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Amplification-free nucleic acid detection method

A technology for amplifying nucleic acid and detection methods, which is applied in the field of amplification-free nucleic acid detection, can solve problems such as aerosol pollution, inability to solve specificity, and inability to stabilize detection signals, so as to improve efficiency, achieve super-specificity, and improve sensitivity Effect

Active Publication Date: 2021-08-06
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

like figure 1 As shown, Ligand / Target molecule is a ligand target molecule, Receptor is a receptor, Non-specific binding is a non-specific binding molecule, and specific binding is a specific binding molecule, which occurs when molecular hybridization or immune reaction reaches an equilibrium state. The number of bound molecules depends on the number of binding sites, sample concentration and affinity, so when the sample concentration is reduced to a certain extent, the number of bound molecules will be less than 1, even if the most advanced single-molecule detection technology is used, it will not be stable signal detected
In principle, PCR technology does not break through the limitation of molecular binding kinetics, nor can it solve the problem of specificity
Finally, based on the amplification strategy, there is another problem that cannot be ignored-aerosol pollution. The nucleic acid aerosol pollution generated by the high amplification efficiency of PCR technology will lead to false positive results, and samples and amplification products often appear in multiple batches. In the same situation, nucleic acid aerosol pollution continues to accumulate in the experimental area, which increases the risk of contamination and the frequency of false positives is also increasing

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0078] The base complementary chain length of the target nucleic acid and the detection molecule is 12base pair (the following detection 12bp) The non-amplification detection method of the target nucleic acid is as follows:

[0079] (1) Use a SPRM rebuilt based on a commercial total internal reflection microscope (Olympus IX-81) as a detection instrument, select an oil lens with a magnification of 100 times, and a numerical aperture N.A=1.49. The light source is an ultra-broadband light source SLED, and the light source controls the intensity of the current At 140mA, the field of view is 512×512pixels (full field of view: 33.28×33.28μm 2 ). A micro manager was used to control the CCD camera (Photometrics) to record the imaging signal of SPRi. Olympus microscope comes with Cell lens software to control the incident angle of the light path.

[0080] (2) On the BK-7 glass slide plated with 3nm chromium and 47nm gold, first wash and dry it with piranha lotion, and then treat it ...

Embodiment 2

[0085] The non-amplification detection method of the target nucleic acid with a base complementary chain length of 11 bp between the target nucleic acid and the detection molecule is as follows:

[0086] (1) Use a SPRM rebuilt based on a commercial total internal reflection microscope (Olympus IX-81) as a detection instrument, select an oil lens with a magnification of 60 times and a numerical aperture N.A=1.49, and the light source is an ultra-broadband light source SLED, and the light source controls the intensity of the current At 150mA, the field of view is 512×512pixels(full field of view:51.2×51.2μm 2 ). A micro manager was used to control the CCD camera (Photometrics) to record the imaging signal of SPRi. Cell lens software is used to control the incident angle of the light path.

[0087] (2) On the BK-7 glass slide coated with 2nm chromium and 47nm gold, first wash and dry it with alcohol and pure water, and then treat it with hydrogen flame to remove surface particl...

Embodiment 3

[0092] The non-amplification detection method of the target nucleic acid with a base complementary chain length of 10 bp between the target nucleic acid and the detection molecule is as follows. In this example, the detection molecule is immobilized on the chip surface, and the capture molecule is combined with the nanoparticle:

[0093] (1) Use a SPRM rebuilt based on a commercial total internal reflection microscope (Olympus IX-81) as a detection instrument, select an oil lens with a magnification of 60 times and a numerical aperture N.A=1.49, and the light source is an ultra-broadband light source SLED, and the light source controls the intensity of the current At 169mA, the field of view is 512×512pixels(full field of view:51.2×51.2μm 2 ). A micro manager was used to control the CCD camera (Photometrics) to record the imaging signal of SPRi. Cell lens software is used to control the incident angle of the light path.

[0094] (2) On the BAK-4 glass slide coated with 2nm c...

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Abstract

The invention relates to an amplification-free nucleic acid detection method. The method comprises the following steps: 1) modifying the surface of a gold film to obtain an amplification-free nucleic acid detection sensing chip; 2) taking gold nanoparticles as reporter molecules and a to-be-detected sample, and adding the gold nanoparticles and the to-be-detected sample into the reaction cavity of the amplification-free nucleic acid detection sensing chip; and 3) realizing amplification-free single particle detection of nucleic acid through a surface plasmon resonance imaging system SPRi which is constructed through wave vector coupling and has an unmarked real-time monitoring capability. Compared with the prior art, through dynamic detection and digital analysis of the single molecule hybridization process, the sensitivity of nucleic acid detection is greatly improved, and the problems that nucleic acid amplification is easily polluted by aerosol and the false positive is high are solved.

Description

technical field [0001] The invention belongs to the field of biosensors, and relates to an amplification-free nucleic acid detection method based on surface plasmon resonance microscopic imaging technology. Background technique [0002] Compared with other technologies, molecular diagnostic technology has the advantages of rapidity, high sensitivity, and high specificity, and is the most important development and research direction of in vitro diagnostic technology. As an important application of molecular diagnostic technology, nucleic acid detection is playing an increasingly important role. As of December 3, 2020, 25 of the 53 new coronavirus detection reagents approved by the State Drug Administration of China It can be seen that single-molecule detection of nucleic acid is still the most widely used and most practical technology. How to improve the speed, sensitivity, and specificity of nucleic acid molecular detection is an important and difficult issue in improving t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6834C12Q1/682G01N21/552
CPCC12Q1/6834C12Q1/682G01N21/553C12Q2563/155C12Q2563/137C12Q2565/628C12Q2565/519Y02A50/30
Inventor 余辉曾强杨玉婷
Owner SHANGHAI JIAO TONG UNIV
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