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Method and kit for realizing multiple detection of nucleic acid through colloidal gold chromatographic technology

A detection kit and multiple detection technology, applied in the field of medical biology, can solve problems such as difficult multiple detection of pathogens, and achieve the effects of reducing detection costs, avoiding trouble, and simple operation.

Pending Publication Date: 2015-12-16
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

Whether it is fluorescent quantitative PCR or isothermal amplification technology, high-end and expensive equipment must be used to qualitatively detect nucleic acids, and it is difficult to achieve multiple detection of pathogens

Method used

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  • Method and kit for realizing multiple detection of nucleic acid through colloidal gold chromatographic technology
  • Method and kit for realizing multiple detection of nucleic acid through colloidal gold chromatographic technology
  • Method and kit for realizing multiple detection of nucleic acid through colloidal gold chromatographic technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] [Example 1] Universal nucleic acid probe labeling colloidal gold particles

[0035] 1. Design the universal probe sequence for sulfhydrylation:

[0036] 5'-SH-CATCTTTCCAGCGGCCTTATGCAGTTGCTCTCCATTTTTAGAAGGCGTCCGTCTTTGAGGC-3'

[0037] 2. Add 20 μl of the designed universal probe (concentration 0.1 mM) to 10 μl TCEP-HCl (concentration 100 mM), make up 110 μl with water, and reduce the thiolated DNA universal probe at room temperature;

[0038] 3. Add the treated universal probe into the colloidal gold solution with 30nm diameter particles, and incubate overnight at room temperature.

[0039] 4. Add 2% SDS solution to make the final concentration 0.01%, incubate at room temperature for 30min.

[0040] 5. Add 2M NaCl dropwise to the solution until the final concentration is 0.15M.

[0041] 6. Centrifugal purification of gold-labeled nucleic acid probes: Centrifuge at 15,000rpm for 15min, wash with washing solution (0.15MNaCl,

[0042] 0.01% SDS) and washed four times, th...

Embodiment 2

[0043] [Example 2] Preparation of nucleic acid detection test strips

[0044] The main raw materials needed in the preparation of nucleic acid test strips: glass fiber membrane, nitrocellulose membrane (NC membrane), sample pad, absorbent paper, PVC bottom plate, etc.

[0045] 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.

[0046] 2. Spray film:

[0047] Detection line (T line): capable of capturing and binding specific probe B sequence, (5uM), spray film volume: 2~3μl / cm;

[0048] Quality control line (C line): capable of capturing and binding specific probe A sequence (5uM), spray film volume: 2~3μl / cm;

[0049] After spraying the film, put the film strip at room temperature to dry, automatically cross-link once in the ultraviolet crosslinker, put it in a clean thermostat at ...

Embodiment 3

[0052] [Example 3] Detection of Mycoplasma pneumoniae (MP) and Chlamydia pneumoniae (Cpn) nucleic acid amplification fragments by universal multiple detection test strips

[0053] Primer sequences for amplifying MP nucleic acids:

[0054] R primer: 5'-TAATACGACTCACTATAGGGAGACTCGTGAACTTGGTGTGGTTT-3'

[0055] Primer F: 5'-GGCAGTCAGACGATGATTACAGGC-3'

[0056] The specific probe A1 and sequence (two pairs) for detecting MP are:

[0057] 5'-GTTCGAGCCACGTCCTCATTAGTTTTTCCTCCAGCTCTGAACGTTTTGCCTCAAAGACGGACGCCTTCT-3'

[0058] 5'-GTTCGAGCCACGTCCTCATTAGTTTTATGATAAGGCTTCAAGTTTTGCCTCAAAGACGGACGCCTTCT-3'

[0059] The specific probe B1 and North sequence (two) for detecting MP are:

[0060] 5'-GGTTCGCCTCGAAGAATTTTCTGTAGGAATGAATGT-3'

[0061] 5'-CCCTCGACCAAGCCAATTTTCTGTAGGAATGAATGT-3'

[0062] The T-line coating sequence for detecting MP is: 5'-ACATTCATTCCTACAG-3'

[0063] Primer sequences for amplifying Cpn nucleic acids:

[0064] R primer: 5'-TAATACGACTCACTATAGGGAGAATACGTGAGCAGCTCTCT...

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Abstract

The invention discloses a method and kit for realizing multiple detection of nucleic acid through a colloidal gold chromatographic technology, and belongs to the field of medical biotechnology. The invention provides a nucleic acid detection test strip, a universal probe is marked with colloidal gold particles to be fixed on a glass cellulose film, the universal probe is designed into a universal sequence, an NC film on the test strip have one or more detection lines and a quality control line, each detection line is coated with a section of nucleic acid sequence which can be in specific hybridization combination with a corresponding specific probe B series; the quality control line is coated with a section of nucleic acid sequence which can in specific hybridization combination with a specific probe A series; the specific probe A series is in complementary pairing hybridization with the universal probe; the probe marked with gold and a nucleic acid amplified fragment are combined in series by virtue of the specific probe A series and the specific probe B series, so that the specific detection of the nucleic acid fragment can be realized. The method has the advantages that the technical requirements of experimenters are low, the required detection time is short, special instruments are not required, and the method is easy to popularize towards grass-roots and remote countryside medical institutions.

Description

technical field [0001] The invention relates to medical biotechnology, in particular to a method and kit for multiple nucleic acid detection using colloidal gold chromatography technology. Background technique [0002] Nucleic acid detection technology is a general term for a series of technologies to detect pathogen nucleic acid (DNA and RNA). Compared with conventional serological detection, nucleic acid detection is the direct detection of pathogenic deoxyribonucleic acid or ribonucleic acid, which has the characteristics of strong specificity and high sensitivity, which can greatly improve the detection rate of pathogens and shorten the detection window period. important supplement. [0003] There are many nucleic acid detection technologies, most of which are developed based on PCR technology, such as the most common one is Realtime fluorescence quantitative PCR (RTFQPCR) technology. There are also some constant temperature amplification techniques applied to the fiel...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6834C12Q2537/143C12Q2563/137C12Q2565/625
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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