A dual RPA-LFD assay for the simultaneous detection of Salmonella typhimurium and Salmonella enteritidis

Chuan Liao; Lele Pan; Meiying Tan; Zihan Zhou; Shaoping Long; Xueli Yi; Xuebin Li; Guijiang Wei; Lina Liang

doi:10.3389/fbioe.2024.1379939

A dual RPA-LFD assay for the simultaneous detection of Salmonella typhimurium and Salmonella enteritidis

Front Bioeng Biotechnol. 2024 Mar 8:12:1379939. doi: 10.3389/fbioe.2024.1379939. eCollection 2024.

Authors

Chuan Liao^{1

2

3}, Lele Pan^{1

2

3}, Meiying Tan^{1

2

3}, Zihan Zhou^{1

2

3}, Shaoping Long⁴, Xueli Yi^{1

2

3}, Xuebin Li^{5

6}, Guijiang Wei^{1

2

3

6}, Lina Liang^{1

2

3}

Affiliations

¹ Center for Medical Laboratory Science, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, China.
² Baise Key Laboratory for Research and Development on Clinical Molecular Diagnosis for High-Incidence Diseases, Baise, China.
³ Key Laboratory of Research on Clinical Molecular Diagnosis for High Incidence Diseases in Western Guangxi, Baise, China.
⁴ Department of Clinical Laboratory, Baise People's Hospital, Baise, China.
⁵ Department of Neurology, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, China.
⁶ Modern Industrial College of Biomedicine and Great Health, Youjiang Medical University for Nationalities, Baise, China.

Abstract

Introduction: Salmonella was one of the most common bacteria that caused foodborne illness, with S. typhimurium (Salmonella typhimurium) and S. enteritidis (Salmonella enteritidis) infections accounting for more than 75% of human salmonella infections. Methods: In this study, we developed a method of dual recombinase polymerase amplification (RPA) combined with a lateral flow dipstick for the rapid detection of S. typhimurium and S. enteritidis in clinical specimens (stool). Results: The entire reaction process, including amplification and result reading, could be completed within 65 min. The detection limits of S. typhimurium and S. enteritidis in pure culture samples were 5.23 × 10¹ CFU/mL and 3.59 × 10¹ CFU/mL, respectively. The detection limits of S. typhimurium and S. enteritidis in artificially contaminated samples were 8.30 × 10¹ CFU/mL and 2.70 × 10² CFU/mL, respectively. In addition, the method had no cross-reaction with other pathogenic microorganisms. The results in clinical samples were fully consistent with those obtained using Bacterial Analysis Manual, with sensitivity and specificity were 100% (8/8) and 100% (17/17) for S. typhimurium and 100% (4/4) and 100% (21/21) for S. enteritidis, respectively. Discussion: The detection limits of S. typhimurium and S. enteritidis in artificially contaminated samples were higher than those in pure culture samples, which might be attributed to the inherent complex composition of artificially contaminated samples. In addition, the detection limits of S. typhimurium and S. enteritidis in the same sample were also different, which might be attributed to different amplification efficiency of two target genes in the same reaction system. Conclusion: This assay had potential application outdoors, as it could be performed within 1 h at 38°C without a complex instrument, and the results could be observed with the naked eye. In conclusion, the dual RPA-LFD assay established in this study had practical significance for the rapid detection of S. typhimurium and S. enteritidis in the future.

Keywords: Salmonella enteritidis; Salmonella typhimurium; dual recombinase polymerase amplification; lateral flow dipstick; simultaneous detection.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. Our research was granted by Natural Science Foundation of China (82260418), Natural Science Foundation of Guangxi (2019JJB140034), First Batch of High-level Talent Scientific Research Projects of the Affiliated Hospital of Youjiang Medical University for Nationalities (R202011701) and Baise Scientific Research and Technology Development Project (20232031).