Development and preliminary evaluation of real-time PCR assays for six lactic acid bacteria

development-and-preliminary-evaluation-of-real-time-pcr-assays-for-six-lactic-acid-bacteria
Development and preliminary evaluation of real-time PCR assays for six lactic acid bacteria

Data availability

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

References

  1. Shehata, H. R., Hassane, B. & Newmaster, S. G. Real-time PCR methods for identification and stability monitoring of Bifidobacterium longum subsp. longum UABl-14 during shelf life. Front. Microbiol. 15, 1360241. https://doi.org/10.3389/fmicb.2024.1360241 (2024).

    Google Scholar 

  2. Khattab, R. A., Ahmed, N. A., Ragab, Y. M. & Rasmy, S. A. Bacteria producing antimicrobials against Clostridium difficile isolated from human stool. Anaerobe 63, 102206. https://doi.org/10.1016/j.anaerobe.2020.102206 (2020).

    Google Scholar 

  3. Kumar, S. et al. Evaluation of the techno-functional properties of lactobacilli strains originated from Bos indicus and Bubalus bubalis calves for probiotic potential. Int. Microbiol. 28, 1649–1668. https://doi.org/10.1007/s10123-025-00641-y (2025).

    Google Scholar 

  4. Feng, M. et al. Lactobacillus agilis SNF7 presents excellent antibacteria and anti-inflammation properties in mouse diarrhea induced by Escherichia coli. Int. J. Mol. Sci. 25, 13660. https://doi.org/10.3390/ijms252413660 (2024).

    Google Scholar 

  5. Yang, Y. et al. Understanding Ligilactobacillus salivarius from probiotic properties to omics technology: A review. Foods 13, 895. https://doi.org/10.3390/foods13060895 (2024).

    Google Scholar 

  6. de Freire, L. Limosilactobacillus fermentum strains as novel probiotic candidates to promote host health benefits and development of biotherapeutics: A comprehensive review. Probiotics Antimicrob. Proteins. 16, 1483–1498. https://doi.org/10.1007/s12602-024-10235-1 (2024).

    Google Scholar 

  7. Zhou, J. et al. Unveiling the potential of Lactobacillus Johnsonii in digestive diseases: a comprehensive review. Front. Microbiol. 16 https://doi.org/10.3389/fmicb.2025.1508382 (2025).

  8. Jiang, S., Cai, L., Lv, L. & Li, L. Pediococcus pentosaceus, a future additive or probiotic candidate. Microb. Cell. Fact. 20 https://doi.org/10.1186/s12934-021-01537-y (2021).

  9. Qi, Y. et al. Pediococcus pentosaceus: screening and application as probiotics in food processing. Front. Microbiol. 12, 762467. https://doi.org/10.3389/fmicb.2021.762467 (2021).

    Google Scholar 

  10. Kim, E., Yang, S. M., Kim, I. S. & Kim, H. Y. Identification of novel molecular targets for Weissella species-specific real-time PCR based on pangenome analysis. Appl. Microbiol. Biotechnol. 106, 4157–4168. https://doi.org/10.1007/s00253-022-12003-z (2022).

    Google Scholar 

  11. Kim, H. B. et al. Development of real-time PCR assay to specifically detect 22 Bifidobacterium species and subspecies using comparative genomics. Front. Microbiol. 11, 2087. https://doi.org/10.3389/fmicb.2020.02087 (2020).

    Google Scholar 

  12. Kim, E., Lee, G. Y., Yang, S. M. & Kim, H. Y. Rapid and accurate on-site identification of Lactobacillus delbrueckii subspecies in dairy products using direct polymerase chain reaction with microfluidic chip. Lwt 179, 114635. https://doi.org/10.1016/j.lwt.2023.114635 (2023).

    Google Scholar 

  13. Zamanpour, S., Eraghi, V., Hashemi, M., Ram, M. & Afshari, A. Optimizing real-time PCR for accurate identification and quantification of common probiotic bacteria in Iranian probiotic dairy products. Indian J. Microbiol. 65, 1891–1905. https://doi.org/10.1007/s12088-024-01325-3 (2024).

    Google Scholar 

  14. Kim, E., Yang, S. M., Kim, D. & Kim, H. Y. Real-time PCR method for qualitative and quantitative detection of Lactobacillus sakei group species targeting novel markers based on bioinformatics analysis. Int. J. Food Microbiol. 355, 109335. https://doi.org/10.1016/j.ijfoodmicro.2021.109335 (2021).

    Google Scholar 

  15. Kim, E., Kim, D. S., Yang, S. M. & Kim, H. Y. The accurate identification and quantification of six Enterococcus species using quantitative polymerase chain reaction based novel DNA markers. Lwt 166, 113769. https://doi.org/10.1016/j.lwt.2022.113769 (2022).

    Google Scholar 

  16. Gaspar, C. et al. Development and validation of a new one step multiplex-PCR assay for the detection of ten Lactobacillus species. Anaerobe 59, 192–200. https://doi.org/10.1016/j.anaerobe.2019.06.004 (2019).

    Google Scholar 

  17. Kim, E. et al. Design of PCR assays to specifically detect and identify 37 Lactobacillus species in a single 96 well plate. BMC Microbiol. 20, 96. https://doi.org/10.1186/s12866-020-01781-z (2020).

    Google Scholar 

  18. Applied Biosystems. Thermofisher Primer Express Version 3.0 Getting Started Guide. https://documents.thermofisher.com/TFS-Assets/LSG/manuals/cms_041902.pdf (Accessed 28 Sept 2025).

