Method and kit for adopting colloidal gold chromatographic technique for detecting mycoplasma pneumoniae nucleic acid

A technology of Mycoplasma pneumoniae and detection kit, applied in the field of medical biology, can solve the problems of cumbersome operation, lack of versatility, etc., and achieve the effects of low technical requirements, reduced requirements, and simple operation

Active Publication Date: 2015-12-30
武汉中帜生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method is cumbersome to operate, and each hybridizatio

Method used

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  • Method and kit for adopting colloidal gold chromatographic technique for detecting mycoplasma pneumoniae nucleic acid
  • Method and kit for adopting colloidal gold chromatographic technique for detecting mycoplasma pneumoniae nucleic acid
  • Method and kit for adopting colloidal gold chromatographic technique for detecting mycoplasma pneumoniae nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] [Example 1] Universal nucleic acid probe labeling colloidal gold particles

[0063] 1. After the design of Universal Probe 1 is completed, carry out thiol modification at its 5' end.

[0064] 2. Add 20 μl of the synthesized universal probe (final concentration 0.1 mM) to 10 μl TCEP-HCl (final concentration 100 mM), and use ddH 2 Make up to 100 μl with O, and reduce the sulfhydrylated DNA universal probe.

[0065] 3. Add the treated universal probe to 500ml of colloidal gold solution with 30nm diameter particles, and incubate overnight at room temperature.

[0066] 4. Add 2% SDS solution to make the final concentration 0.01%, incubate at room temperature for 30min.

[0067] 5. Add 2M NaCl dropwise to the solution until the final concentration is 0.15M.

[0068] 6. Centrifugal purification of gold-labeled nucleic acid probes: Centrifuge at 15,000rpm for 15min, wash with washing solution (0.15MNaCl,

[0069] 0.01% SDS) and washed four times, the colloidal gold precipit...

Embodiment 2

[0070] [Example 2] Preparation of nucleic acid detection test strips

[0071] The main raw materials needed in the preparation of nucleic acid detection test strips: glass fiber membrane, nitrocellulose membrane (NC membrane), sample pad, PVC bottom plate, etc.

[0072] 1. Preparation of colloidal gold pads: cut the glass fiber membrane into small modules of 0.5×1 cm square, and evenly drop 10 μl of gold-labeled nucleic acid probe solution on each module, let it dry at room temperature, and seal it for future use.

[0073] 2. Spray film (coated with NC film):

[0074] Detection line (T line): avidin (about 0.5~1.0mg / ml), spray film volume: 1.5~3μl / cm;

[0075] Quality control line (C line): anti-digoxigenin antibody (about 0.5~1.0mg / ml), spray film volume: 1.5~3μl / cm;

[0076] After spraying the film, put the film strip in a clean incubator at 37°C to dry for 3 to 4 hours, and store it in a dry environment for later use.

[0077] 3. Assembly of test strips:

[0078] Cut ou...

Embodiment 3

[0079] [Example 3] Detection of Mycoplasma pneumoniae (MP) NASBA amplification products

[0080] a. Amplify Mycoplasma pneumoniae nucleic acid:

[0081] components Volume (μl) Mycoplasma pneumoniae nucleic acid (or lysate) 2 Amplification reaction solution: containing dNTPs, NTPs, primer R&F and various salt ions 17 total 19

[0082] 95°C, 2min, 42°C, 2min, add 1μl of amplification enzyme mixture (AMV, T7 polymerase and RNaseH), react at 42°C for 45min, and wait for detection.

[0083] b. Detect the specific amplification product of Mycoplasma pneumoniae obtained in a:

[0084] Amplified product: 10μl

[0085] Specific probe A1 (5μM): 1μl

[0086] Specific probe A2 (5μM): 1μl

[0087] Specific probe B1 (5 μM): 1 μl

[0088] Specific probe B2 (5 μM): 1 μl

[0089] Universal Probe 2 (1 μM): 1 μl

[0090] Chromatography solution (4×SSC containing 5% formamide) to a total volume of 100 μl

[0091] After 10 minutes at 42°C, point on the test ...

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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 a kit for detecting mycoplasma pneumoniae nucleic acid by colloidal gold chromatography. Background technique [0002] Mycoplasma pneumoniae (Mycoplasmalpneumonia, MP) is a microorganism between viruses and bacteria, and is the pathogen of human mycoplasma pneumonia. Mycoplasma pneumonia accounts for about 10% of various pneumonias and has become a communicable disease with a high incidence rate. Severe mycoplasma pneumonia can also lead to 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 and culture is the most traditional detection method. Although this method is reliable, it is demanding, has low sensitivity, and takes a long time (2 to 3 weeks), so this method cannot be effectively applied clinically. Serological tests mainl...

Claims

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Application Information

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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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