaya, Nakhon Si Thammarat, Thailand

https://doi.org/10.30466/vrf.2025.2065775.4838
Abstract
Streptococcus agalactiae is a Gram-positive bacterial pathogen accountable for disease outbreaks in tilapia, resulting in substantial economic losses in aquaculture. In this study, a 10-round whole-cell Systemic Evolution of Ligands by Exponential Enrichment procedure was conducted to isolate and characterize single-stranded DNA aptamers with specific binding affinity to S. agalactiae. The enriched single-stranded DNA libraries were sequenced by high-throughput sequencing, yielding a total of 180,987 reads, of which 126,933 included unique aptamer sequences. The most frequent sequences were selected for further analysis. Among them, SA1 had the highest frequency, with 231 reads, whereas SA2 showed a lower number of 16 reads. These findings underscored the validity of combining whole-cell Systemic Evolution of Ligands by Exponential Enrichment, high-throughput sequencing and aptamer-structure analysis to explore the interaction between aptamers and bacterial cell surfaces. The selected aptamers not only showed promise for the detection of S. agalactiae but also served as molecular probes for identifying surface-exposed proteins, studying host-pathogen interactions and guiding targeted drug delivery in aquaculture-related applications.

Keywords

Aptamer
Aquatic pathogens
High-throughput sequencing
Streptococcus agalactiae
Whole-cell SELEX
Subjects
1.     Tacon AGJ, Shumway SE. Critical need to increase aquatic food production and food supply from aquaculture and capture fisheries: trends and outlook. Rev Fish Sci Aquac 2024; 32(3): 389-395.
2.     Yadav NK, Patel AB, Singh SK, et al. Climate change effects on aquaculture production and its sustainable management through climate-resilient adaptation strategies: a review. Environ Sci Pollut Res Int 2024; 31(22): 31731-31751.
3.     Leung TL, Bates AE. More rapid and severe disease outbreaks for aquaculture at the tropics: implications for food security. J Appl Ecol 2013; 50: 215-222.
4.     Rahman MM, Rahman MA, Monir MS, et al. Isolation and molecular detection of Streptococcus agalactiae from popped eye disease of cultured Tilapia and Vietnamese koi fishes in Bangladesh. J Adv Vet Anim Res 2021; 8(1): 14-23.
5.     Abdallah ESH, Metwally WGM, Abdel-Rahman MAM, et al. Streptococcus agalactiae infection in Nile Tilapia (Oreochromis niloticus): a review. Biology (Basel) 2024; 13(11): 914. doi: 10.3390/biology13110914.
6.     Johri AK, Paoletti LC, Glaser P, et al. Group B Streptococcus: global incidence and vaccine development. Nat Rev Microbiol 2006; 4(12): 932-942.
7.     Lusiastuti AM, Suhermanto A, Hastilestari BR, et al. Impact of temperature on the virulence of Streptococcus agalactiae in Indonesian aquaculture: a better vaccine design is required. Vet World 2024; 17(3): 682-689.
8.     Wang L, Wang R, Wei H, et al. Selection of aptamers against pathogenic bacteria and their diagnostics application. World J Microbiol Biotechnol 2018; 34(10): 149. doi: 10.1007/s11274-018-2528-2.
9.     Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature 1990; 346(6287): 818-822.
10. Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990; 249(4968): 505-510.
11. Zhou J, Rossi J. Aptamers as targeted therapeutics: current potential and challenges. Nat Rev Drug Discov 2017; 16(3): 181-202.
12. Beloborodov SS, Bao J, Krylova SM, et al. Aptamerfacilitated purification of functional proteins. J Chromatogr B Analyt Technol Biomed Life Sci 2018; 1073: 201-206.
13. Kolovskaya OS, Savitskaya AG, Zamay TN, et al. Development of bacteriostatic DNA aptamers for salmonella. J Med Chem 2013; 56(4): 1564-1572.
14. Ning Y, Cheng L, Ling M, et al. Efficient suppression of biofilm formation by a nucleic acid aptamer. Pathog Dis 2015; 73(6): ftv034. doi: 10.1093/femspd/ftv034.
15. Zhou L, Wang S, Yu Q, et al. Characterization of novel aptamers specifically directed to red-spotted grouper nervous necrosis virus (RGNNV)-infected cells for mediating targeted siRNA delivery. Front Microbiol 2020; 11: 660. doi: 10.3389/fmicb.2020.00660.
16. Zhang X, Wang L, Liu J, et al. Generation and identification of novel DNA aptamers with antiviral activities against largemouth bass virus (LMBV). Aquaculture 2022; 547: 737478. doi: 10.1016/ j.aquaculture.2021.737478.
