CN101162201A - TSA- nano-gold making silver-staining testing method of gene chip - Google Patents

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

A kind of TSA-nano gold mark silver dyeing detection method of genetic chip

Technical field

The present invention relates to a kind of detection method of genetic chip, specifically, relate to a kind of TSA (Tyramine Signal Amplification, junket ammonia signal amplifies)-gold label silver stain visible detection method that increases substantially the genetic chip detection sensitivity.

Background technology

Genetic chip claims DNA chip or dna microarray again, has been one of science and technology progress that high-tech area is the most great since the nineties, and being microelectronics, physics, chemistry, material science intersects the new and high technology that combines with life science.Genetic chip is based on the principle of the complementary hybridization of nucleic acid and develops; be by the fixing gene probe that is latticed dense arrangement in a large number of specific arrangement mode on solid-phase matrix; sample to be analyzed by with chip in the complementary hybridization of dna fragmentation of conventional base base sequence; thereby determine nucleotide sequence and character in the sample; amount and characteristic to gene expression are analyzed, and are the important means of obtaining associated biomolecule information efficiently on a large scale.Biochip technology has the feature of high flux, parallelization, robotization, shows wide application value and development prospect in biomedical numerous research fields such as gene expression profile research, single nucleotide polymorphism analysis, disease Molecular Detection and large-scale medicine screenings.

The correlation technique of genetic chip comprises: the analytical technology of the technology of preparing of the processing of target and probe sample and labelling technique, chip, hybridization and detection technique and hybridization image etc.

Fluorescence detection method is generally adopted in the detection of genetic chip at present, and soon fluorescent material such as fluorescein (FITC), cyanine (Cy3, Cy5) directly or indirectly are marked on DNA or the RNA molecule, and fluorescence signal adopts laser confocal scanning to detect.Fluorescence detection highly sensitive compared with isotope-labelling method, avoided using harmful radiomaterial; But there is the defective that is difficult to overcome in fluoroscopic examination self, as easily saturated, the easy cancellation of fluorescence signal, poor stability etc., cost an arm and a leg and the greatest problem that faces is a detecting instrument, thereby seriously limited applying of biochip, especially applying in medium and small medical institutions.

The gold label silver stain technology is the special visible detection method of a kind of sensitivity, is derived from modern camera technique, tissue chemical technology and immune technology for gold.The gold mark is that this mark has very high labeling effciency and stability with nano level gold grain physisorption or covalently bound on biomacromolecule, has brought difficulty but particle is too little to input; The silver dyeing technique also is the autography technology, and by reducing silver-colored specificity deposition at golden marker site, the sensitivity that significantly improves input makes the gold label silver stain technology become the visible or low magnification method for visualizing under optical microscope of a kind of naked eyes.

Over more than 20 year, the gold label silver stain technology is widely used in situ hybridization and immunohistochemical study; In recent years, the existing report of the application of this technology in genetic chip research.The gold label silver stain technology makes chip realize visual detection, has avoided fluoroscopic examination to use expensive scanner, greatly reduces the detection cost.The gold label silver stain detection method of genetic chip mandate (the ZL patent No.: 02113147.3) that patented.

As a kind of visible detection method, the detection sensitivity of gold label silver stain method can't satisfy the requirements at the higher level of disease Molecular Detection at present, especially for the detection of minute quantity in the sample to be tested or single bacterium.The sensitivity that how further to improve visual detection is the important topic that faces during biochip is applied.

The TSA method also is CARD (Catalyzed Reporter Deposition, catalysis reporter molecule deposition) technology, is proposed first in 1989 by people such as Bobrow.It is a phenolic group compound that signal amplifies molecule junket ammonia, can be used as the effect substrate of horseradish peroxidase (HRP).The principle of TSA is under HRP catalysis, connects the deposition of haptenic junket amino molecule in a large number.1992, Adams at first introduced SABC with TSA, was used for antigen or detection of antibodies.Compare with the biotin-streptavidin method of routine, this technology can improve detection sensitivity 1000 times in theory.Now, the TSA technology has all obtained using widely at numerous areas such as Western blotting, Enzyme Linked Immunoadsorbent Assay and in situ hybridizations.

Summary of the invention

The technical problem to be solved in the present invention be at the sensitivity of the gold label silver stain detection method of present biochip need further to improve problem, a kind of technology that increases substantially the detection sensitivity of gold label silver stain method is provided.

