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Aptamer-based detection of Salmonella enteritidis using double signal amplification by Klenow fragment and dual fluorescence

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Abstract

This article describes a sensitive and selective fluorometric method for the determination of Salmonella enteritidis by exploiting the polymerase activity of the Klenow fragment and dual fluorescence. First, one end of a target-selective aptamer was labeled with the fluorophore 6-carboxyfluorescein (FAM). Once the labeled aptamer binds to graphene oxide (GO) via π-stacking interaction, the fluorescence of FAM is quenched. However, the addition of target (16S rRNA) leads to the restoration of fluorescence due to the binding of probe and target which shifts the FAM fluorophore away from the quenching GO. By using the Klenow fragment and by exploiting the synergistic effect of FAM and the DNA probe SYBR Green I (which is strongly fluorescent in presence of dsDNA only), fluorescence is strongly amplified and sensitivity improved. The analyte 16SrRNA can be determined by this method in the 60 pM to 100 nM concentration range, and the detection limit is 60 pM. It is also shown that Salmonella enteritidis can be determined in milk samples by this method in concentrations between 102 to 105 cfu⋅mL‾1, with a detection limit of 300 cfu⋅mL‾1. This assay displays high sensitivity and selectivity and may possess wide applications in pathogen detection.

If labeled aptamer against Salmonella enteritidis 16S rRNA binds to graphene oxide GO) by π-interaction, the fluorescence of the label is quenched. The addition of target rRNA leads to the restoration of fluorescence, and this effect can be used to quantify Salmonella enteritidis.

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References

  1. Rodrigue DC, Tauxe RV, Rowe B (1990) International increase in salmonella enteritidis: a new pandemic? Epidemiol Infect 105:21–27

    Article  CAS  Google Scholar 

  2. Pui CF, Wong WC, Chai LC, Tunung R, Jeyaletchumi P, Noor Hidayah MS, Ubong A, Farinazleen MG, Cheah YK, Son R (2011) Salmonella: a foodborne pathogen. International Food Research Journal 18:465–473

    Google Scholar 

  3. Herikstad H, Motarjemi Y, Tauxe RV (2002) Salmonella surveillance: a global survey of public health serotyping. Epidemiol Infect 129:1–8

    CAS  Google Scholar 

  4. Allen MJ, Edberg SC, Reasoner DJ (2004) Heterotrophic plate count bacteria-what is their significance in drinking water? Int J Food Microbiol 92(3):265–274

    Article  Google Scholar 

  5. Uyttendaele M, Vanwildemeersch K, Debevere J (2003) Evaluation of real-time PCR vs automated ELISA and a conventional culture method using a semi-solid medium for detection of salmonella. Lett Appl Microbiol 37:386–391

    Article  CAS  Google Scholar 

  6. Martin B, Garriga M, Aymerich T (2012) Pre-PCR treatments as a key factor on the probability of detection of Listeria monocytogenes and salmonella in readyto-eat meat products by real-time PCR. Food Control 27(1):163–169

    Article  CAS  Google Scholar 

  7. Sanvicens N, Pastells C, Pascual N, Marco MP (2009) Nanoparticle-based aptasensors for detection of pathogenic bacteria. Trends Anal Chem 28(11):1243–1252

    Article  CAS  Google Scholar 

  8. Sanvicens N, Pascual N, Fernández-Argüelles MT, Adrián J, Costa-Fernández JM, Sánchez-Baeza F, et al. (2011) Quantum dot-based array for sensitive detection of Escherichia coli. Anal Bioanal Chem 399(8):2755–2762

    Article  CAS  Google Scholar 

  9. Lungu B, Waltman WD, Berghaus RD, Hofacre CL (2012) Comparison of a real-time PCR method with a culture method for the detection of Salmonella enterica serotype enteritidis in naturally contaminated environmental samples from integrated poultry houses. J Food Prot Apr. 75(4):743–747. doi:10.4315/0362-028X.JFP-11-297