17. Sarkar DJ, Biswas A, Mondal S, et al. Aeromonas veronii specific aptamer and peroxidase mimic tyrosine-capped gold NanoZymes enable highly specific sensing of fish pathogenic bacteria. Biosens Bioelectron: X 2024; 19: 100505. doi: 10.1016/j.biosx.2024.100505.
18. Moon J, Kim G, Lee S, et al. Identification of Salmonella typhimurium-specific DNA aptamers developed using whole-cell SELEX and FACS analysis. J Microbiol. Methods 2013; 95(2): 162-166.
19. Dwivedi HP, Smiley RD, Jaykus LA. Selection of DNA aptamers for capture and detection of Salmonella typhimurium using a whole-cell SELEX approach in conjunction with cell sorting. Appl Microbiol Biotechnol 2013; 97(8): 3677-3686.
20. Hoon S, Zhou B, Janda KD. Aptamer selection by high-throughput sequencing and informatic analysis. Biotechniques 2011; 51(6): 413-416.
21. Levay A, Brenneman R, Hoinka J, et al. Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment. Nucleic Acids Res 2015; 43(12): e82. doi: 10.1093/nar/gkv534.
22. Laith AA, Ambak MA, Hassan M, et al. Molecular identification and histopathological study of natural Streptococcus agalactiae infection in hybrid tilapia (Oreochromis niloticus). Vet World 2017; 10(1): 101-111.
23. Geng Y, Wang KY, Huang XL, et al. Streptococcus agalactiae, an emerging pathogen for cultured ya‐fish, Schizothorax prenanti, in China. Transbound Emerg Dis 2012; 59(4): 369-375.
24. Andrew S. FASTQC: a quality control tool for high throughput sequence data. Babraham Institute; 2010. Available at: http://www.bioinformatics.babraham.ac. uk/projects/fastqc/. Accessed June 07, 2026.
25. Ewels P, Magnusson M, Lundin S, et al. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics 2016; 32(19): 3047-3048.
26. Magoc̆ T, Salzberg SL: FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 2011; 27(21): 2957-2963.
27. Caporaso JG, Kuczynski J, Stombaugh J, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods 2010; 7(5): 335-336.
28. Rognes T, Flouri T, Nichols B, et al. VSEARCH: a versatile open-source tool for metagenomics. PeerJ 2016; 4: e2584. doi: 10.7717/peerj.2584.
29. Layman B, Mandella B, Carter J, et al. Isolation and characterization of a ssDNA aptamer against major soluble antigen of Renibacterium salmoninarum. Molecules 2022; 27(6): 1853. doi: 10.3390/molecules 27061853.
30. Moon J, Kim G, Park SB, et al. Comparison of whole-cell SELEX methods for the identification of Staphylococcus aureus-specific DNA aptamers. Sensors (Basel) 2015; 15(4): 8884-8897.
31. Oliveira R, Pinho E, Barros MM, et al. In vitro selection of DNA aptamers against staphylococcal enterotoxin A. Sci Rep 2024; 14(1): 11345. doi: 10.1038/s41598-024-61094-3.
32. Afanasyeva A, Nagao C, Mizuguchi K. Prediction of the secondary structure of short DNA aptamers. Biophys Physicobiol 2019; 16: 287-294.
33. Chen Z, Hu L, Zhang BT, et al. Artificial intelligence in Aptamer–Target binding prediction. Int J Mol Sci 2021; 22(7): 3605. doi: 10.3390/ijms22073605.
34. Sakamoto T, Ennifar E, Nakamura Y. Thermodynamic study of aptamers binding to their target proteins. Biochimie 2018; 145: 91-97.
35. Chen F, Zhou J, Luo F, et al. Aptamer from whole-bacterium SELEX as new therapeutic reagent against virulent Mycobacterium tuberculosis. Biochem Biophys Res Commun 2007; 357(3): 743-748.
36. Renders M, Miller E, Lam CH, et al. Whole cell-SELEX of aptamers with a tyrosine-like side chain against live bacteria. Org Biomol Chem 2017; 15(9): 1980-1989.
37. Yılmaz D, Muslu T, Parlar A, et al. SELEX against whole-cell bacteria resulted in lipopolysaccharide binding aptamers. J Biotechnol 2022; 354: 10-20.
38. Cesarini V, Scopa C, Silvestris DA, et al. Aptamer-based in vivo therapeutic targeting of glioblastoma. Molecules 2020; 25(18): 4267. doi: 10.3390/molecules25184267.
39. Nosaz Z, Rasoulinejad S, Mousavi Gargari SL. Development of a DNA aptamer to detect Brucella abortus and Brucella melitensis through cell SELEX. Iran J Vet Res 2020; 21(4): 294-300.
Veterinary Research Forum
Volume 17, Issue 4
April 2026
Pages 225-231

  • Receive Date 16 July 2025
  • Revise Date 04 November 2025
  • Accept Date 15 November 2025

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