Technical solution of the present invention is that the TSA technology is combined with gold label silver stain, the chip that hybridization is good at first carries out TSA to be handled, by a large amount of depositions of HRP catalysis junket amino molecule and the specific bond of biotin and streptavidin, a large amount of nanogold particles is connected on the target molecules, dyes reaction by silver again and obtain visual signals clearly.This method can increase substantially the sensitivity of the visual detection of chip.

Specifically, the TSA-nano gold mark silver dyeing detection method of genetic chip, its characteristics are to comprise the steps:

1. the purifying of testing gene, amplification and mark: the isogeneity method is referring to " molecular cloning experiment guide ", Science Press. author, J. Sa nurse Brooker; Gene amplification method adopts polymerase chain reaction (PCR) or post transcription cloning polymerase chain reaction (RT-PCR); Nucleic acid labeling methods is that the downstream primer employing end-labelling of PCR is introduced biotin, carries out the PCR reaction then.

2. the preparation of chip and hybridization: design the probe of gene to be checked, some system genetic chip; PCR product and gene chip hybridization with above-mentioned gene to be checked.In order to improve the efficient of hybridization reaction, use hybridization solution to mix during hybridization with the PCR product.

3.TSA process: the chip that hybridization is good is at first put the horseradish peroxidase (Streptavidin-HRP) that adds the streptavidin mark, by the biotin specific bond that streptavidin is connected with target, introduces HRP on target; Select then and add biotin labeled junket ammonia (Biotin-Tyramine), the catalytic action by HRP is deposited on around the reaction site a large amount of junket ammonia.

Biotin-Tyramine reagent prepares according to document: Tyramine.HCl and Biotin-NHS are dissolved in DMF respectively, and mixed room temperature reaction in certain proportion then, reactant liquor becomes prescribed concentration with ethanol dilution, 4 ℃ of preservations.

4. gold label silver stain process: the chip point adds the nm of gold (Streptavidin-Gold) of streptavidin mark, and the specific bond by Streptavidin and Biotin is connected to nm of gold on the target molecules once more; Add silver-colored transfection reagent then, utilize silver-colored particle deposition that the redox reaction of silver-colored transfection reagent forms on nm of gold, form macroscopic hybridization signal.

5. signal processing: hybridization signal can scan detection with common optical scanner, and can adopt image analysis software to obtain the gray-scale value of signal, carries out quantitative test.

This method has following advantage and effect:

1. this method can be carried out highly sensitive visual detection to bacterium or virus, compares with single step gold label silver stain method, and detection sensitivity improves 10 times to 100 times.

2. the sample mark of this method is handled the method that adopts terminal primer mark biotin, has advantages such as with low cost, that the reagent term of validity is long, easy and simple to handle.

3. the signal display method of this method is silver-colored particle, and the result is easy to preserve, contrast and standardization; Simultaneously, compare advantages such as the environmental baseline that this method has requirement is low, reaction time weak point with the enzyme development process.

4. the Biotin-Tyramine reagent that uses of this method, have the preparation method simple, exempt purifies and separates, stable performance, the advantage that is easy to preserve.

Description of drawings

Fig. 1 detects the chip visual signals scintigram of shigella dysenteriae for adopting TSA-gold label silver stain and single step gold label silver stain respectively.

Fig. 2 detects the chip visual signals scintigram of Escherichia coli O 157 for adopting TSA-gold label silver stain and single step gold label silver stain respectively, comparison be the intensity of hybridization signal of pcr template two kinds of detection methods when getting a series of dilution gradient.

Fig. 3 relatively schemes for the signal intensity that adopts TSA-gold label silver stain and single step gold label silver stain to detect the chip visual signals of Escherichia coli O 157 respectively.

Embodiment

Embodiment 1

Detect the testing process of the TSA-gold label silver stain of shigella dysenteriae with genetic chip

1. make important pathogenic microorganism by oneself and detect genetic chip

2. the reagent that provides by kit carries out the gene extracting with lauryl sodium sulfate (SDS) boiling method: the bacterium liquid of getting 40 microlitre shigella dysenteriaes adds 10 microlitre DNA extraction liquid, boiled after mixing 20 minutes, left the heart 1 minute with per minute 13000, get the template of supernatant as pcr amplification.

3. carry out gene magnification according to the used gene segment of chip, its primer adopts the subsidiary primer of chip agent box, wherein the 5` of reverse primer end mark biotin.

