Abstract
Salmonella spp. can cause animal and human salmonellosis. In this study, we established a simple method to detect all Salmonella species by amplifying a specific region within the flgE gene encoding the flagellar hook protein. Our preliminary sequence analysis among flagella-associated genes of Salmonella revealed that although Salmonella Gallinarum and Salmonella Pullorum are lacking flagella, they did have flagella-associated genes, including flgE. To investigate in detail, a comparative flgE sequence analysis was conducted using different bacterial strains including flagellated and non-flagellated Salmonella as well as non-Salmonella strains. Two unique regions (481–529 bp and 721–775 bp of the reference sequence) within the flgE open reading frame were found to be highly conserved and specific to all Salmonella species. Next, we designed a pair of PCR primers (flgE-UP and flgE-LO) targeting the above two regions, and performed a flgE-tailored PCR using as template DNA prepared from a total of 76 bacterial strains (31 flagellated Salmonella strains, 26 non-flagellated Salmonella strains, and 19 other non-Salmonella bacteria strains). Results showed that specific positive bands with expected size were obtained from all Salmonella (including flagellated and non-flagellated Salmonella) strains, while no specific product was generated from non-Salmonella bacterial strains. PCR products from the positive bands were confirmed by DNA sequencing. The minimum detection amount for genomic DNA and bacteria cells reached 18.3 pg/μL and 100 colony-forming unit (CFU) per PCR reaction, respectively. Using the flgE-PCR method to detect Salmonella in artificially contaminated milk samples, as low as 1 CFU/mL Salmonella was detectable after an 8-h pre-culture. Meanwhile, the flgE-tailored PCR method was applied to evaluate 247 clinical samples infected with Salmonella from different chicken breeding farms. The detection results indicated that flgE-PCR could be used to specifically detect Salmonella in concordance with the traditional bacterial culture-based detection method. It is worthwhile noticed that identification results using flgE-tailored PCR should be completed within less than 1 day, expanding the result of much faster than the standard method, which took more than 5 days. Overall, the flgE-tailored PCR method can specifically detect flagellated and non-flagellated Salmonella and can serve as a powerful tool for rapid, simple, and sensitive detection of Salmonella species.
Key points
• Targeting flgE gene for all Salmonella spp. found.
• The established PCR assay is used to specifically detect all Salmonella spp.
• The PCR method is applied to detect clinical Salmonella spp. samples within less than 1 day.
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Acknowledgments
The authors gratefully acknowledge Professor Xinan Jiao in Yangzhou University, Professor Chengping Lu in Nanjing Agricultural University, Professor Shulin Liu from Harbin Medical University and the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences for providing the bacteria strains for this study.
Funding
This work was supported by the grants from the no. 2016YFD0500905 and 2017YFD0500105 from the National Key Research and Development Program of China, and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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YY and PW performed the experiments, analyzed the data, and wrote the manuscript. PX, BY, PD, TH, and JL participated in the data analysis and wrote the paper. QS contributed to the experiments designing, manuscript writing, and language polishing. GZ and XM conceived and designed the study, participated in experimental work, and wrote the paper. All authors read and approved the final manuscript.
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Yang, Y., Wang, P., Xia, P. et al. Rapid detection of flagellated and non-flagellated Salmonella by targeting the common flagellar hook gene flgE. Appl Microbiol Biotechnol 104, 9719–9732 (2020). https://doi.org/10.1007/s00253-020-10925-0
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DOI: https://doi.org/10.1007/s00253-020-10925-0