Molecule detection signal amplification technique

A technology for detecting signals and signal amplification, applied in biological testing, material inspection products, etc., can solve the problems of expensive detection instruments, easy saturation of detection signals, poor signal stability, etc., to achieve visual detection, stable detection signals, and high detection. Effects of sensitivity and specificity

Inactive Publication Date: 2012-06-13
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a stable detection signal, high detection sensitivity and no need for expensive detection instruments in view...

Method used

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  • Molecule detection signal amplification technique
  • Molecule detection signal amplification technique
  • Molecule detection signal amplification technique

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Detection of Ev71 virus and rotavirus

[0031] 1. Self-made gastrointestinal virus detection gene chip.

[0032] 2. The Ev71 virus and rotavirus cell culture medium were operated according to the instructions of the QIAamp Viral RNA Mini Kit Nucleic Acid Extraction Kit (QIAGEN) to obtain purified RNA. Use the reverse transcription kit PrimeScript One Step RT-PCR Kit Ver.2 (Bao Biological Engineering Co., Ltd.) for reverse transcription, the reaction system is 20 μL: 2 × One Step Buffer 10 μL, PrimeScript 1 Step Enzyme Mix 0.8 μL, 0.2 μL ( Upstream primer, 10 μM), 1 μL (downstream primer, 10 μM), RNA template 5 μL, nuclease-free water to make up the volume to 20 μL system. Reaction conditions: reverse transcription: 50°C, 30min; pre-denaturation: 94°C, 2min; PCR amplification: 94°C, 30s; 50°C, 30s; 72°C, 30s; extension: 72°C, 10min. A total of 45 cycles were performed in PCR amplification.

[0033] 3. Wash the prepared gene chip with 0.2% SDS for 20 seconds, then with...

Embodiment 2

[0039] Detection of Coxsackie B3, Coxsackie B4, Coxsackie B5, Polio1, Polio2, Polio3, Eike 30, Coxsackie A16, Enterovirus Virus type 71, rotavirus.

[0040] Self-made gene chip for gastrointestinal virus detection.

[0041] 1. Coxsackie B3 virus, Cossackie B4 virus, Cossackie B5 virus, polio1 virus, polio2 virus, polio3 virus, Eike 30 virus, Cossackie A16 virus, Enterovirus 71 and rotavirus cell culture medium were operated according to the instructions of the QIAamp Viral RNA Mini Kit Nucleic Acid Extraction Kit (QIAGEN) to obtain a purified RNA solution. Use the reverse transcription kit PrimeScript One Step RT-PCR Kit Ver.2 (Bao Biological Engineering Co., Ltd.) for reverse transcription, the reaction system is 20 μL: 10 μL (2×One Step Buffer), 0.8 μL (PrimeScript 1Step Enzyme Mix) , 0.2 μL (upstream primer, 10 μM), 1 μL (downstream primer, 10 μM), 5 μL (LRNA template), and nuclease-free water to make up the volume to 20 μL. Reaction conditions: reverse transcription: 50...

Embodiment 3

[0048] Detection of hepatitis B surface antigen HBsAg in patients' serum

[0049] Chip preparation: HBsAb (0.5 mg / mL) was spotted on the aldehyde-modified glass slide; BSA (1 mg / mL) negative control. After being placed at 4°C for 12h, it was blocked with blocking solution (1×PBS+25% calf serum) for 2h, then washed three times with PBST (1×PBS+0.5% Tween) and dried at 4°C for later use.

[0050] 1. Take the prepared chip and add sample serum (1:5 dilution) in sequence, incubate at 37°C for 30 minutes, take out the chip and wash it three times with PBST (1×PBS+0.05% Tween20) for 20 seconds each time, and dry it in the air.

[0051] 2. Add HbsAb-Biotin (1:1000, 1×PBS-0.5%BSA, 1mg / mL) to the corresponding area and incubate at 37°C for 30min, take out the chip and wash it three times with PBST (1×PBS+0.05%Tween20) repeatedly 20s, dry.

[0052] 3. Add Streptavidin-QDs (diluted 1:50, 2XPBS-0.1% BSA-0.01% thimerosal) in the corresponding area, incubate at 37°C for 30min, wash with P...

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Abstract

The invention relates to novel molecule detection signal amplification technique, which is characterized in that the molecule detection signal amplification technique is a biomolecule detection method with high sensitivity which is formed by streptavidin, an antigen, an antibody, polypeptide and the like which are marked by quantum dots through tyramine signal amplification and silver strengthening dyeing signal amplification. The detection method comprises the following steps: the biomolecule to be detected is combined with nucleic acid, the antibody, the antigen, the polypeptide and the like which are fixed on a solid phase material, the quantum dots are introduced to molecule to be detected through biomolecule specificity combination, silver strengthening dyeing is carried out on the quantum dots by using silver dyeing reagents, and signals are amplified. Obtained signals can be scanned through common optical scanners, analyzed through common imaging instruments or observed through naked eyes. The detection method achieves high sensitivity detection of the biomolecule on one hand, and reduces application cost of biomolecule detection on the other hand.

Description

technical field [0001] The invention relates to a molecular detection technology. Background technique [0002] Biomolecules include proteins, nucleic acids, lipids, and carbohydrates. Molecular detection methods mainly include analytical immunodiafiltration, immunochromatography, biochip technology, PCR technology, etc. Among them, biochip technology includes gene chips, protein chips, cell chips, tissue chips, etc. It is an important means of large-scale acquisition of biological information such as nucleic acids and proteins, and has broad application prospects in many fields such as gene expression profiling, disease molecular detection, and large-scale drug screening. At present, fluorescence detection is commonly used in biochip detection. The materials used, such as Cy3, Cy5, FITC, etc., are directly or indirectly labeled on DNA or RNA, antibodies, proteins and other molecules, and the fluorescent signals generated are detected by laser confocal scanning. The disad...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/53
Inventor 王升启张春刘琪琦陈苏红彭贤慧
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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