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Visualization method for rapidly detecting neisseria gonorrhoeae by utilizing recombinase polymerase isothermal amplification technology

A technology of Neisseria gonorrhoeae and primer set, which is applied in the field of molecular biology detection, can solve the problems of false positives, increase detection costs, aerosol pollution, etc., achieve good operability, avoid false positive results, and increase experimental operation steps. Effect

Inactive Publication Date: 2019-03-19
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

When performing agarose gel electrophoresis detection on RPA products, product purification is required, because components such as the reaction system buffer will affect the judgment of gel imaging results; real-time fluorescent quantitative RPA technology using exo probes for detection, amplification reactions and The interpretation of the results requires the use of a fluorescent detection system, which increases the detection cost; and the lateral flow chromatography RPA detection technology combined with the nfo probe for detection, the RPA amplification product needs to be diluted, otherwise the reaction substrate will interfere with the antibody on the test paper. Aerosol pollution is easily generated during the detection process, resulting in false positive results

Method used

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  • Visualization method for rapidly detecting neisseria gonorrhoeae by utilizing recombinase polymerase isothermal amplification technology

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1. Clinical diagnosis in hospital

[0047] 1) Use the sample collection tube provided in the kit to collect patient secretions or urine samples.

[0048] 2) For urine samples, centrifuge at 8,000 rpm for 10 minutes, discard the supernatant, and add an appropriate amount of Lysis buffer to the sample collection tube; for swab samples of secretions, add an appropriate amount of Lysis buffer to the sample collection tube. Stir and soak the swab in Lysis buffer for 5 minutes.

[0049] 3) Place the sample collection tube in a metal bath or water bath, heat at 95°C for 10 minutes, and stand still at room temperature to complete the extraction of genomic DNA from the sample. (The above steps can be used to complete the extraction of genomic DNA with other nucleic acid extraction kits or methods)

[0050] 4) Using the above steps to obtain the sample genomic DNA as a template, perform the RPA amplification reaction under the guidance of any one of the two sets of primer sets. ...

Embodiment 2

[0053] Example 2. Example of individual patient screening:

[0054] 1) Use the sample collection tube provided in the kit to collect patient secretions or urine samples.

[0055] 2) For urine samples, add the same amount of crude extraction reagent Lysis buffer to the sample collection tube; for swab samples of secretions, add an appropriate amount of crude extraction reagent Lysis buffer to the sample collection tube, stir, and put the swab Soak in Lysis buffer for 5 minutes.

[0056] 3) Place the sample collection tube in a boiling water bath, heat it for 10 minutes, and let it stand at room temperature to complete the extraction of the sample genomic DNA.

[0057] 4) Using the above steps to obtain the sample genomic DNA as a template, perform the RPA amplification reaction under the guidance of any one of the two sets of primer sets. The 27μl reaction system includes: 1.5μl forward and reverse primers at a concentration of 10μmol / L, 14.75μl RPA reaction buffer, 2μl sample genomic...

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Abstract

The invention belongs to the technical field of molecular biological detection, relates to a detection method for pathogenic microorganisms, and in particular relates to a visualization method for rapidly detecting neisseria gonorrhoeae by utilizing a recombinase polymerase isothermal amplification technology. According to the method, by combining the recombinase polymerase isothermal amplification technology and fluorescent dye developing, rapid amplification of neisseria gonorrhoeae nucleic acids can be realized under the condition that a PCR (Polymerase Chain Reaction) instrument and any other heat cycle equipment are not used. The results are identified by observing the color change of the reaction system with naked eyes. According to related reagents provided by the invention, the neisseria gonorrhoeae in samples can be rapidly detected by virtue of simple treatment of to-be-detected samples, and the detection method has the advantages of being simple in operation, high in specificity, capable of determining the results with naked eyes, and the like, and is applicable to clinical diagnosis and individual early screening.

Description

Technical field [0001] The invention belongs to the technical field of molecular biology detection, and relates to a detection method of pathogenic microorganisms, in particular to a visualization method for rapid detection of Neisseria gonorrhoeae using recombinant enzyme polymerase isothermal amplification technology. It can be used for clinical diagnosis and early personal screening. Background technique [0002] Gonorrhoea is a common sexually transmitted disease with the fifth largest number of reported cases among my country's Class A and B infectious diseases. In 2017, the number of reported cases of gonorrhea nationwide exceeded 130,000. Neisseria gonorrhoeae (NG) is the causative bacterium of gonorrhoea. At present, there is no effective vaccine for gonorrhea, and it has become an important global public health problem. Since the symptoms of Neisseria gonorrhoeae infection are not obvious in the early stage, if timely diagnosis and treatment will lead to serious genitou...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6844C12Q2521/507C12Q2522/101C12Q2531/119C12Q2563/107
Inventor 彭俊平修乐山
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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