Nucleic acid isothermal amplification reaction detecting method and detection kit based on nucleic acid isothermal amplification reaction detecting method

A technology of isothermal amplification and amplification reaction, applied in biochemical equipment and methods, determination/inspection of microorganisms, etc., can solve the problems of long nucleic acid amplification reaction time, high reaction environment requirements, and high temperature, and achieve convenient use and high temperature. The effect of promotion, system complexity reduction, and reagent cost reduction

Inactive Publication Date: 2015-06-10
杜文红
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  • Description
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  • Application Information

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Problems solved by technology

[0006] It can be seen that the existing nucleic acid detection methods have the disadvantages of long nucleic acid amplification reaction time, high reaction temperature, high requirements for the reaction environment, inconvenient detection of reactants, inaccurate detection results, and incapable of quantitative detection and detection methods. The defect of high cost needs to be further improved

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  • Nucleic acid isothermal amplification reaction detecting method and detection kit based on nucleic acid isothermal amplification reaction detecting method
  • Nucleic acid isothermal amplification reaction detecting method and detection kit based on nucleic acid isothermal amplification reaction detecting method
  • Nucleic acid isothermal amplification reaction detecting method and detection kit based on nucleic acid isothermal amplification reaction detecting method

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Embodiment 1

[0052] Embodiment 1. Design of Molecular Beacons

[0053] For successful detection of nucleic acid samples, molecular beacons must bind to target DNA, while unbound probes remain closed and non-fluorescent. The loop loop (ie, the probe region) should have target specificity, and complementary sequences that can form a hairpin structure on both sides. When designing beacons, the following principles should generally be followed:

[0054] 1. The probe region should be 15~33bp long, and the loop sequence must be complementary to the target sequence;

[0055] 2. The Tm value of the probe region should be 7~10°C higher than the binding temperature during the reaction (refer to the GC content prediction rules), and the probe sequence should be estimated separately before adding the stem structure sequence;

[0056] 3. In order to ensure that the beacon preferentially hybridizes to the target sequence, the probe must be complementary to a region with less secondary structure in the...

Embodiment 2

[0066] Embodiment 2, bacterial DNA detection

[0067] Taking the detection of Acinetobacter baumannii DNA as an example, the nucleic acid isothermal amplification reaction detection method mediated by recombinase of the present invention is used for qualitative detection of bacterial DNA. The specific process is as follows:

[0068] Step 1: Acinetobacter baumannii DNA Extraction

[0069] The standard strain of Acinetobacter baumannii (purchased from ATCC, the strain number is ATCC19606) was inoculated and cultured overnight, and the DNA was extracted using Tiangen TIANamp Bactercia DNA Kit Bacterial Genomic DNA Extraction Kit (spin column type).

[0070] Step 2: Primer 1, Primer 2 and Molecular Beacon Design

[0071] Design the detection primer 1, primer 2 and specific molecular beacon according to the nucleic acid fragment of Acinetobacter baumannii inherently specific gene OXA51, and entrust Beijing Sanbo Polygala Biotechnology Co., Ltd. to synthesize it. For the specific s...

Embodiment 3

[0082] Embodiment 3, viral RNA detection

[0083] Taking the detection of hepatitis C virus RNA as an example below, the nucleic acid isothermal amplification reaction detection method mediated by recombinase of the present invention is used to carry out qualitative detection of viral RNA. The specific process is as follows:

[0084] Step 1: Sample HCV RNA Extraction

[0085] Collect the clinical sera positive for hepatitis C virus, and use the TIANamp Virus RNA Kit virus RNA extraction kit (spin column type) for RNA extraction, and pay attention to personal safety protection at all times during the operation.

[0086] Step 2, Primer 1, Primer 2 and Molecular Beacon Design

[0087] Design the detection primer 1, primer 2 and specific molecular beacon according to the nucleic acid fragment of the hepatitis C virus 5'UTR gene, and entrust Beijing Sanbo Yuanzhi Biotechnology Co., Ltd. to synthesize it. The specific sequence is as sequence 2: >gi|221612: 1-341 Hepatitis C virus ...

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Abstract

The invention discloses a recombinase-mediated nucleic acid isothermal amplification reaction detecting method. The nucleic acid isothermal amplification reaction detecting method comprises the following step that 1, an amplification reaction system is prepared. The amplification reaction system mainly comprises a, a nucleic acid sample to be detected; b, a pair of oligonucleotides primer 1 and primer 2, wherein the primer 1 and the primer 2 are cross-fertilized with the nucleic acid sample; c, a fluorescent probe, wherein the fluorescent probe is one or multiple of a molecular beacon, a fluorescent resonator, a scorpion probe and a molecular torch, and the concentration of the fluorescent probe ranges from 50-900 nM / L; d, recombinase; e, DNA polymerase; f, single-stranded DNA binding protein; g, amplification reaction buffer. The invention further provides a detection kit based on the nucleic acid isothermal amplification reaction detecting method. According to the nucleic acid isothermal amplification reaction detecting method and the detection kit based on the nucleic acid isothermal amplification reaction detecting method, the complexity of the system is reduced, reagent cost is reduced, sequence synthesis of the probe can be achieved easily, the probe can be designed easily, and actual application and popularization are facilitated.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a nucleic acid isothermal amplification reaction detection method and a detection kit using the method. Background technique [0002] Polymerase chain reaction (referred to as PCR) is a revolutionary technology developed since 1983, which can realize the efficient amplification of trace nucleic acid, and amplify a very small amount of specific nucleic acid sequence molecules to the level detectable by instruments. It is widely used in modern agriculture, medicine, veterinary medicine and biosafety and other fields. PCR consists of three basic reaction steps: denaturation-annealing (annealing)-extension. Denaturation of template DNA: After the template DNA is heated to 90-95°C for a certain period of time, the double-stranded DNA of the template or the double-stranded DNA formed by PCR amplification is dissociated to make it single-stranded, so that it can combine w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2521/507C12Q2521/101C12Q2522/101
Inventor 不公告发明人
Owner 杜文红
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