Nucleic acid liquid-phase hybridization capture and detection method based on magnetic separation and enzyme catalysis

Abstract

The invention relates to a method for carrying out magnetic separation capture after nucleic acid liquid-phase hybridization and carrying out signal amplification detection by utilizing a bio-enzyme catalysis substrate. The method comprises two core elements of magnetic separation and enzyme catalysis. According to the magnetic separation element, a double-stranded complex after nucleic acid hybridization is captured from a liquid phase under the action of magnetic field force through a probe connected to a magnetic microparticle, and specific enrichment is performed on a target nucleic acid sequence to be detected; according to the enzyme catalysis element, a signal is amplified through the bio-enzyme catalysis substrate connected to the probe, so as to judge whether the target nucleic acid sequence to be detected exists or not. The target nucleic acid sequence can be a PCR amplification product and can also be nucleic acid which is not amplified. One of the core values of the invention is that a full-automatic nucleic acid detection device is developed by matching with the method, so that seamless joint of full-automatic nucleic acid extraction and full-automatic nucleic acid detection can be realized, and complete full-automatic nucleic acid detection is realized.

Description

technical field [0001] The invention relates to a nucleic acid liquid-phase hybridization capture detection method. The method is realized by two core elements of magnetic separation and enzymatic catalysis. The magnetic separation is achieved by connecting the magnetic particles with the hybridization probe nucleic acid sequence. After the probe and the target nucleic acid sequence base are complementary to form a hybrid, magnetic The collection of microparticles achieves the purpose of selectively separating and enriching the target nucleic acid sequence from the liquid phase. At the same time, another or more nucleic acid probes that are complementary to other parts of the target nucleic acid sequence are connected to the biological enzyme. After hybridization, the biological enzyme and the target nucleic acid sequence are connected together, and the signal is transmitted through the enzyme-catalyzed substrate color development or luminescence. Amplification can achieve hi...

AI Technical Summary

Problems solved by technology

Due to the limited contact area between the microwell plate and the sample to be tested, the probability of intermolecular collisions is reduced, resulting in limited sensitivity

To make the reaction more complete, the reaction time needs to be prolonged, which affects the efficiency of detection

(2) Since microporous plates are usually a row of multi-well plates arranged in series, the operating cost of testing only one sample at a time is greatly increased. This type of testing usually needs to collect enough samples before performing unified testing. Poor timeliness from sample to implementation of testing

(3) Due to the need for the whole plate operation of the enzyme plate, once the operation is started, the sample arrangement is locked, and the random insertion test of the emergency position of the sample cannot be performed, and the flexibility of sample detection is poor

The detection sensitivity of this scheme is limited due to the low photon efficiency and short decay period of the fluorescent signal

Invention patent CN106755460A "A single-base mutation detection method" discloses a method for detecting single-base mutations in nucleic acids by hybridization capture. The technical path of probe separation and fluorescence signal output. The purpose of magnetic separation is to transfer the nucleic acid sequence to be tested from the liquid phase of the sample to the artificial buffer. The target nucleic acid sequence has been separated from the magnetic particles during detection. The solution also has the characteristic that the fluorescent signal decays and weakens with time, and at the same time, the magnetic particles are separated from the target nucleic acid sequence during detection, which limits the applicable scenarios of this technical solution

Patent CN107058584A "A Nucleic Acid Hybridization Chemiluminescence Detection Method" achieves nucleic acid hybridization and luminescence detection by linking capture probes to microspheres of organic substrates such as polystyrene. Although this scheme uses signal amplification technology at the signal end However, at the capture end, there is the defect that microspheres with organic matter as the capture matrix are inconveniently separated from the liquid phase, and it is also difficult to achieve fully automatic detection

Patent CN101351564B "Detecting Nucleic Acids by Target-Specific Hybridization Capture Method" provides a hybridization capture detection method similar to the CN107058584A carrier, the capture carrier used is still organic microspheres, and the signal output provides fluorescence, gold labeling There are three pathways catalyzed by substances and enzymes, and the separation of microspheres from the liquid phase is also inconvenient.

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