CN109853045B - Gene chip for high-flux detection - Google Patents
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Abstract
The application discloses a high-flux detection gene chip, which comprises a solid phase carrier layer and a gene chip area arranged on the solid phase carrier layer, wherein a probe array is arranged on the gene chip area, the probe array is a concentric array, and probe points on two adjacent circular rings forming the concentric array are staggered. The probe array of the high-flux detection gene chip is a concentric circle array, and the probe array points of two adjacent circles are staggered, so that the mutual interference between the two adjacent points in the probe synthesis process can be avoided, the final signal identification is influenced, and the detection accuracy is further improved; the structure is succinct, convenient to use has reduced the experiment degree of difficulty, has improved the degree of accuracy of testing result.
Description
Technical Field
The application relates to a gene chip for high-flux detection, and belongs to the field of gene detection.
Background
With the implementation of the human genome (sequencing) program and the rapid development of molecular biology-related disciplines, more and more animal, plant and microorganism genome sequences are being determined, and the speed of gene sequence data is rapidly increasing. The gene chip detection has been widely used in the fields of disease diagnosis and treatment, drug screening, judicial identification, environmental detection, etc. because of its high throughput and rapid and efficient detection characteristics. The gene chip adopts a large number of specific oligonucleotide fragments or gene fragments as probes, and is regularly fixed on a silicon wafer, a glass sheet, a plastic sheet or a nylon substrate and other solid supports combined with a photoelectric measuring device to form a two-dimensional array, and the two-dimensional array is hybridized with genes of a marked sample to be detected according to a base pairing principle, so that the specific genes are detected.
The experimental steps for detecting by using the gene chip mainly comprise: firstly, carrying out PCR amplification on genetic materials in a nucleic acid amplification reaction container, sucking out amplification products after a certain enrichment amount is reached, adding the amplification products to a gene chip for chip probe hybridization analysis, eluting the chip after hybridization to remove unbound reactants, then placing the chip into a detector for signal detection, and finally carrying out data analysis on detection signals. The existing two-dimensional array of gene chips is a matrix array, and because the reaction pores on the front, back, left and right sides are all adjacent, the interference between adjacent probes is large when the probes are fixed on a support, and particularly oligonucleotide probes are synthesized by using an in-situ synthesis method, the technology mainly comprises the following steps: the support is first hydroxylated and then protected with a photoactive protecting group. The light shielding film is selected appropriately every time to make the portions to be polymerized pass light, and the other portions are not passed light. Thus, light is irradiated to the support through the light shielding film, and the hydroxyl groups in the light-receiving portion are deprotected and then participate in the reaction. Because the distance between two adjacent probes of the matrix array is too short, the distance between the light transmission holes of the light shielding film is very small, and the problem of light diffraction exists, so that the final hybridization signal is blurred, and the signal to noise ratio is reduced. At the same time, the matrix array can make elution after hybridization difficult and can easily interfere with detection signals.
Disclosure of Invention
In order to solve the problems in the prior art, the application provides a high-throughput detection gene chip.
In order to solve the technical problems, the technical scheme adopted by the application is as follows:
the gene chip for high flux detection includes solid carrier layer and gene chip area on the solid carrier layer, and the gene chip area has probe array in the concentric array and with staggered probe points.
The probe array on the gene chip is a concentric array, and the probe array points of two adjacent circular rings are staggered, so that the mutual interference between the two adjacent points in the probe synthesis process can be avoided, and the final signal identification is influenced.
The gene chip of the application can obviously improve the accuracy of gene detection.
As a matter of common sense, a concentric circle array is made up of a plurality of concentric circles.
In order to improve hybridization efficiency and facilitate elution, the gene chip region comprises a base film layer and a bearing layer which are connected up and down, wherein the base film layer is connected with a solid phase carrier layer, the probe array is arranged on the bearing layer, and the height difference exists between two adjacent circular rings forming a concentric circular array on the bearing layer. In the hybridization process, the reaction liquid is kept at a position which is higher than the position at which the reaction liquid is left to a position which is lower than the position at which the reaction liquid is left, so that the hybridization efficiency is improved; during elution, the eluent is conveniently left from high to low, and then the elution is convenient.
