Marking probe of nano microparticle and affinity element and its preparation method as well as application

A nanoparticle and labeled probe technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of low sensitivity, complex reaction process, expensive detection equipment, etc., and achieve high sensitivity and good specificity. Effect

Inactive Publication Date: 2003-05-07
上海华冠生物芯片有限公司
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  • Application Information

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Problems solved by technology

Due to the complex reaction process of the enzyme-labeled method, the enzyme-catalyzed substrate reacts too quickly, and the color area is not concentrated, which limits its use, especially not suitable for applications in the field of biochips
Fluorescence detection has the disadvantages of complex process, expensive detection equipment, operators need to be trained, which makes it difficult to popularize and apply.
[0004] Chemiluminescent labeling, common silver staining and some other labeling methods have limited applications due to their low sensitivity
So far, the relatively mature gene chip detection method is laser scanning confocal microscopy, but its procedure is complicated, the detection equipment is expensive, and it is difficult to promote and apply
[0005] Chinese patent CN1339609A discloses a nanoparticle-labeled gene probe and its application in gene detection. Due to the instability and easy aggregation of the prepared nanoparticle, this technology has the disadvantages of high production cost, poor stability and the use of inconvenient defect
However, since the problem of connection between nanoparticles and DNA and its preservation have not been fundamentally resolved, there are still practical difficulties in its promotion and use.

Method used

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  • Marking probe of nano microparticle and affinity element and its preparation method as well as application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Preparation of nano gold (colloidal gold) (100ml):

[0036] 0.01% HAuClO 4 (Auric chloride) heated to boiling. Add 4ml under stirring, 1wt% trisodium citrate (Na 3 C 6 h 5 o 7 2H 2 (0), continue to heat and boil for 15 minutes, cool to room temperature under stirring, return to the original volume with distilled water;

[0037] Preparation of streptavidin-colloidal gold:

[0038] With 0.2M of K 2 CO 3 (potassium carbonate) or 1M Tris base to adjust the pH of the colloidal gold solution to 6.5;

[0039] Take the prepared nanometer or colloidal gold and centrifuge at 10,000 rpm for 5 minutes to remove the precipitate, add 60ug of streptavidin to 10ml of colloidal gold solution, leave it at room temperature for 4 hours, and then 20,000 rpm Centrifuge for 40 minutes, remove the supernatant, add a solution containing PBS and glycerol stabilizer to resuspend, and repeat 3 times to remove impurities such as unstable nano-gold / colloidal gold and free protein.

[0040...

Embodiment 2

[0041]Example 2 Detection of Mycobacterium tuberculosis resistance to rifampicin

[0042] Put the immobilized solid phase carrier into a hybridization tube, add 6×SSPE / 0.5% SDS hybridization solution containing the PCR amplification product after alkali denaturation, and hybridize for 2 hours at 45-60°C. Take out the hybridized solid-phase carrier, wash it with 1×PBS and Tween-20 at room temperature for 10 minutes, then place it in the solution of Example 1 diluted 200 times with 1×PBS / 0.1% BSA solution, and incubate at room temperature for 1-2 Hour. Then place the solid phase carrier in a hybridization tube, and use 1×SSPE eluent to elute at 50-65° C. for 10-20 minutes.

[0043] Take out the solid-phase carrier and wash it with 1×PBS for 3 times, each time for 5 minutes, add the prepared silver enhancer (the mixture of silver nitrate and reducing agent prepared now), react for 10 minutes, and finally wash it with distilled water for several times and then naturally dry. Re...

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Abstract

A nanopartide and its avidin probe are disclosed. Said gene probe for detecting the mutation or non-mutation of DNA is prepared from nanoparticles or colloidal particles and avidin, streptavidin, or antibiotin through preparing colloidal gold and preparing the compound. Its advantges are high sensitivity, specificity and speed.

Description

technical field [0001] The invention relates to a gene detection technology, more specifically, to a nanometer or colloid particle and avidin complex labeling probe and a preparation method thereof, and a method for detecting DNA by combining the probe with silver enhancement / gold enhancement. Background technique [0002] Gene research has driven the rapid development of the entire life science in the 20th century; in the 21st century, gene research will still promote the in-depth development of life science research. Gene detection is not only crucial to biological research, but also to clinical medicine, pharmacy, agricultural technology, Environmental monitoring, legal identification and other fields are also of great significance. [0003] Genetic testing generally uses direct sequencing or hybridization testing. Hybridization detection includes radiation method, enzyme labeling method, chemiluminescence labeling method and so on. Radiation method has high sensitivity...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 苏蓓胡志能费西金维荣赵国屏
Owner 上海华冠生物芯片有限公司
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