Table 1: the primer sequence of the shigella dysenteriae genetic fragment that is used to increase

Condition by the chip appointment is carried out the PCR temperature cycles: 94 ℃, and 5 minutes; 94 ℃, 30 seconds; 55 ℃, 30 seconds; 72 ℃, 30 seconds; 72 ℃, 5 minutes; 35 circulations.After the loop ends immediately with the product sex change: 94 ℃, 5 minutes, ℃ freezing preservation then-20.

4. the genetic chip that will put system places 0.2% SDS solution and distilled water washing and airing successively, the PCR product dilution (the PCR product dilutes 10 times with hybridization solution) that adds 10 microlitres at each district's point of chip, chip is placed moist hybridizing box, in 50 ℃ of hybridization 1 hour, biotin labeled DNA sample is combined with specific probe on the chip; (C:0.1 * SSC) wash successively also dries for A:1 * standard citric acid salt solution (SSC)+0.2%SDS, B:0.2 * SSC with A, B, C cleaning fluid with chip then.

5.TSA process: each distinguishes a Streptavidin-HRP dilution that adds 10 microlitres (1: 1500 chip, 1 * PBS-BSA1% dilution), chip was placed 37 ℃ of incubations 30 minutes then, (1 * PBS+0.05%Tween20) washs and dries with the PBST washing lotion to take out the back; Each district point add 10 microlitres a Biotin-Tyramine dilution (1: 200,1 * PBS-BSA1%+0.5%H ₂O ₂Dilution), 37 ℃ of incubations 30 minutes take out the back and wash with the PBST washing lotion and dry.

6. each district point of chip adds the Streptavidin-Gold dilution (1: 50,1 * PBS-BSA1% dilution) of 10 microlitres, 37 ℃ of incubations 30 minutes, after the taking-up successively with PBST washing lotion and deionized water wash and dry; Silver-colored transfection reagent A liquid and B liquid are mixed, and each is distinguished immediately lucifuge and selects A, the B mixed liquor that adds 20 microlitres, finishes through colour developing in about 20 minutes, removes the silver-colored transfection reagent of chip surface with washed with de-ionized water, dries.

7. macroscopic grey clearly of hybridization signal or black round dot use the ordinary optical scanning instrument record, can use various chips hybridization signal analysis software that hybridization signal is carried out quantitative test.

The testing process that detects the single step gold label silver stain of shigella dysenteriae with genetic chip is that the 5th step of above-mentioned steps is omitted, and other steps are identical.

The intensity of hybridization signal of two kinds of detection methods when relatively the PCR product is got different extension rate respectively calculates corresponding gray-scale value and sees Table 1 and table 2.

The signal gray-scale value of gold label silver stain under the PCR product serial dilution gradient of table 2. shigella dysenteriae

The signal gray-scale value of TSA-gold label silver stain under the PCR product serial dilution gradient of table 3. shigella dysenteriae

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Embodiment 2

Detect the TSA-gold label silver stain of Escherichia coli O 157 and the testing process of single step gold label silver stain with genetic chip.Identical with the process that detects shigella dysenteriae.

Table 4: the primer sequence of the Escherichia coli O 157 genetic fragment that is used to increase

Claims (7)

1. the detection method of a genetic chip is characterized in that comprising the TSA process.

2. detection method according to claim 1 is characterized in that method is the TSA-gold mark silver dyeing detection method.

3. detection method according to claim 2 is characterized in that may further comprise the steps:

(1) purifying of testing gene, amplification are introduced biotin to the downstream primer of amplification with end-labelling and are carried out nucleic acid marking;

(2) preparation of chip and hybridization;

(3) TSA process;

(4) gold mark silver dyeing process;

(5) signal Processing.

4. detection method according to claim 3 is characterized in that step (3) TSA process is:

Point adds the horseradish peroxidase of streptavidin mark on the good chip of hybridization, by the biotin specific bond that streptavidin is connected with target, introduces HRP on target; Select then and add biotin labeled junket ammonia, the catalytic action by HRP is deposited on around the reaction site a large amount of junket ammonia.

5. detection method according to claim 3 is characterized in that step (4) gold mark silver dyeing process is:

The chip point adds the nanoscale gold grain of streptavidin mark, and the specific bond by streptavidin and biotin is connected to gold grain on the target molecules; Add silver-colored transfection reagent then, utilize silver-colored particle deposition that the redox reaction of silver-colored transfection reagent forms on nm of gold, form macroscopic hybridization signal.

6. detection method according to claim 3 is characterized in that step (5) signal Processing adopts optical scanner to scan detection.

7. detection method according to claim 3 is characterized in that step (5) signal Processing adopts image analysis software to obtain the gray-scale value of signal, carries out quantitative test.

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