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A method and kit for detecting mycoplasma pneumoniae nucleic acid using colloidal gold chromatography

A technology of mycoplasma pneumoniae and a detection kit, which is applied in the field of medical biology, can solve the problems of cumbersome operation and lack of versatility, and achieve the effect of low technical requirements, reduced requirements, and simple operation

Active Publication Date: 2017-03-01
武汉中帜生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is cumbersome to operate, and each hybridization needs to label a specific target molecule, which is not universal.

Method used

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  • A method and kit for detecting mycoplasma pneumoniae nucleic acid using colloidal gold chromatography
  • A method and kit for detecting mycoplasma pneumoniae nucleic acid using colloidal gold chromatography
  • A method and kit for detecting mycoplasma pneumoniae nucleic acid using colloidal gold chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] [Example 1] Colloidal gold particles labeled with universal nucleic acid probes

[0062] 1. After the universal probe 1 is designed, carry out sulfhydryl modification at its 5'end.

[0063] 2. Add 20μl of the synthesized universal probe (final concentration 0.1mM) to 10μl TCEP-HCl (final concentration 100mM), use ddH 2 Make up O to 100μl to reduce the thiolated DNA universal probe.

[0064] 3. Add the treated universal probe to 500ml of colloidal gold solution with 30nm diameter particles, and incubate overnight at room temperature.

[0065] 4. Add 2% SDS solution to make the final concentration 0.01%, and incubate at room temperature for 30 minutes.

[0066] 5. Add 2M NaCl dropwise to the solution to a final concentration of 0.15M.

[0067] 6. Purification of gold-labeled nucleic acid probe by centrifugation: Centrifuge at 15000 rpm for 15 minutes, wash the precipitate with washing solution (0.15M NaCl, 0.01% SDS) four times, and resuspend the colloidal gold pellet in the resuspe...

Embodiment 2

[0068] [Example 2] Preparation of nucleic acid detection test strips

[0069] The main raw materials needed when preparing nucleic acid detection test strips: glass fiber membrane, nitrocellulose membrane (NC membrane), sample pad, PVC base plate, etc.

[0070] 1. Preparation of colloidal gold pad: Cut the glass fiber membrane into 0.5×1cm square modules, and evenly drop 10μl of gold-labeled nucleic acid probe solution on each module with a gun, dry it at room temperature, and store it in a sealed storage for later use.

[0071] 2. Spray film (coated with NC film):

[0072] Detection line (T line): Avidin (about 0.5~1.0mg / ml), spray film volume: 1.5~3μl / cm;

[0073] Quality control line (line C): anti-digoxigenin antibody (about 0.5~1.0mg / ml), spray film volume: 1.5~3μl / cm;

[0074] After spraying the film, place the film strip in a clean thermostat at 37°C for 3 to 4 hours, and store it in a dry environment for later use.

[0075] 3. Test strip assembly:

[0076] Cut out 2cm long absorben...

Embodiment 3

[0077] [Example 3] Detection of Mycoplasma Pneumoniae (MP) NASBA Amplification Products

[0078] a. Amplify the nucleic acid of Mycoplasma pneumoniae:

[0079]

[0080]

[0081] 95℃, 2min, 42℃, after 2min, add 1μl of the amplification enzyme mixture (AMV, T7 polymerase and RNaseH), react at 42℃ for 45min, and wait for testing.

[0082] b. Detection of specific amplification products of Mycoplasma pneumoniae obtained in a:

[0083]

[0084] Chromatography solution (4×SSC containing 5% formamide) to a total volume of 100μl

[0085] 42℃10min later, point on the test strip for 5~10min and observe the result (such as Figure 4 ).

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Abstract

The invention discloses a method and a kit for adopting a colloidal gold chromatographic technique for detecting mycoplasma pneumoniae nucleic acid and belongs to the technical field of medical biochemistry. According to the method, a colloidal gold grain is directly marked on a nucleic acid probe; the sequence for the marked nucleic acid probe is designed as a universal sequence; the nucleic acid probe also can be used for detecting other pathogens. During the design process for the kit provided by the invention, the introduced special probe A and special probe B have the functions of bridge molecular components and a gold marked probe and an MP (Mycoplasma Pneumoniae) nucleic acid amplified fragment are successively combined with each other in series by the two probes, so that the special detection for the MP nucleic acid fragment is realized. More than two probes can be designed in each set of probes; such a design is beneficial to the increasing of the sensitivity of the test strip; the advantages of the amplification technique for the depending nucleic acid sequence of MP and the colloidal gold marked detection for the products after amplification are integrated; the technical demand on the experimenter is low; no special instrument is required; the popularization of the MP nucleic acid detection in basic and faraway rural medical institutions is easily realized.

Description

Technical field [0001] The invention relates to medical biotechnology, in particular to a method and kit for detecting Mycoplasma pneumoniae nucleic acid by using colloidal gold chromatography technology. Background technique [0002] Mycoplasmal pneumonia (Mycoplasmal pneumonia, MP) is a microorganism between viruses and bacteria, and is the pathogen of human Mycoplasmal pneumonia. Mycoplasma pneumonia accounts for about 10% of various pneumonias and has become a spreading disease with a high incidence. Severe mycoplasma pneumonia can also cause death. [0003] There are many laboratory diagnostic methods for MP infection, which can generally be divided into MP isolation and culture, serological examination and PCR diagnosis. MP isolation culture is the most traditional detection. Although this method is reliable, it is demanding, has low sensitivity, and takes a long time (2 to 3 weeks). Therefore, this method cannot be effectively applied in clinical practice. Serological test...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569
CPCG01N33/569
Inventor 李先强姜昕陈巨
Owner 武汉中帜生物科技股份有限公司
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