Optical system and implementation method for real-time detection of nucleic acid melting temperature

Abstract

The invention discloses an optical system and a realization method for real-time detection of nucleic acid melting temperature, wherein the system includes: an optical microscope; a light source is arranged on one side of the optical microscope, and an optical fiber spectrometer is connected to the other side through an optical fiber, and the optical fiber spectrometer is connected to a computer There is a substrate for curing the sample to be tested under the objective lens of the optical microscope, the substrate is fixed on a temperature control platform, and the sample is kept warm by the temperature control platform; one side of the temperature control platform is provided with a pairing for injection on the substrate The microflow pump of the sample; the light emitted by the light source is irradiated on the sample to be tested and the substrate through the objective lens of the optical microscope, and the beams generated by the reflection of the light on the upper surface and the lower surface of the substrate form interference, and the interference light converges on the objective lens of the optical microscope First, after the conversion is transmitted to the fiber optic spectrometer through the optical fiber, the conversion information is transmitted to the computer, and the computer analyzes the conversion information and outputs spectral data, and the melting temperature of nucleic acid is obtained from the spectral data.

Description

technical field [0001] The invention relates to the fields of reflection interference spectroscopy, temperature control, microfluidic control and analysis, in particular to an optical system and a realization method for real-time detection of nucleic acid melting temperature. Background technique [0002] The optical correlation detection method has the advantages of fast speed, high sensitivity, and non-destructive detection of tiny samples, making it widely used in the detection of biomolecular interactions. Among them, the detection of DNA sequences and oligonucleotide fragments has been widely used in many fields such as medical diagnosis, biological research, food industry monitoring and environmental monitoring. However, the hybridization of nucleic acid molecules with different sequences has different melting temperatures, so accurately determining the Tm of oligonucleotide hybridization will be of more practical significance and practical value. [0003] At present,...

AI Technical Summary

Problems solved by technology

[0003] At present, there are reports at home and abroad on the use of optical means to analyze the melting temperature of nucleic acid. Darby’s research group obtained the melting temperature of DNA molecules of different strands by using fluorescent labeling. The disadvantage of this scheme is that it needs to DNA molecules are labeled, the experimental results are inaccurate, and the melting temperature of DNA molecules cannot be monitored in real time (Darby, R.A.J., et al., High throughput measurement of duplex, triplex and quadruplex melting curves using molecular beacons and a LightCycler.Nucleic AcidsResearch, 2002.30(9):p.e39); Robert Miller's research group can quickly detect and identify plant viruses by using surface plasmon resonance to analyze the melting temperature of different DNA probes. The disadvantage of this scheme is that the surface plasmon Bulk optical devices are sensitive to temperature, so the detection results are inaccurate and the anti-interference ability is poor (Zhou, J.C., et al., Immobilization-mediated reduction in melting temperatures of DNA–DNA and DNA–RNA hybrids: Immobilized DNA probe hybridization studied by SPR .Colloids&Surfaces A Physicochemical&Engineering Aspects,2015.481:p.72-79); In addition, the Florian Prol research group used the optical device combined with the reflection interference spectroscopy system and the composite plate substrate to detect the melting temperature of DNA molecules. The disadvantage of this scheme is The stability of the biosensor chip is poor, and the detection results are not accurate ( F., et al., Label-free characterization of oligonucleotide hybridisation using reflectometric interference spectroscopy. Analytical & Bioanalytical Chemistry, 2005.382(8): p.1889-1894)

In the detection of the melting temperature of nucleic acid, the above-mentioned optical method needs to mark the sample, has poor anti-interference ability, and cannot perform real-time, fast and accurate detection of the melting temperature of nucleic acid.

Images