Difference between revisions of "Part:BBa K4613206"

Latest revision as of 14:28, 12 October 2023

Molecular Beacon

Molecular Beacon usually formed a double fluorophore-labeled and quencher-labeled dual-strand structure. Through DNA hybridization, resulting in F-Q proximity and concomitant fluorescence quenching of the fluorophore. When the target specifically binds to fluorophore (F), quencher (Q) is released and the fluorescence intensity increases. We chose a single-strand oligonucleotide, which combined with a fluorophore (F) and a quencher (Q), as our molecular beacon. When the rolling circle amplification (RCA) reaction was triggered and the long single DNA strands with repeated sequences were produced, the molecular beacon would hybridize with the RCA product and molecular beacon probes open, resulting in a target-specific RCA-amplified increase of fluorescence intensity. As shown in Fig. 2, the fluorescence intensity rises with the increase of concentrations of OTA. Europe and China have established a maximum allowable limit of 2 μg/L for OTAs. In our experiments, 2 μg/L (5000 pM/L) OTA had a significant variation in florescence intensity compared to the blank control. MBs of different diameters gave a different performance in detection. MBs of 1 μm in diameter showed a more rapid and obvious upward trends than MBs of 300 nm in diameter.

Fig. 1 The principle of rolling circle amplification (RCA) induced molecular beacon probes open and increase of fluorescence intensity.

Fig. 2 The response of the method to OTA at varied concentrations (0 pM to 20000 pM). The fluorescence values were detected by quantitative PCR instrument.

Reference

1. Xu G, Zhao J, Yu H, et al. Structural insights into the mechanism of high-affinity binding of ochratoxin A by a DNA aptamer[J]. Journal of the American Chemical Society, 2022, 144(17): 7731-7740.
2. Zhang J, Lu Y, Gao W, et al. Structure-switching locked hairpin triggered rolling circle amplification for ochratoxin A (OTA) detection by ICP-MS[J]. Microchemical Journal, 2023, 186: 108365.

Sequence and Features