Self-primer Molecular Beacon For MiRNA Amplification Detection

Changes in micro RNA(miRNA)expression levels show a great effect on the initiation and progression of the disease.Studies have shown that the overexpression of miR-24 is related to the occurrence and poor prognosis of human oral cancer,and can be used as a marker of the occurrence and development of oral cancer for disease diagnosis,so its detection is of great significance.However,they are often present at low concentrations in complex physiological environments such as body fluids and cells,so their detection methods need to be highly sensitive and selective.The molecular beacon,an oligonucleotide sequence with a stem-loop structure,is marked with fluorescent and quenching groups at both ends respectively.It has the advantages of high specificity and low background and has been widely used in the analysis and detection of biological molecules.However,there are still problems such as insufficient sensitivity and susceptibility to being intervened by other substances when used for miRNA detection.This thesis addresses the problems in miRNA detection,designs the sequences of molecular beacons,constructs a novel molecular beacon,and develops a novel nucleic acid amplification method for miRNA detection by combining isothermal amplification and magnetic separation techniques.The details are as follows:(1)A nucleic acid amplification method for miR-24 detection was developed,which is based on the self-primer molecular beacon.In the natural state,the 3’ end of the self-primer molecular beacon probe has a partially suspended single strand,and the fluorescent and quenching groups in the corresponding position of the stem are close to each other,and fluorescence quenching occurs.When miR-24 is combined with the stem-loop region of the self-primer molecular beacon,the molecular beacon structure is opened and the fluorescence is recovered.At the same time,the suspended single strand can be hybridized with some regions of the same side sequence,and the intramolecular configuration conversion occurs to form a primer.With the existence of polymerase,an isothermal amplification reaction occurs,and the target is released.The hybridization and release cycle of miR-24 produced a large number of fluorescence recovery amplification products.This method avoided the use of exogenous primers,enabling reactions to be carried out at a constant temperature under simple and convenient conditions.The detection limit for miR-24 was as low as34.91 p M.And the structure of the self-primer molecular beacon was versatile and can be used for the detection of other nucleic acid molecules by changing the sequence of probes.(2)A magnetic bead-assisted self-primer molecular beacon nucleic acid amplification method was constructed to detect miR-24.The self-primer molecular beacon is coupled to the magnetic beads,which can be separated from the sample by a magnetic field after binding to the target.At the same time,intramolecular configuration conversion occurs.Under the action of strand displacement polymerase,an isothermal amplification reaction occurs to make the miR-24 cycle,resulting in a large number of amplification products of fluorescence recovery,and the fluorescence signal on the surface of the magnetic beads in the post-reaction samples is collected using flow cytometry.In this method,the target was captured by magnetic beads modified with self-primer molecular beacon probes,and miR-24 was separated from complex samples to realize the detection of miR-24 in serum samples.By combining the amplification method with the magnetic beads carrier,the local concentration for the probe was increased.And compared with the previous work,the detection limit of miR-24 was reduced to 0.95 p M,which improved the detection sensitivity.