Reaction device and method for visual detection of isothermal amplification of nucleic acid

A constant temperature amplification and reaction device technology, which is applied in the fields of chemical analysis and nucleic acid analysis, can solve the problems of unfavorable naked eye observation, the influence of metal indicators, and the background signal reduces the detection sensitivity, so as to achieve the effect of low background interference and accurate judgment

Active Publication Date: 2014-12-10
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the current method for visual detection of nucleic acid amplification has inevitable disadvantages: 1. The metal indicator is easily affected by other metal ions in the sample, causing errors; 2. The positive reaction result is from one color to another color, It is not conducive to naked eye observation; 3. The detection object is a metal ion with a high initial concentration in the reaction solution, so it will have a high background signal and reduce the detection sensitivity

Method used

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  • Reaction device and method for visual detection of isothermal amplification of nucleic acid
  • Reaction device and method for visual detection of isothermal amplification of nucleic acid
  • Reaction device and method for visual detection of isothermal amplification of nucleic acid

Examples

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example 1

[0036] Example 1: The present invention is used to visualize the cross-primer constant temperature amplification (CPA) to detect the transgene terminator T-Nos gene.

[0037] Reagent preparation before the reaction: in the a region of the reactor in Example 1, add 9ul reaction amplification solution (containing primers, dNTP, Tris-HCl buffer, Mg 2+ , NH 4 + , template, from Hangzhou Ustar Biotechnology Company Transgenic NOS Sequence Constant Temperature Amplification Kit, Cat. No. I016-01), and 1ul 0.1U / ul thermostable inorganic pyrophosphatase (from New England Biolabs, Cat. No. M0296s); b Add 2ul 10% ascorbic acid solution and 8ul water to area; add 4ul ammonium molybdate-potassium antimony tartrate solution (containing 21mM ammonium molybdate, 2mM potassium antimony tartrate, 5M sulfuric acid) and 76ul water to area c.

[0038] Nucleic acid isothermal amplification reaction: put the above device in a 63°C water bath and heat for 1 hour.

[0039] Visual detection of cons...

example 2

[0041] Example 2: The present invention is used to visualize the cross-primer constant temperature amplification (CPA) to detect the transgene terminator T-Nos gene.

[0042] Nucleic acid constant temperature amplification reaction: put the above device in a 63°C metal bath and heat for 1h. Others are the same as embodiment 1.

[0043]

example 3

[0044] Example 3: The present invention is used to detect Staphylococcus aureus by visual cross-primer constant temperature amplification (CPA).

[0045] Reaction amplification solution (including primers, dNTPs, Tris-HCl buffer, Mg 2+ , NH 4 + , template, from Hangzhou Ustar Biotechnology Co., Ltd. Staphylococcus aureus Isothermal Amplification Nucleic Acid Kit, Cat. No. I011-01). Others are the same as embodiment 1.

[0046]

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Abstract

The invention discloses a method and a reaction device for visual detection of isothermal amplification of nucleic acid. The method comprises the following steps: in a special hermetic reaction device, adding a reagent capable of decomposing pyrophosphoric acid into phosphoric acid into a nucleic acid amplified reaction liquid in advance; after reaction, under the condition of not uncapping, mixing the nucleic acid amplified reaction liquid with other selected color reagents which are additionally stored in the device in advance to react and observing the color of the mixture after further reaction; and judging whether nucleic acid amplified reaction is carried out on a sample or not, wherein the selected color reagents are reagent combination capable of being in color reaction with phosphoric acid (radicals). The method disclosed by the invention is simple to operate and is quick and sensitive. The test result can be detected by virtue of naked eyes. The method is low in cost and makes up the defects in the existing technology for detecting nucleic acid amplification.

Description

technical field [0001] The invention belongs to the fields of chemical analysis and nucleic acid analysis, and in particular relates to a reaction device and method for visually detecting nucleic acid isothermal amplification products. Background technique [0002] With the development of biotechnology, nucleic acid constant temperature amplification technology, as a new nucleic acid amplification technology, is widely used in the fields of agriculture, medicine and food industry. At present, the commonly used constant temperature amplification techniques include cross-primed constant temperature amplification (CPA), loop-mediated constant temperature amplification (LAMP), strand displacement amplification (SDA), and helicase-dependent amplification (HDA). Since this new type of amplification technology can achieve nucleic acid amplification at a relatively low constant temperature (most constant temperature amplification technologies require a reaction temperature of about ...

Claims

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Application Information

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IPC IPC(8): C12M1/34C12Q1/68
CPCB01L3/502B01L2200/10C12Q1/6844C12Q2521/525
Inventor 吴坚张芳
Owner ZHEJIANG UNIV
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