Rolling circle amplification based colorimetric determination of Staphylococcus aureus
- PDF / 1,712,782 Bytes
- 10 Pages / 595.276 x 790.866 pts Page_size
- 90 Downloads / 263 Views
ORIGINAL PAPER
Rolling circle amplification based colorimetric determination of Staphylococcus aureus Yanan Li 1 & Junying Wang 2 & Shuo Wang 3 & Junping Wang 1 Received: 29 June 2019 / Accepted: 10 December 2019 # Springer-Verlag GmbH Austria, part of Springer Nature 2020
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. Keywords Rolling circle amplification (RCA) . Colorimetric microplate assay . Multifunctional gold nanoparticles . Biotin– streptavidin system . Staphylococcus aureus
Introduction Staphylococcus aureus (S. aureus) can be found in many foods and can cause food poisoning, skin infections and even sepsis, and being considered as an important food-borne Junying Wang contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00604-019-4082-5) contains supplementary material, which is available to authorized users. * Shuo Wang [email protected] * Junping Wang [email protected] 1
State Key Laboratory for Food Nutrition and Safety, College of Food Science and Engineering, Tianjin University of Science and Technology, 29 The Thirteenth Road, Tianjin Economy and Technology Development Area, Tianjin 300457, People’s Republic of China
2
Chinese Academy of Agricultural Sciences, Biotechnology Research Institute, Haidian District, Beijing 010010, People’s Republic of China
3
Medical college, Nankai University, No.38 Tongyan Road, Jinnan District, Tianjin 300350, People’s Republic of China
pathogen [1–5]. The gold standard for determination of S. aureus is direct microscopic examination and culture, but it requires 4–7 days and is tedious. It is necessary to develop quickly and accurately method for determination of S. aureus, which will be helpful to control pathogens [6]. Some rapid methods have been developed, among which the molecular recognition method is the most important. Nucleic acid amplification plays a major role in the determination of bacterial pathogens [7], many methods of nucleic acid amplification have been developed such as nested-
Data Loading...