  19. Benchling, P. Design. https://test.benchling.com/primer-design-for-pcr (Accessed 28 Sept 2025).

  20. Premier Biosoft. PCR Primer Design Guidelines. http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html (Accessed 28 Sept 2025).

  21. Kralik, P. & Ricchi, M. A. Basic guide to real time PCR in microbial diagnostics: Definitions, parameters, and everything. Front. Microbiol. 8, 108. https://doi.org/10.3389/fmicb.2017.00108 (2017).

    Google Scholar 

  22. Xiang, X. et al. Rapid identification of novel specific molecular targets for PCR detection of four Enterococcus species. Lwt 173, 114356. https://doi.org/10.1016/j.lwt.2022.114356 (2023).

    Google Scholar 

  23. Shehata, H. R. et al. Guidelines for validation of qualitative real-time PCR methods for molecular diagnostic identification of probiotics. J. AOAC Int. 102, 1774–1778. https://doi.org/10.5740/jaoacint.18-0320 (2019).

    Google Scholar 

  24. Svec, D., Tichopad, A., Novosadova, V., Pfaffl, M. W. & Kubista, M. How good is a PCR efficiency estimate: recommendations for precise and robust qPCR efficiency assessments. Biomol. Detect. Quantification. 3, 9–16. https://doi.org/10.1016/j.bdq.2015.01.005 (2015).

    Google Scholar 

  25. Dung, T. T. N. et al. Development and validation of multiplex real-time PCR for simultaneous detection of six bacterial pathogens causing lower respiratory tract infections and antimicrobial resistance genes. BMC Infect. Dis. 24, 164. https://doi.org/10.1186/s12879-024-09028-2 (2024).

    Google Scholar 

  26. Downs, S. L., Madhi, S. A., van der Merwe, L., Nunes, M. C. & Olwagen, C. P. Optimization of a high-throughput nanofluidic real-time PCR to detect and quantify of 15 bacterial species and 92 Streptococcus pneumoniae serotypes. Sci. Rep. 13, 4588. https://doi.org/10.1038/s41598-023-31820-4 (2023).

    Google Scholar 

  27. Broeders, S. et al. Guidelines for validation of qualitative real-time PCR methods. Trends Food Sci. Technol. 37, 115–126. https://doi.org/10.1016/j.tifs.2014.03.008 (2014).

    Google Scholar 

Download references

Funding

This research was supported by Key Scientific Research Platforms and Projects for Universities in Guangdong Province (Grant Number: 2024GCZX011), Youth Science Fund Project of National Natural Science Foundation of China (Grant Number: 32502380), Guangdong Provincial Key Discipline Construction Program for Enhancing Research Capacity (Grant Number: 2024ZDJS089), Foshan Self-Financed Science and Technology Innovation Project (Grant Number: 2420001004679), Guangzhou College of Technology and Business Scientific Research Project (Grant Number: KYYB202431), Practical Quality Engineering Construction Project of Guangzhou College of Technology and Business (Grant Number: SYKC2024042).

Author information

Authors and Affiliations

  1. Department of Food Quality and Safety, School of Engineering, Guangzhou College of Technology and Business, Guangzhou, China

    Shao-Ji Li, Kaidi Luo, Shiting He, Wanqiong Lu, Keqing Xu, Xintong Zhu, Xiaoyan Sun, Yuxuan Xie, Lu Han, Yin Zheng, Lei Qian, Shiyi Ou & Chunmin Yang

  2. College of Food Science and Engineering, Foshan University, Foshan, China

    Biyan Cui

  3. College of Food Science and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou, China

    Wenhua Li

  4. College of Resources and Environment, Zhongkai University of Agriculture and Engineering, Guangzhou, China

    Fuqing Lu

  5. College of Food Science and Technology, Guangdong Ocean University, Zhanjiang, China

    Lirong Yuan

  6. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China

    Guangzhi Zhang

  7. Department of Food and Engineering, Jinan University, Guangzhou, China

    Shiyi Ou

Authors

  1. Shao-Ji Li
  2. Biyan Cui
  3. Wenhua Li
  4. Fuqing Lu
  5. Lirong Yuan
  6. Kaidi Luo
  7. Shiting He
  8. Wanqiong Lu
  9. Keqing Xu
  10. Xintong Zhu
  11. Xiaoyan Sun
  12. Yuxuan Xie
  13. Lu Han
  14. Yin Zheng
  15. Lei Qian
  16. Shiyi Ou
  17. Guangzhi Zhang
  18. Chunmin Yang

Contributions

Conceptualization, S.-J. Li, C. Yang, G. Zhang and S. Ou; methodology, S.-J. Li and G. Zhang; software, S.-J. Li; validation, B. Cui, W. Li, F. Lu and L. Yuan; formal analysis, L. Han, Y. Xie, X. Sun, Y. Zheng and L. Qian; investigation, K. Luo, S. He, W. Lu, K. Xu, X. Zhu; resources, S.-J. Li, Y. Zheng, and G. Zhang.; data curation, S.-J. Li; writing—original draft preparation, S.-J. Li; writing—review and editing, L. Han, Y. Xie, X. Sun, Y. Zheng and L. Qian; visualization, S.-J. Li; supervision, S.-J. Li; project administration, L. Han, Y. Xie, Y. Zheng, C. Yang and S. Ou; funding acquisition, L. Han, Y. Xie, Y. Zheng, C. Yang and S. Ou.

Corresponding authors

Correspondence to Shao-Ji Li, Guangzhi Zhang or Chunmin Yang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, SJ., Cui, B., Li, W. et al. Development and preliminary evaluation of real-time PCR assays for six lactic acid bacteria. Sci Rep (2026). https://doi.org/10.1038/s41598-026-37047-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41598-026-37047-3

Keywords