CN110577989A - probe, gene chip and kit for detecting genes related to sudden exercise death - Google Patents

Abstract

The invention belongs to the technical field of biological detection, and discloses a probe, a gene chip and a kit for detecting genes related to exercise sudden death. The probe comprises a probe I736I probe, an I736T probe, a P731P probe, a P731S probe, a R723R probe, a R723G probe, a R663R probe, a R663H probe, a Y842Y probe, a Y842X probe, a R190R probe, a R190W probe, a W305W probe, a W305L probe, a R2401R probe and a R2401H probe. The gene chip contains the probe. The kit comprises the probe and a gene chip. The invention combines the gene chip method through the selection of the specific probe and the primer, and has the advantages of good specificity, high accuracy, rapidness and convenience; the visible light optical amplification of the gene chip can achieve higher signal resolution; meanwhile, one-time multi-site determination of the gene related to the sudden exercise death is realized, and the detection time and cost are reduced; the detection result can be directly read by photographing or naked eyes, and is simple and convenient.

Description

Probe, gene chip and kit for detecting genes related to sudden exercise death

Technical Field

the invention belongs to the technical field of biological detection, and particularly relates to a probe, a gene chip and a kit for detecting genes related to exercise sudden death.

background

Violent exercise or emotional overstimulation can induce syncope or sudden death. Of these, Hypertrophic Cardiomyopathy (HCM) and the QT interval (total time course of ventricular depolarization and repolarization processes) prolongation syndrome (LQTS) are the major causes of motion-related syncope or sudden death, and these disorders are genetically related.

according to the European and American study, HCM is the most common cause of sudden death among young people or athletes under 35 years old, accounting for about 1/500. Up to 900 mutations have been found or identified in HCM-associated genes distributed over 13 cardiac sarcomere-encoding genes, the major genes being MYH7 (encoding β -myosin heavy chain), MYBPC3 (encoding myosin-binding protein C), and TNNT2 (encoding cardiac troponin T). For HCM, clinical or forensic reports are individual case or family reports, and at present, no single gene or single mutation can specifically and accurately predict the clinical phenotype and the risk degree of the carrier, and some individual case cases even exist in combination of multiple genes and multiple mutations. But double mutations are more dangerous than single mutations; the hazard of the MYH7 mutation is significantly greater than the hazard of the MYBPC3 mutation. Now in connection with the report on the chinese population retrieved on PubMed, suggested sites for detection are as follows:

MYH7 gene has been reported to be associated with syncope or sudden death gene mutations: N391T, I736T, P731S, R723G, R719Q, R663H and the like.

MYBPC3 is a crude myofilament-binding protein with a relative molecular mass of approximately 150000, HCM caused by MYBPC3 mutations account for 20% -25% of cases, and MYBPC3 gene has been reported to be associated with mutations in syncope or sudden death: Y842X, and the like.

in LQTS, multiple mutations in multiple genes can lead to LQTS. Wherein, LQT1 (type 1 long QT syndrome) affects about 50% of LQT gene mutation carriers, KCNQ1 is a pathogenic gene thereof and can cause syncope, sudden death or drowning of male teenagers in the process of exercise, particularly swimming; LQT2 (type 2 Long QT syndrome) is the most common LQT subtype among Chinese people, affects about 40% of carriers, has a pathogenic gene KCNH2, mainly affects women, and shows that syncope or sudden death can be induced at night under loud noise; LQT3 (type 3 long QT syndrome) affects about 10% of carriers, the causative gene is SCN5A, which is commonly found in adult males and manifests as syncope or sudden death in sleep. It can be seen that most of the genes causing syncope or sudden death in rest or sleep are LQT2 and LQT3, so both of them are not associated much with exercise-induced syncope or sudden death. Now in connection with the report on the chinese population retrieved on PubMed, suggested sites for detection are as follows:

Mutations in KCNQ1 that can induce syncope or sudden death are: R190W, R380S, W305L, and the like.

catecholamine-derived (or sympathetic-derived) multiple ventricular tachycardia (CPVT) is also associated with the occurrence of syncope or sudden death. Mutations in the RyR2 receptor (cardiac calcium ion release channel) gene (or CPVT-1-type associated gene) are associated with more than 50% of CPTV cases. The disease begins in adolescent period, and sudden syncope recurrent attacks, consciousness loss and even sudden cardiac death appear under stress conditions such as exercise or emotional agitation. The reported SNP sites of the RyR2 gene mainly comprise R2401H.

at present, although partial research on sudden cardiac death mutation gene detection is carried out, more sudden death-related gene detection is not carried out. Therefore, it is necessary to provide a more excellent probe, gene chip and kit for detecting a gene related to sudden exercise death.

disclosure of Invention

aiming at the defects of the prior art, the invention provides a probe, a gene chip and a kit for detecting a gene related to sudden exercise death. The invention has the advantages of high specificity, good accuracy and capability of rapidly detecting multiple sites at one time.

