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Microchimica Acta

, Volume 183, Issue 2, pp 643–649 | Cite as

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

  • Keyi Liu
  • Xing Yan
  • Biyao Mao
  • Sheng Wang
  • Le Deng
Original Paper

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.

Graphical Abstract:

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.

Keywords

FAM SYBR green I Graphene oxide Foodborne pathogen 16S rRNA 

Notes

Acknowledgments

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

Supplementary material

604_2015_1692_MOESM1_ESM.pdf (305 kb)
ESM 1 (PDF 305 kb)

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Copyright information

© Springer-Verlag Wien 2015

Authors and Affiliations

  • Keyi Liu
    • 1
  • Xing Yan
    • 1
  • Biyao Mao
    • 1
  • Sheng Wang
    • 1
  • Le Deng
    • 1
  1. 1.The Co-construction Laboratory of Microbial Molecular Biology of Province and Ministry of Science and Technology, College of Life ScienceHunan Normal UniversityChangshaChina

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