Abstract
［Objective］ To develop a novel fluorescence polarization (FP) detection strategy and achieve highly sensitive Salmonella detection by incorporating recombinase polymerase amplification (RPA ) technology.［Methods］ The detection mechanism relies on homologous sequence recognition between target DNA and specific primers:When Salmonella targets exist,primer and target DNA binding triggers strand displacement and initiates DNA amplification.To enhance sensitivity,two key design features are implemented:5'-end modification of primers with 6-carboxyfluorescein (6-FAM ) as a fluorescent reporter,and selective digestion of unbound primers by exonuclease Ⅰ (Exo Ⅰ).［Results］］ During RPA amplification,the substantially increased molecular weight of amplicons restricts the rotational freedom of 6-FAM -labeled DNA compounds,resulting in significantly enhanced FP signals.After optimization,this method achieves a detection limit of 11 CFU/mL for Salmonella with excellent specificity.［Conclusion］ This method provides an efficient Salmonella detection.Its modular design principle can also be readily adapted for rapid diagnosis of other foodborne pathogens.
Publication Date
7-3-2025
First Page
44
Last Page
50
DOI
10.13652/j.spjx.1003.5788.2025.80343
Recommended Citation
Pengpeng, XUE; Wei, CHEN; Jianguo, XU; Jia, TU; and Wei, QU
(2025)
"Salmonella detection based on fluorescence polarization enhanced by recombinase polymerase amplification,"
Food and Machinery: Vol. 41:
Iss.
6, Article 6.
DOI: 10.13652/j.spjx.1003.5788.2025.80343
Available at:
https://www.ifoodmm.cn/journal/vol41/iss6/6
References
[1] GALLO M,FERRARA L,CALOGERO A,et al.Relationships between food and diseases:what to know to ensure food safety[J].Food Research International,2020,137:109414.
[2] HAMAIDEH S,OLAIMAT A,AL-HOLY M,et al.The influence of technological shifts in the food chain on the emergence of foodborne pathogens:an overview [J].Applied Microbiology,2024,4(2):594-606.
[3] SUNARTI L S.Bacterial contamination in food:sources,risks,and prevention strategies [J].International Journal of Pathogen Research,2024,13(6):90-100.
[4] LI C,CHEN X,WEN R Q,et al.Immunocapture magnetic beads enhanced the LAMP-CRISPR/Cas 12a method for the sensitive,specific,and visual detection of Campylobacter jejuni[J].Biosensors,2022,12(3):154.
[5] 费鹏,赵胜娟,姜亦超,等.东北市售鸡肉中空肠弯曲杆菌的分离鉴定、分型及耐药性分析 [J].食品与机械,2018,34(6):46-49.FEI P,ZHAO S J,JIANG Y C,et al.Isolation,identification,multilocus sequence typing analysis and antimicrobial resistance of Campylobacter jejuni isolated from commercial chicken in northeast China [J].Food & Machinery,2018,34(6):46-49.
[6] HAZEN T H,MICHALSKI J M,TENNANT S M,et al.Genomic diversity of non-diarrheagenic fecal Escherichia coli from children in sub-Saharan Africa and south Asia and their relatedness to diarrheagenic E.coli [J].Nature Communications,2023,14(1):1 400.
[7] 曹文凯,山珊,刘道峰,等.基于 dsDNA-CuNCs 的荧光 ELISA检测牛奶中的 E.coli O157:H7[J].食品与机械,2023,39(9):38-43,94.CAO W K,SHAN S,LIU D F,et al.A fluorescence ELISA based on dsDNA-CuNCs for the detection of E.coli O157:H7 in milk [J].Food & Machinery,2023,39(9):38-43,94.
[8] HOWDEN B P,GIULIERI S G,WONG FOK LUNG T,et al.Staphylococcus aureus host interactions and adaptation [J].Nature Reviews Microbiology,2023,21(6):380-395.
[9] 陈小敏,黄泽璇,谭礼浩,等.表没食子儿茶素没食子酸酯对食源性致病菌抑菌机理及应用研究 [J].食品与机械,2024,40(8):127-134.CHEN X M,HUANG Z X,TAN L H,et al.Study on the inhibitory mechanism of epigallocatechin gallate against foodborne pathogens and its application [J].Food & Machinery,2024,40(8):127-134.
[10] PENG Y B,XUE P P,WANG R J,et al.Engineering of an adaptive tandem CRISPR/Cas 12a molecular amplifier permits robust analysis of Vibrio parahaemolyticus [J].Talanta,2024,266:125061.
[11] 于晨,杨露,管峰,等.抑制副溶血性弧菌的乳酸菌筛选鉴定及其生物学特性研究 [J].食品与机械,2023,39(6):12-18.YU C,YANG L,GUAN F,et al.Screening,identification and biological characteristics of lactic acid bacteria inhibiting Vibrio parahaemolyticus [J].Food & Machinery,2023,39(6):12-18.
[12] XU J G,ZHANG X L,YAN C,et al.Trigging isothermal circular amplification-based tuning of rigorous fluorescence quenching into complete restoration on a multivalent aptamer probe enables ultrasensitive detection of Salmonella [J].Analytical Chemistry,2022,94(2):1 357-1 364.
[13] ZHANG X L,PENG Y B,YAO L,et al.Self-assembly of multivalent aptamer-tethered DNA monolayers dedicated to a fluorescence polarization-responsive circular isothermal strand displacement amplification for Salmonella assay [J].Analytical Chemistry,2023,95(4):2 570-2 578.