A probe for detecting a gene associated with sudden exercise death, the probe having the sequence:

I736I probe: GTTCATTGATAGCAGGA, respectively;

I736T probe: GTTCACTGATAGCAGGA, respectively;

P731P probe: AGCGGCCATCCCTGAGGG, respectively;

P731S probe: AGCGGCCATCTCTGAGGG, respectively;

R723R probe: CCTCAGGTATCGCATCCT, respectively;

R723G probe: CCTCAGGTATGGCATCCT, respectively;

R663R probe: CAACTTGCGCTCCACCCA, respectively;

R663H probe: CAACTTGCACTCCACCCA, respectively;

Y842Y probe: GTGTACGAGATGCGCGTC, respectively;

Y842X probe: GGTGTAGGAGATGCGCGT, respectively;

R190R probe: CTCTGGGGGCGGCTGCGC, respectively;

R190W probe: CTCTGGGGGTGGCTGCGC, respectively;

W305W probe: CGCTGTGGTGGGGGGTGG, respectively;

W305L probe: CGCTGTGGTTGGGGGTGG, respectively;

R2401R probe: CTCTTGGGACGCTGTGCTC, respectively;

R2401H probe: CTCTTGGGACACTGTGCTC are provided.

a gene chip for detecting a gene related to sudden exercise death comprises the probe.

A preparation method of a gene chip for detecting a gene related to sudden exercise death comprises the following steps:

(1) Coating a film on a substrate to obtain a biosensor;

(2) Covering a coating on the biosensor, treating the biosensor with 6-hydrazino nicotinic acid succinimide ester hydrochloride, and washing the biosensor with water;

(3) And (3) spotting the modified probe on a biosensor to prepare the gene chip.

Preferably, the substrate in the step (1) is a silicon wafer (Si) with a thickness of 2.5mm and a diameter of 20-25 cm.

Preferably, the film coating in the step (1) is to coat a silicon nitride film with the thickness of 45-50nm and a TSPS (T structure polydimethylsiloxane) film with the thickness of 12-15nm on a silicon wafer.

preferably, the coating in the step (2) is a polyphenylalanine-lysine coating, and the thickness of the polyphenylalanine-lysine coating is 15 nm; the concentration of the 6-hydrazino nicotinic acid succinimide ester hydrochloride is 1-10 mu mol/L, and the treatment time of the 6-hydrazino nicotinic acid succinimide ester hydrochloride is 15-25 min.

preferably, in the step (3), one end of the probe is modified by aldehyde group or amino group.

A kit for detecting genes related to exercise-induced sudden death comprises a gene chip, a PCR reaction system, a hybridization buffer solution, a washing solution, a BW reaction solution (horseradish peroxidase + sodium citrate buffer solution) and a TMB (3,3',5,5' -tetramethylbenzidine) color development solution.

Preferably, the PCR reaction system comprises the following components: 5 × Buffer, dUTP mix, F-primer, R-primer, Hot taq, UNG enzyme (uracil DNA glycosylase) and dd H₂o (double distilled water).

The UNG enzyme is added into a PCR reaction system, so that the pollution of amplification products can be removed, and false positive results are reduced.