  10. Sokolov DM, Sokolov MS (2013) Rapid methods for the genus salmonella bacteria detection in food and raw materials. Vopr Pitan 82:33–40

    CAS  Google Scholar 

  11. Zhi Y, Sasai D, Okubo Y, Shinozaki M, Nakayama H, Yamagata Murayama S, Wakayama M, Ide T, Zhang Z, Shibuya K (2013) Comparison between the effectiveness of polymerase chain reaction and in situ hybridization in detecting the presence of pathogenic fungi by using the preserved DNA in formalin-fixed and paraffin-embedded tissues. Jpn J Infect Dis 66:173–179

    Article  CAS  Google Scholar 

  12. Lei P, Tang H, Ding S, et al. (2015) Determination of the invA gene of salmonella using surface Plasmon resonance along with streptavidin aptamer amplification. Microchim Acta 182:289–296

    Article  CAS  Google Scholar 

  13. Jia F, Duan N, Wu S, Dai R, Wang Z, Li X (2015) Impedimetric salmonella aptasensor using a glassy carbon electrode modified with an electrodeposited composite consisting of reduced graphene oxide and carbon nanotubes. Microchim Acta. doi:10.1007/s00604-015-1649-7

    Google Scholar 

  14. Fei J, Dou W, Zhao G (2015) A sandwich electrochemical immunosensor for salmonella pullorum and salmonella gallinarum based on a screen-printed carbon electrode modified with an ionic liquid and electrodeposited gold nanoparticles. Microchim Acta. doi:10.1007/s00604-015-1573-x

    Google Scholar 

  15. Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925

    CAS  Google Scholar 

  16. Harvey I, Cormier Y, Beaulieu C, Akimov VN, Meriaux A, Duchaine C (2001) Random amplified ribosomal DNA restriction analysis for rapid identification of thermophilic actinomycete-like bacteria involved in hypersensitivity pneumonitis. Syst Appl Microbiol 24:277–284

    Article  CAS  Google Scholar 

  17. Robinson JT, Perkins FK, Snow ES, Wei ZQ, Sheehan PE (2008) Reduced graphene oxide molecular sensors. Nano Lett 8:3137–3140

    Article  CAS  Google Scholar 

  18. Wu J, Wang YS, Yang XY, Liu YY, Yang JR, Yang R, Zhang N (2012) Graphene oxide used as a carrier for Adriamycin can reverse drug resistance in breast cancercells. Nanotechnology 23:355101. doi:10.1088/0957-4484/23/35/355101

    Article  CAS  Google Scholar 

  19. Yin D, Li Y, Lin H, Guo B, Du Y, Li X, Jia H, Zhao X, Tang J, Zhang L (2013) Functional graphene oxide as a plasmid-based Stat3 siRNA carrier inhibits mouse malignant melanoma growth in vivo. Nanotechnology 24:105102. doi:10.1088/0957-4484/24/10/105102

    Article  CAS  Google Scholar 

  20. Pérez-López B, Merkoçi A (2012) Carbon nanotubes and graphene in analytical sciences. Microchim Acta 179:1–16

    Article  CAS  Google Scholar 

  21. Duan YF, Ning Y, Song Y, Deng L (2014) Fluorescent aptasensor for the determination of salmonella typhimurium based on a graphene oxide platform. Microchim Acta 181:647–653

    Article  CAS  Google Scholar 

  22. Xiang DS, Zhou GH, Luo M, Ji XH, He ZK (2012) Dual color fluorescence quantitative detection of specific single-stranded DNA with molecular beacons and nucleic acid dye SYBR green I. Analyst 137:3787–3793

    Article  CAS  Google Scholar 

  23. Cordeiro M, Giestas L, Lima JC, Baptista PJ (2013) Coupling an universal primer to SBE combined spectral codification strategy for single nucleotide polymorphism analysis. Biotechnol. 168(1):90–94