[14] SCHLECH W F.Epidemiology and clinical manifestations of Listeria monocytogenes infection [J].Microbiology Spectrum,2019,7(3):2 018.
[15] REYNOLDS D,KOLLEF M.The epidemiology and pathogenesis and treatment of Pseudomonas aeruginosa infections:an update [J].Drugs,2021,81(18):2 117-2 131.
[16] 王芳妹,徐越,林丹,等.包装饮用水中铜绿假单胞菌实时荧光定量 PCR快速检测 [J].食品与机械,2023,39(6):75-80.WANG F M,XU Y,LIN D,et al.Study on the rapid detection of Pseudomonas aeruginosa by real-time fluorescence quantitative PCR in packaged drinking water [J].Food & Machinery,2023,39(6):75-80.
[17] EHUWA O,JAISWAL A K,JAISWAL S.Salmonella,food safety and food handling practices [J].Foods,2021,10(5):907.
[18] POPA G L,POPA M I.Salmonella spp.infection –a continuous threat worldwide [J].Germs,2021,11(1):88-96.
[19] KUEHN B.Multidrug-resistant salmonella [J].Jama,2019,322(14):1 344.
[20] KAAVYA R,PANDISELVAM R,ABDULLAH S,et al.Emerging non-thermal technologies for decontamination of Salmonella in food [J].Trends in Food Science & Technology,2021,112:400-418.
[21] GILCHRIST J J,MACLENNAN C A.Invasive nontyphoidal Salmonella disease in Africa [J].EcoSal Plus,2019,8(2):1-23.
[22] 宋晟,高晗,严礼,等.生鲜畜禽肉中沙门氏菌全基因组分析与分子溯源 [J].食品与机械,2020,36(9):55-62.SONG S,GAO H,YAN L,et al.Whole genome analysis and molecular traceability study of Salmonellain fresh livestock and poultry meat [J].Food & Machinery,2020,36(9):55-62.
[23] BONNET M,LAGIER J C,RAOULT D,et al.Bacterial culture through selective and non-selective conditions:the evolution of culture media in clinical microbiology [J].New Microbes and New Infections,2020,34:100622.
[24] ZHU H L,ZHANG H Q,XU Y,et al.PCR past,present and future [J].BioTechniques,2020,69(4):317-325.
[25] JOSHI M,DESHPANDE J D.Polymerase chain reaction:methods,principles and application [J].International Journal of Biomedical Research,2011,2(1):81-97.
[26] XU L L,DUAN J X,CHEN J M,et al.Recent advances in rolling circle amplification-based biosensing strategies-a review [J].Analytica Chimica Acta,2021,1148:238187.
[27] YAN C,CUI J,HUANG L,et al.Rapid and visual detection of 2019 novel coronavirus (SARS-CoV- 2) by a reverse transcription loop-mediated isothermal amplification assay [J].Clinical Microbiology and Infection,2020,26(6):773-779.
[28] LOBATO I M,O'SULLIVAN C K.Recombinase polymerase amplification:basics,applications and recent advances [J].TrAC Trends in Analytical Chemistry,2018,98:19-35.
[29] TOMAR S,LAVICKOVA B,GUIDUCCI C.Recombinase polymerase amplification in minimally buffered conditions [J].Biosensors and Bioelectronics,2022,198:113802.
[30] LI J,MACDONALD J,VON STETTEN F.Review:a comprehensive summary of a decade development of the recombinase polymerase amplification [J].The Analyst,2018,144(1):31-67.
[31] LIU Y J,LIU H,YU G L,et al.One-tube RPA-CRISPR Cas12a/Cas 13a rapid detection of methicillin-resistant Staphylococcus aureus [J].Analytica Chimica Acta,2023,1 278:341757.
[32] JI T,ZHANG J L,GAO Y Z,et al.A rapid and visual detection of Staphylococcus haemolyticus in clinical specimens with RPA-LFS [J].Analytica Chimica Acta,2023,1 273:341534.
[33] LUAN H R,WANG S J,JU L,et al.KP177R-based visual assay integrating RPA and CRISPR/ Cas12a for the detection of African swine fever virus [J].Frontiers in Immunology,2024,15:1358960.
[34] ZHAO C J,YANG L H,ZHANG X,et al.Rapid and sensitive genotyping of SARS-CoV- 2 key mutation L 452R with an RPA- Pf ago method [J].Analytical Chemistry,2022,94(49):17 151-17 159.
[35] ZHANG H Y,YANG S P,DE RUYCK K,et al.Fluorescence polarization assays for chemical contaminants in food and environmental analyses [J].TrAC Trends in Analytical Chemistry,2019,114:293-313.
[36] FANG X,WANG X Y,LI Y X,et al.Fluorescence detection of trace disinfection byproducts by Ag nanoprism-modulated lanthanide MOFs [J].Analytical Chemistry,2023,95(4):2 436-2 444.
[37] AFZALINIA A,MIRZAEE M.Ultrasensitive fluorescent miRNA biosensor based on a "sandwich" oligonucleotide hybridization and fluorescence resonance energy transfer process using an ln (III)-MOF and Ag nanoparticles for early cancer diagnosis:application of central composite design [J].ACS Applied Materials & Interfaces,2020,12(14):16 076-16 087.
[38] ZHAO Q,TAO J,UPPAL J S,et al.Nucleic acid aptamers improving fluorescence anisotropy and fluorescence polarization assays for small molecules [J].TrAC Trends in Analytical Chemistry,2019,110:401-409.