Preferably, the sequence of the F-primer is:

663-F：5'-TGCATCTCTTTCTGGCATT-3'；

723/731/736-F：5'-TCTATGGGCAGAGCAGATCAC-3'；

842-F：5'-TGCGGCTGAACTTCGACC-3'；

190-F：5'-GAGTACGTGGTCCGCCT-3'；

305-F：5'-CATCTTCTCCTCGTACTTTGTG-3'；

2401-F：5'-GGAACGCGATGACC-3'。

Preferably, the sequence of the R-primer is:

663-R：5'-CCTCACCTGGAGACTTTG-3'；

723/731/736-R：5'-ACTGGTTGTGATCAATGTCCAG-3'；

842-R：5'-AGGCTCACCGATAGGCAT-3'；

190-R：5'-TCCACAGGGCAGGCATGACTC-3'；

305-R：5'-CACCAGTGCCCAGATGTC-3'；

2401-R：5'-ATGCTGTTATGCTCTTTGAC-3'。

Preferably, the hybridization buffer comprises 0.3mol/L trisodium citrate, 3mol/L sodium chloride, 0.3mol/L sodium dodecyl sulfate and Triton X-1000.1% aqueous solution.

Preferably, the washing solution is a 0.1 XSSC solution.

A detection method of a gene related to sudden exercise death adopts the kit for detection, and comprises the following steps:

(1) Adding the substance to be detected into a PCR reaction system for amplification to obtain a PCR amplification product;

(2) Adding PCR amplification product and hybridization buffer solution on the gene chip for reaction;

(3) washing the gene chip with 0.1 XSSC solution, and drying in the air after washing;

(4) taking BW reaction liquid to react on a gene chip at room temperature;

(5) adding TMB color developing solution for color development, washing the gene chip with 0.1 XSSC solution before and after adding the TMB color developing solution, air drying, and taking pictures or reading the detection result by naked eyes.

the sample contains DNA fragments.

the detection of the kit and the object to be detected is hybridization reaction, and when the object to be detected contains SNP (single nucleotide polymorphism) sites of a target gene which can be specifically combined with the probe, the gene chip area corresponding to the probe is blue.

Preferably, the conditions for amplification in step (1) are: reacting at 95 ℃ for 10 min; denaturation at 94 ℃ for 30s, renaturation at 56 ℃ for 30s, and extension at 72 ℃ for 30s, and repeating 40 cycles; extension at 72 ℃ for 5 min.

preferably, the addition amount of the PCR amplification product in the step (2) is 10. mu.L, and the hybridization buffer is 100. mu.L; the reaction conditions were: reacting for 30-60min at 50-60 ℃.

Preferably, the temperature of the 0.1 XSSC solution in step (3) is 50 ℃ and the washing time is 1 min.

preferably, the amount of BW reaction solution added in step (4) is 100. mu.L, and the reaction time is 10 min.

preferably, the addition amount of the TMB color developing solution in the step (5) is 100 mu L, and the reaction time is 5 min; the mixture was washed 3 times with 0.1 XSSC solution before and after addition of TMB developing solution, each for 1 min.

compared with the prior art, the invention has the following beneficial effects:

(1) By adopting a gene chip method, the one-time multi-site determination of the genes related to the sudden exercise death is realized, and the detection time and the detection cost are reduced;

(2) the invention combines the gene chip method through the selection of the specific probe and the primer, and has the advantages of good specificity, high accuracy, rapidness and convenience;

(3) the visible light optical amplification of the gene chip can achieve higher signal resolution;

(4) The detection result can be directly read by photographing or naked eyes, and is simple and convenient.

drawings

FIG. 1 is a diagram showing the results of detecting mixed plasmids containing plasmids 1, 3, 5, 7, 9, 11, 13 and 15 using the gene chip and the kit prepared in examples 1-2 of the present invention;

FIG. 2 shows the results of detecting mixed plasmids containing plasmids 2, 4, 6, 8, 10, 12, 14 and 16 using the gene chip and the kit prepared in example 1-2 of the present invention;

FIG. 3 shows the results of testing the plasmid mixture containing plasmids 1-16 using the gene chip and the kit prepared in examples 1-2 of the present invention.