    CAS  Google Scholar 

  24. Singer VL, Lawlor TE, Yue S (1999) Comparison of SYBR green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the salmonella/mammalian microsome reverse mutation assay (ames test). Mutat Res 439(1):37–47

    Article  CAS  Google Scholar 

  25. Xiang D, Zhai K, Xiang W, Wang L (2014) Highly sensitive fluorescence quantitative detection of specific DNA sequences with molecular beacons and nucleic acid dye SYBR green I. Talanta 129:249–253

    Article  CAS  Google Scholar 

  26. Song Y, Li WK, Duan YF, Li ZJ, Deng L (2014) Nicking enzyme-assisted aptasensor for salmonella enteritidis detection based on fluorescence resonance energy transfer. Biosens Bioelectron 55:400–404

    Article  CAS  Google Scholar 

  27. Li S, Li Y, Chen H, Horikawa S, Shen W, Simonian A, Chin BA (2010) Direct detection of salmonella typhimurium on fresh produce using phage-based magnetoelastic aptasensors. Biosens Bioelectron 26:1313–1319

    Article  CAS  Google Scholar 

  28. Ohk S-H, Bhunia AK (2013) Multiplex fiber optic aptasensor for detection of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica from ready-to-eat meat samples. Food Microbiol 33:166–171

    Article  CAS  Google Scholar 

  29. Duan N, Wu S, Yu Y, Ma X, Xia Y, Chen X, Huang Y, Wang Z (2013) A dual-color flow cytometry protocol for the simultaneous detection of vibrio parahaemolyticus and salmonella typhimurium using aptamer conjugated quantum dots as labels. Anal Chim Acta 804:151–158

    Article  CAS  Google Scholar 

  30. Wu X, Xu C, Tripp RA, Huang YW, Zhao Y (2013) Detection and differentiation of foodborne pathogenic bacteria in mung bean sprouts using field deployable label-free SERS devices. Analyst 138:3005–3012

    Article  CAS  Google Scholar 

  31. Wu WH, Li M, Wang Y, Ouyang HX, Wang L, Li CX, Cao YC, Meng QH, Lu JX (2012) Aptasensors for rapid detection of Escherichia coli O157:H7 and salmonella typhimurium. Nanoscale Res Lett 7:658

    Article  CAS  Google Scholar 

  32. Nguyen P-D, Tran TB, Nguyen DTX, et al. (2014) Magnetic silica nanotube-assisted impedimetric immunosensor for the separation and label-free detection of salmonella typhimurium. Sensors Actuators B Chem 197:314–320

    Article  CAS  Google Scholar 

  33. Wu W, Li J, Pan D, Li J, Song S, RongM LZ, Gao J, Lu J (2014) Gold nanoparticle-based enzyme-linked antibody-aptamer sandwich assay for detection of salmonella typhimurium. ACS Appl Mater Interfaces 6(19):16974–16981

    Article  CAS  Google Scholar 

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Acknowledgments

This work was financially supported by the National Natural Science Foundation (81271660), Doctoral Program of Higher Education from the Ministry of Education (20114306110006).

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Authors and Affiliations

  1. The Co-construction Laboratory of Microbial Molecular Biology of Province and Ministry of Science and Technology, College of Life Science, Hunan Normal University, Changsha, Hunan, 410081, China

    Keyi Liu, Xing Yan, Biyao Mao, Sheng Wang & Le Deng

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  1. Keyi Liu
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  2. Xing Yan
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  3. Biyao Mao
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  4. Sheng Wang
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  5. Le Deng
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Correspondence to Le Deng.

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Liu, K., Yan, X., Mao, B. et al. Aptamer-based detection of Salmonella enteritidis using double signal amplification by Klenow fragment and dual fluorescence. Microchim Acta 183, 643–649 (2016). https://doi.org/10.1007/s00604-015-1692-4

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  • DOI: https://doi.org/10.1007/s00604-015-1692-4

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