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TSA- nano-gold making silver-staining testing method of gene chip

A technology of gene chip and detection method is applied in the field of TSA-gold-labeled silver staining visual detection, and achieves the effects of simple preparation method, long validity period of reagents and improved detection sensitivity

Inactive Publication Date: 2008-04-16
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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AI Technical Summary

Problems solved by technology

[0009] The technical problem to be solved in the present invention is to provide a technology for greatly improving the detection sensitivity of the gold-labeled silver staining method aimed at the problem that the sensitivity of the gold-labeled silver staining detection method of the current biochip needs to be further improved

Method used

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  • TSA- nano-gold making silver-staining testing method of gene chip
  • TSA- nano-gold making silver-staining testing method of gene chip
  • TSA- nano-gold making silver-staining testing method of gene chip

Examples

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Embodiment 1

[0027] Detection process of TSA-gold-labeled silver staining for detection of Shigella with gene chip

[0028] 1. Self-made important pathogenic microorganism detection gene chip

[0029] 2. Carry out gene extraction with sodium dodecyl sulfate (SDS) boiling method according to the reagents provided in the kit: take 40 microliters of bacteria liquid of Shigella dysenteriae and 10 microliters of DNA extraction solution, mix well and boil for 20 minutes to Centrifuge at 13,000 rpm for 1 minute, and take the supernatant as a template for PCR amplification.

[0030] 3. Carry out gene amplification according to the gene fragments used in the chip, and the primers attached to the chip kit are used as primers, wherein the 5' end of the reverse primer is labeled with biotin.

[0031] Table 1: Primer sequences used to amplify Shigella dysenteriae gene fragments

[0032]

[0033] Perform PCR temperature cycle according to the conditions specified by the chip: 94°C, 5 minutes; 94°C,...

Embodiment 2

[0045] The detection process of TSA-gold-labeled silver staining and single-step gold-labeled silver staining for the detection of Escherichia coli O157 by gene chip. Same process as for Shigella.

[0046] Table 4: Primer sequences used to amplify Escherichia coli O157 gene fragments

[0047]

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Abstract

The invention relates to a detection method with a gene chip, in particular to a visualized detection method which can improve the sensitivity of gold label silver stain of a gene chip to a great extent. The detection method mainly comprises the following steps that: gene marker biotin molecule of a sample to be tested is used to hybridize with the gene chip. At first, TSA treatment is performed on the hybridized chip; vast Tyramine is precipitated around a reaction site under the catalysis of enzymes; gold nanoparticles with streptavidin are added and nanoscale gold particles are connected to target molecules; silver stain reagent is used to color the hybridized gene chips to observe and analyze signals. A TSA procedure is added in the method provided by the invention based on a single gold label silver stain detection, the Biotin-Tyramine reagent used for TAS is simple to prepare, avoids purification and is easy to preserve and stable in performance. Compared with the single gold label silver stain detection method, the detecting sensitivity of the gene chips can be improved by ten to one hundred times.

Description

technical field [0001] The invention relates to a detection method of a gene chip, in particular to a TSA (Tyramine Signal Amplification, tyramine signal amplification)-gold standard silver staining visual detection method which greatly improves the detection sensitivity of the gene chip. Background technique [0002] Gene chip, also known as DNA chip or DNA microarray, is one of the most significant scientific and technological advances in the high-tech field since the 1990s. It is a high-tech cross-combination of microelectronics, physics, chemistry, material science and life science. The gene chip is developed based on the principle of nucleic acid complementary hybridization. It immobilizes a large number of gene probes densely arranged in a grid on a solid-phase substrate in a specific arrangement. Complementary hybridization of DNA fragments, so as to determine the nucleic acid sequence and properties in the sample, and analyze the quantity and characteristics of gene ...

Claims

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Application Information

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IPC IPC(8): G01N21/77C12Q1/68
Inventor 王升启戚红卷陈苏红金大智
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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