Detailed Description

in order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.

example 1

A probe for detecting a gene related to sudden exercise death, which has the sequence as follows:

I736I probe: GTTCATTGATAGCAGGA, respectively;

I736T probe: GTTCACTGATAGCAGGA, respectively;

P731P probe: AGCGGCCATCCCTGAGGG, respectively;

P731S probe: AGCGGCCATCTCTGAGGG, respectively;

R723R probe: CCTCAGGTATCGCATCCT, respectively;

R723G probe: CCTCAGGTATGGCATCCT, respectively;

R663R probe: CAACTTGCGCTCCACCCA, respectively;

R663H probe: CAACTTGCACTCCACCCA, respectively;

Y842Y probe: GTGTACGAGATGCGCGTC, respectively;

Y842X probe: GGTGTAGGAGATGCGCGT, respectively;

R190R probe: CTCTGGGGGCGGCTGCGC, respectively;

R190W probe: CTCTGGGGGTGGCTGCGC, respectively;

W305W probe: CGCTGTGGTGGGGGGTGG, respectively;

W305L probe: CGCTGTGGTTGGGGGTGG, respectively;

R2401R probe: CTCTTGGGACGCTGTGCTC, respectively;

R2401H probe: CTCTTGGGACACTGTGCTC are provided.

a gene chip containing the probe for detecting the gene related to the sudden exercise death comprises the following steps:

A silicon nitride film with the thickness of 47.5nm and a TSPS film with the thickness of 13.5nm are coated on a silicon wafer (Si) with the thickness of about 2.5mm and the diameter of 20cm by a rotary vacuum coating machine and a vacuum vapor deposition method to prepare a corresponding biosensor, a polyphenylalanine-lysine coating with the thickness of 15nm is covered on the biosensor, finally, the biosensor is treated by 10 mu mol/L6-hydrazino nicotinic acid succinimide ester hydrochloride for 20 minutes, and the chip is cleaned by clear water. Modifying the 5' end of the probe for detecting the genes related to the sudden exercise death by aldehyde group, spotting the probe on a processed chip, and reacting at room temperature to prepare the gene chip containing the probe.

Each probe was placed in three parallel arrays on the chip, so the arrangement of the probes on the chip is shown in Table 1:

table 1: gene chip probe sample application arrangement

The quality control probe is PolyA, and the sequence is AAAAAAAAAAAAAAAAAA.

The quality control probe not only plays a role in positioning but also plays a role in quality control on the gene chip. When the detection result does not develop color at the point of sample application of the quality control probe, the gene chip is proved to be invalid.

Example 2

a kit for detecting folate gene polymorphism, which comprises the gene chip, PCR reaction system, hybridization buffer, washing solution, BW reaction solution and TMB color solution of example 1.

Wherein the composition of the PCR reaction system is shown in Table 2:

table 2: PCR reaction System composition

Wherein the sequence of the F-primer in the PCR reaction system is as follows:

663-F：5'-TGCATCTCTTTCTGGCATT-3'；

723/731/736-F：5'-TCTATGGGCAGAGCAGATCAC-3'；

842-F：5'-TGCGGCTGAACTTCGACC-3'；

190-F：5'-GAGTACGTGGTCCGCCT-3'；

305-F：5'-CATCTTCTCCTCGTACTTTGTG-3'；

2401-F：5'-GGAACGCGATGACC-3'。

wherein the sequence of the R-primer in the PCR reaction system is as follows:

663-R：5'-CCTCACCTGGAGACTTTG-3'；

723/731/736-R：5'-ACTGGTTGTGATCAATGTCCAG-3'；

842-R：5'-AGGCTCACCGATAGGCAT-3'；

190-R：5'-TCCACAGGGCAGGCATGACTC-3'；

305-R：5'-CACCAGTGCCCAGATGTC-3'；

2401-R：5'-ATGCTGTTATGCTCTTTGAC-3'。

wherein the hybridization buffer comprises 0.3mol/L trisodium citrate, 3mol/L sodium chloride, 0.3mol/L sodium dodecyl sulfate and Triton X-1000.1% aqueous solution.

Wherein the washing solution is a 0.1 XSSC solution.

Wherein the Hot-Taq enzyme, dUTP and UNG enzyme are purchased from Shenzhen Fenpeng biological member GmbH.

wherein the primer and the probe are designed by self and synthesized by Shanghai biological engineering Co.

Example 3

the gene chip and the kit prepared in example 1-2 were used for detection, and the object to be detected was a plasmid containing the SNP site of the target gene, and the types of the plasmid are shown in Table 3:

Table 3: plasmid type

plasmids	Containing the target gene	SNP site
			Plasmid 1	MYH7	R663R
plasmid 2	MYH7	R663H
			plasmid 3	MYH7	R723R
Plasmid 4	MYH7	R723G
			Plasmid 5	MYH7	P731P
Plasmid 6	MYH7	P731S
			plasmid 7	MYH7	I736I
Plasmid 8	MYH7	I736T
			Plasmid 9	MYBPC3	Y842Y
Plasmid 10	MYBPC3	Y842X
			plasmid 11	KCNQ1	R190R
plasmid 12	KCNQ1	R190W
			Plasmid 13	KCNQ1	W305W
Plasmid 14	KCNQ1	W305L
			plasmid 15	RyR2	R2401R
plasmid 16	RyR2	R2401H

the above plasmids are synthesized and provided by Shanghai Bioengineering Co., Ltd.

