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A nucleic acid isothermal amplification reaction system, preparation method and application thereof

A technology of isothermal amplification and reaction system, applied in nucleic acid amplification reaction system, warm amplification reaction system and its preparation field, can solve the problems of inability to realize miniaturization, inability to effectively seal, high manufacturing cost, etc., and achieve high throughput Detection, prevention of solution evaporation, low cost effect

Inactive Publication Date: 2016-04-20
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Therefore, the object of the present invention is to provide a nucleic acid isothermal amplification reaction system and its preparation method and application for the current nucleic acid isothermal amplification system with complex operation and high manufacturing cost, which cannot be truly miniaturized and cannot be effectively sealed. In order to achieve nucleic acid isothermal amplification, the operation is simple, the manufacturing cost is low, and it can realize real miniaturization and effective sealing

Method used

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  • A nucleic acid isothermal amplification reaction system, preparation method and application thereof
  • A nucleic acid isothermal amplification reaction system, preparation method and application thereof
  • A nucleic acid isothermal amplification reaction system, preparation method and application thereof

Examples

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Effect test

Embodiment 1

[0055] Preparation of plasmid amplification reaction solution:

[0056] 1× ThermoPol buffer (containing 20.0mM Tris-HCl, 10.0mM KCl, 10.0mM (NH 4 ) 2 SO 4 , 0.1%TritonX-100, pH8.8), 8.0mM MgSO 4 , 0.5mMMnCl 2 , 25.0 μM calcein, 0.4 mM dNTPs, the amounts of each primer in the Pandemic H1N1 (Pandemic H1N1) conserved sequence and FluA conserved sequence are as follows: F3 primer and B3 primer each 0.2 μM, FIP primer and BIP primer each 2.0 μM, LF primer 0.8 μM each of primers and LB primers, 1.0M betaine, 0.32U / μl BstDNA polymerase, a little pUC57 vector inserted with PandemicH1N1 conservative sequence and pUC57 vector inserted with FluA conservative sequence (both of these plasmids were purchased from Shanghai Sangong Bioengineering Technology Services Co., Ltd.) DNA samples were used as templates.

[0057] There are two types of plasmids, which respectively amplify two target sequences related to influenza A virus:

[0058] Epidemic H1N1 conserved sequence:

[0059] GAAA...

Embodiment 2

[0080] The composition of reaction solution, reaction condition, reaction time are all identical with embodiment 1, just pass into the liquid order in glass capillary different, concrete situation of passing into is as follows image 3Shown in (A), and heated with a flexible electric heating plate, reacted at 65°C for 1 hour. Its characteristic is that there are plasmid-containing reaction solutions at both ends of the reaction solution that does not contain plasmids, but they are separated by two water droplets. Under the irradiation of ultraviolet light from an ultraviolet flashlight, the test results that emit purple fluorescence are observed as follows: image 3 In (B) shown.

[0081] Also, using the same plasmid amplification reaction solution in a traditional PCR vial, and performing a LAMP reaction under the same conditions, the same visualization results were obtained.

[0082] This example proves that two sections of water columns are used to separate adjacent reacti...

Embodiment 3

[0084] The composition of reaction solution, reaction condition, reaction time are all identical with embodiment 1, just pass into the liquid order in glass capillary different, concrete situation of passing into is as follows Figure 4 Shown in (A), and heated with a hot plate, reacted at 65°C for 1 hour. It is characterized in that there are two sections of reaction solution on one side of the reaction solution containing the plasmid, neither of which contains the target sequence, one of which does not contain DNA at all, and the other contains DNA of a non-target sequence. Separated by two water droplets, under the irradiation of ultraviolet light from the ultraviolet money detector pen, observe the test results of purple fluorescence as follows: Figure 4 In (B) shown.

[0085] Also, using the same plasmid amplification reaction solution in a traditional PCR vial, and performing a LAMP reaction under the same conditions, the same visualization results were obtained.

[0...

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Abstract

The invention provides a nucleic acid isothermal amplification reaction system, and a making method and applications thereof. The nucleic acid isothermal amplification reaction system comprises a capillary main body, at least one sample amplification reaction liquid layer is filled in the capillary main body, and two ends of the sample amplification reaction liquid layer are respectively sequentially filled with a gas layer and at least one liquid layer; and the applications comprise an application of the nucleic acid isothermal amplification reaction system in nucleic acid amplification and an application of the nucleic acid isothermal amplification reaction system in the preparation of disease detection medicines. The system eliminates a problem of the leakage pollution of an aerosol in a nucleic acid isothermal amplification reaction, and substantially improves the credibility of an isothermal amplification technology; and the system has the advantages of extremely simple operation, low cost, easy integration, diversified heating mode and diversified signal exciting mode, makes the whole flow completely divorced from an external power supply, and allows a final signal to be directly observed by naked eyes.

Description

technical field [0001] The invention relates to a nucleic acid amplification reaction system, in particular to a nucleic acid isothermal amplification reaction system and its preparation method and application, belonging to the field of biotechnology. Background technique [0002] Nucleic acid isothermal amplification technology is very attractive in the field of nucleic acid detection because of its constant temperature characteristics. For example, loop-mediated isothermal amplification technology (loop-mediated isothermal amplification, LAMP) is a typical representative. However, this type of technology has long been subject to the problem of aerosol pollution, which greatly limits its practical application. LAMP is much more sensitive than the traditional polymerase chain reaction (polymerasechainreaction, PCR), so that it is also very sensitive to trace contamination, and false positives often occur. [0003] At present, some studies have shown that microreactors made ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12M1/00C12M1/34C12N15/10C12Q1/70C12Q1/68
Inventor 蒋兴宇张翼孙佳姝
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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