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Method for detecting nucleic acid by colloidal gold chromatography technology and reagent kit

A detection kit and colloidal gold technology, applied in the field of medical biology, can solve the problems of lack of versatility and cumbersome operation

Inactive Publication Date: 2016-02-03
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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  • Method for detecting nucleic acid by colloidal gold chromatography technology and reagent kit
  • Method for detecting nucleic acid by colloidal gold chromatography technology and reagent kit
  • Method for detecting nucleic acid by colloidal gold chromatography technology and reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] [Example 1] Universal nucleic acid probe labeling colloidal gold particles

[0071] 1. Design the universal probe sequence for sulfhydrylation:

[0072] 5'-SH-CATCTTTCCAGCGGCCTTATGCAGTTGCTCTCCATTTTTAGAAGGCGTCCGTCTTTGAGGC-3', (SeqNo.1)

[0073] 2. Add the designed universal probe (final concentration 0.1mM) to TCEP-HCl (final concentration 100mM) to reduce the thiolated DNA universal probe;

[0074] 3. Add the treated universal probe to the colloidal gold solution with 30nm diameter particles, and incubate overnight at room temperature

[0075] education.

[0076] 4. Add 2% SDS solution to make the final concentration 0.01%, incubate at room temperature for 30min.

[0077] 5. Add 2M NaCl dropwise to the solution until the final concentration is 0.15M.

[0078] 6. Centrifugal purification of gold-labeled nucleic acid probes: centrifuge at 15,000rpm for 15min, wash with washing solution (0.15M

[0079] NaCl, 0.01%SDS) washed four times, the colloidal gold precipitate ...

Embodiment 2

[0082] [Example 2] Preparation of nucleic acid detection test strips

[0083] 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.

[0084] 1. Preparation of colloidal gold pad: cut the glass fiber membrane into small modules of 0.5×1cm 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.

[0085] 2. Spray film:

[0086] Detection line (T line): avidin (about 0.5~1.0mg / ml), spray film volume: 1.5~3μl / cm;

[0087] Quality control line (C line): anti-digoxigenin antibody (about 0.5~1.0mg / ml), spray film volume: 1.5~3μl / cm;

[0088] After spraying the film, put the film strip in a clean incubator at 37°C to dry for 5-6 hours, and store it in a dry environment for later use.

[0089] 3. Assembly of test strips

[0090] Cut out 2cm-long absorbent paper, ...

Embodiment 3

[0091] [Example 3] Universal nucleic acid probe direct gold labeling method (double universal probe), detection of respiratory syncytial virus (RSV) T7 linear amplification product

[0092] Primer sequences for amplifying RSV nucleic acid:

[0093] R primer: 5'-TAATACGACTCACTATAGGGAGAATGCAGGTGTAACTACACCTGTAAG-3'

[0094] Primer F: 5'-GTGGTAATTGTACTACATATGCTAAG-3'

[0095] The specific probe A sequence for detecting RSV is:

[0096] 5'-Dig-ATCATTGATTAATGATTTTTGCCTCAAAGACGGACGCCTTCT-3'

[0097] The specific probes B1 and B2 (two) for detecting RSV are:

[0098] 5'-TTAACATATAAGTGCTTTTTGGCCTCTAAGTCGTAGCCCA-3'

[0099]5'-TACAGGTGTAGTTACATTTTGGCCTCTAAGTCGTAGCCCA-3'

[0100] The universal probe 2 sequence labeled with biotin is:

[0101] 5'-Biotin-TGGGCTACGACTTAGAGGCC-3'

[0102] a. T7 linear amplification of respiratory syncytial virus nucleic acid:

[0103] components Volume (μl) Respiratory syncytial virus nucleic acid (or lysate) 2 Amplification rea...

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Abstract

The invention discloses a method for detecting nucleic acid by a colloidal gold chromatography technology and a reagent kit, and belongs to the technical field of medical biology. A universal test paper strip for nucleic acid detection is provided; colloidal gold particles are marked on a universal probe 1, and then, the universal probe is fixed on a glass cellulose membrane; the probe sequence is designed into a universal sequence; the test paper strip is fixed on a PVC (polyvinyl chloride) bottom plate; a sample pad, glass fiber, an NC membrane and water absorption paper are in sequential arrangement from the left side to the right side, wherein a T line (detection line) and a C line (quality control line) are arranged on the NC membrane; antibodies to labels b cover the detection line; antibodies to labels a cover the quality control line; the specificity probe A series and the specificity B series successfully combine a gold mark probe and nucleic acid amplification fragments in series; the specificity detection of nucleic acid amplification fragments is realized. The method has the advantages that the technical requirement on the experiment personnel is low; the required detection time is short; special instrument equipment is not needed; the popularization to basic levels and remote rural area medical institutions is easy.

Description

technical field [0001] The invention relates to medical biotechnology, in particular to a method and a kit for detecting nucleic acid by colloidal gold chromatography. Background technique [0002] Colloidal gold technology is a solid-phase labeling immunoassay technology developed after the three major labeling technologies (fluorescein, radioisotope and enzyme) in the 1980s. Initially, this method was only used for immunoelectron microscopy, but with the continuous development of biotechnology, immunospot filtration and immunochromatographic techniques have appeared. Colloidal gold immunochromatography technology has been widely used in clinical diagnosis, such as detection of early pregnancy, hepatitis B, parasites, sexually transmitted diseases and virus bacteria. The biggest feature of this method is that it is cheap, simple, fast, specific and sensitive, does not require any equipment and reagents, and can observe brightly colored experimental results with the naked e...

Claims

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Application Information

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IPC IPC(8): G01N33/558
CPCG01N33/5308G01N33/558
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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