Plasmids 1, 3, 5, 7, 9, 11, 13 and 15 were mixed to obtain a mixed plasmid, and the mixed plasmid was dissolved in 1mL of TE buffer so that the concentration of the plasmid was 2. mu.g/mL, i.e., 2000 ng/mL. And adding 5 mu L of plasmid solution serving as a substance to be detected into a PCR reaction system to ensure that the total reaction volume is 50 mu L, and preparing a PCR reaction mixed solution.

Putting the PCR reaction mixed solution into a PCR instrument for PCR amplification, wherein the conditions of the PCR amplification are as follows:

reacting at 95 ℃ for 10 min; denaturation at 94 ℃ for 30s, renaturation at 56 ℃ for 30s, and extension at 72 ℃ for 30s, and repeating 40 cycles; extension at 72 ℃ for 5 min.

After the amplification is finished, heating the PCR amplification product for 3 minutes at 95 ℃, and quickly placing the PCR amplification product on ice;

Taking 10 mu L of PCR amplification product to the prepared chip;

100 mu L of hybridization buffer solution is taken to be arranged on a chip and reacted for 60 minutes at 55 ℃;

washing the chip in 0.1 XSSC solution at 50 deg.C for 1min, and air drying the chip surface;

Taking 100 mu L of BW reaction solution on a chip, and reacting for 10 minutes at room temperature;

washing the chip with 0.1 XSSC solution for 3 times, each time for 1 minute, and air-drying the surface of the chip;

Taking 100 mu L of TMB color developing solution on the surface of the chip, and reacting for 5 minutes;

the chip was washed 3 times with 0.1 XSSC solution for 1 minute each time, the chip surface was air dried, and the results were read by photographing or by naked eye.

As shown in FIG. 1, the detection results were that the I736I probe, P731P probe, R723R probe, R663R probe, Y842Y probe, R190R probe, W305W probe and R2401R probe all showed blue color at the spot location of the gene chip (zones 1-8, respectively), and the spot location of the quality control probe (zone A) also showed blue color. It was confirmed that the gene chip and the kit prepared in example 1-2 can accurately detect the R663R, R723R, P731P, I736I, Y842Y, R190R, W305W, and R2401R sites. The color development at the probe proves that the substance to be detected contains the SNP sites specifically bound with the probe, for example, parallel sites in the regions 3 and 5 have light color development but still have color development, and the light color development does not influence the detection result.

example 4

example 4 is substantially the same as example 3 except that the test substance used is a mixed plasmid containing plasmids 2, 4, 6, 8, 10, 12, 14, and 16.

the test substance was detected using the gene chip and the kit prepared in example 1-2.

As shown in FIG. 2, the detection results were confirmed that the I736T probe, P731S probe, R723G probe, R663H probe, Y842X probe, R190W probe, W305L probe and R2401H probe all showed blue color at the spot of the gene chip (regions 9-16, respectively), and the spot of the quality control probe (region A) also showed blue color, and that the gene chip and the kit prepared in example 1-2 could accurately detect the I736T, P731S, R723G, R663H, Y842X, R190W, W305L and R2401H sites.

example 5

example 5 is substantially the same as example 3 except that the specimen used is a mixed plasmid containing plasmids 1 to 16.

the test substance was detected using the gene chip and the kit prepared in example 1-2.

as shown in FIG. 3, the detection results were confirmed to be excellent in specificity even when 16 types of plasmids were mixed by using the gene chip and the kit prepared in examples 1 to 2, and accurate detection results were obtained in the same manner as the I736I probe, the P731P probe, the R723R probe, the R663R probe, the Y842Y probe, the R190R probe, the W305W probe, the R2401R probe, the I736T probe, the P731S probe, the R723G probe, the R663H probe, the Y842X probe, the R190W probe, the W305L probe, and the R2401H probe all showed blue colors at the spot location (regions 1 to 16, respectively) on the gene chip.

