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Rolling circle amplification based colorimetric determination of Staphylococcus aureus

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Abstract

A colorimetric microplate assay for determination of Staphylococcus aureus DNA is described. Linear padlock probes were designed to recognize target sequences. After DNA binding, the linear padlock probes were circularized by ligation and then hybridize with biotin-labeled capture probes. Biotin-labeled capture probes act as primers to initiate the RCA. The biotin-labeled RCA products hybridize with digoxin-labeled signal probes fixed on streptavidin-functionalized wells of a 96-well plate. To enhance sensitivity, an AuNP-anti-digoxigenin-POx-HRP conjugate was added to the wells and then bound to digoxin-labeled signalling probes. The oxidation of tetramethylbenzidine (TMB) by H2O2 produces a color change from colorless to blue via HRP catalysis. After the reaction was terminated, absorbance is measured at 450 nm. For target sequences of Staphylococcus aureus, the detection limit is 1.2 pM. For genomic DNA, the detection limit is 7.4 pg.μL−1. The potential application of the method was verified by analyzing spiked food samples.

Schematic representation of rolling circle amplification and functionalized AuNP-based colorimetric determination of Staphylococcus aureus. The method uses streptavidin-functionalized 96-well plates and RCA as a molecular tool and AuNP-anti-digoxigenin-POx-HRP as signal transduction markers to increase sensitivity.

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Acknowledgements

The authors are grateful for providing language help from the American Journal Experts.

Funding information

This work was supported by “The National Key R&D Program of China” (No.2016YFD0401202), Special Project of Tianjin Innovation Platform (No.17PTGCCX00230), Tianjin science and technology planning project(No. 18PTSYJC00130).

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Correspondence to Shuo Wang or Junping Wang.

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Li, Y., Wang, J., Wang, S. et al. Rolling circle amplification based colorimetric determination of Staphylococcus aureus. Microchim Acta 187, 119 (2020). https://doi.org/10.1007/s00604-019-4082-5

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Keywords

  • Rolling circle amplification (RCA)
  • Colorimetric microplate assay
  • Multifunctional gold nanoparticles
  • Biotin–streptavidin system
  • Staphylococcus aureus