Claims (10)

1. a probe for detecting a sudden kinetic death-related gene, wherein the sequence of the probe is:

I736I probe: GTTCATTGATAGCAGGA, respectively;

I736T probe: GTTCACTGATAGCAGGA, respectively;

P731P probe: AGCGGCCATCCCTGAGGG, respectively;

P731S probe: AGCGGCCATCTCTGAGGG, respectively;

R723R probe: CCTCAGGTATCGCATCCT, respectively;

R723G probe: CCTCAGGTATGGCATCCT, respectively;

R663R probe: CAACTTGCGCTCCACCCA, respectively;

R663H probe: CAACTTGCACTCCACCCA, respectively;

Y842Y probe: GTGTACGAGATGCGCGTC, respectively;

Y842X probe: GGTGTAGGAGATGCGCGT, respectively;

R190R probe: CTCTGGGGGCGGCTGCGC, respectively;

R190W probe: CTCTGGGGGTGGCTGCGC, respectively;

W305W probe: CGCTGTGGTGGGGGGTGG, respectively;

W305L probe: CGCTGTGGTTGGGGGTGG, respectively;

R2401R probe: CTCTTGGGACGCTGTGCTC, respectively;

R2401H probe: CTCTTGGGACACTGTGCTC are provided.

2. a gene chip for detecting a gene associated with sudden exercise death, which comprises the probe of claim 1.

3. A method for preparing the gene chip of claim 2, comprising the steps of:

(1) coating a film on a substrate to obtain a biosensor;

(2) covering a coating on the biosensor, treating the biosensor with 6-hydrazino nicotinic acid succinimide ester hydrochloride, and washing the biosensor with water;

(3) And (3) spotting the modified probe on a biosensor to prepare the gene chip.

4. The preparation method according to claim 3, wherein one end of the probe is modified with aldehyde or amino in the step (3).

5. a kit for detecting a gene associated with sudden exercise death, comprising the gene chip of claim 2, a PCR reaction system, a hybridization buffer, a washing solution, a BW reaction solution and a TMB color developing solution.

6. The kit of claim 5, wherein the PCR reaction system comprises the following components: 5 XBuffer, dUTP mix, F-primer, R-primer, Hot taq, UNG enzyme and dd H2O.

7. The kit of claim 6, wherein the F-primer has the sequence:

663-F：5'-TGCATCTCTTTCTGGCATT-3'；

723/731/736-F：5'-TCTATGGGCAGAGCAGATCAC-3'；

842-F：5'-TGCGGCTGAACTTCGACC-3'；

190-F：5'-GAGTACGTGGTCCGCCT-3'；

305-F：5'-CATCTTCTCCTCGTACTTTGTG-3'；

2401-F：5'-GGAACGCGATGACC-3'。

8. the kit according to claim 6, wherein the sequence of the R-primer is:

663-R：5'-CCTCACCTGGAGACTTTG-3'；

723/731/736-R：5'-ACTGGTTGTGATCAATGTCCAG-3'；

842-R：5'-AGGCTCACCGATAGGCAT-3'；

190-R：5'-TCCACAGGGCAGGCATGACTC-3'；

305-R：5'-CACCAGTGCCCAGATGTC-3'；

2401-R：5'-ATGCTGTTATGCTCTTTGAC-3'。

9. The kit of claim 5, wherein the hybridization buffer comprises 0.3mol/L trisodium citrate, 3mol/L sodium chloride, 0.3mol/L sodium dodecyl sulfate, and Triton X-1000.1% aqueous solution.

10. a method for detecting a sudden exercise death-related gene, which comprises the steps of using the kit according to any one of claims 5 to 9 for detection, comprising:

(1) Adding the substance to be detected into a PCR reaction system for amplification to obtain a PCR amplification product;

(2) Adding PCR amplification product and hybridization buffer solution on the gene chip for reaction;

(3) Washing the gene chip with 0.1 XSSC solution, and drying in the air after washing;

(4) taking BW reaction liquid to react on a gene chip at room temperature;

(5) adding TMB color developing solution for color development, washing the gene chip with 0.1 XSSC solution before and after adding the TMB color developing solution, air drying, and taking pictures or reading the detection result by naked eyes.