In order to further improve hybridization efficiency and facilitate elution, two adjacent rings forming a concentric circular array are arranged on the bearing layer, and the height of the inner ring is higher than that of the outer ring. On the one hand, the design can enable the reaction solution to flow to the outer ring in sequence in the hybridization process, improve the hybridization efficiency, and on the other hand, the eluent can flow to the outer ring in the elution process, so that the elution is convenient and quick.
According to the application, the direction of the circle center of the concentric circular array pointing to the periphery of the concentric circular array is regarded as the direction from inside to outside, namely two adjacent circular rings, and the radius of the inner circular ring is smaller than that of the outer circular ring.
For convenient control and use, the height difference of any two adjacent circular rings forming the concentric circular array on the bearing layer is equal.
In order to improve the detection accuracy, the height difference between the innermost circular ring and the outermost circular ring forming the concentric circular array on the bearing layer is 1/5-1/2 of the radius of the concentric circular array.
For convenient use, 2-10 gene chip areas are arranged on the same solid phase carrier layer; the bearing layer is a slide layer or a plastic layer.
Further preferably, the support layer is at least one of a nitrocellulose layer or a nylon layer or a polystyrene layer.
In order to realize the measurement of a plurality of targets, the probe array consists of more than two groups of test units, and each group of test units comprises a detection probe, a positive quality control probe, a negative quality control probe and a blank reference point.
As a preferable scheme of the application, the detection probes, the positive quality control probes, the negative quality control probes and the blank reference points of the same group of test units are arranged on the same ring.
As another preferable scheme of the application, the detection probes, the positive quality control probes, the negative quality control probes and the blank reference points of the same group of test units are arranged on the same sector or on the same radial direction.
The technology not mentioned in the present application refers to the prior art.
The beneficial effects of the application are as follows:
the probe array of the high-flux detection gene chip is a concentric circle array, and the probe array points of two adjacent circles are staggered, so that the mutual interference between the two adjacent points in the probe synthesis process can be avoided, the final signal identification is influenced, and the detection accuracy is further improved; the structure is simple, the use is convenient, the experiment difficulty is reduced, and the accuracy of the detection result is improved; further, the circular rings forming the concentric circular array are gradually reduced from inside to outside, so that on one hand, the reaction liquid can flow outwards in sequence in the hybridization process, the hybridization efficiency is improved, and the eluent can flow outwards again in the other direction during elution, so that the elution is convenient and rapid.
Drawings
FIG. 1 is a schematic diagram of the structure of a high throughput assay gene chip of the present application;
FIG. 2 is a schematic diagram of a probe array on a gene chip region for high throughput detection according to the present application (in the figure, the connection line between two adjacent probe points is an auxiliary line);
FIG. 3 is a cross-sectional view of a gene chip region according to the present application;
FIG. 4 is a diagram showing the arrangement of probes in the application example, wherein the left diagram is a control group (square), and the right diagram is a circle of the present application;
FIG. 5 is a scan of a 5 adenovirus nucleic acid gene chip of the application example, the left panel is a control group, and the right panel is the application;
in the figure, 1: solid phase carrier, 2: gene chip, 21 probe spots.
Detailed Description
For a better understanding of the present application, the following examples are further illustrated, but are not limited to the following examples.
Example 1
The gene chip for high flux detection includes solid carrier layer and 6 gene chip areas on the solid carrier layer, and probe arrays in the probe areas are set on the gene chip areas and are concentric arrays with staggered probe points in two adjacent rings.
The probe array on the gene chip is a concentric circle array, and the probe points of two adjacent circles are staggered, so that the mutual interference between the two adjacent points in the probe synthesis process can be avoided, the final signal identification is influenced, and the accuracy of gene detection is obviously improved.
Example 2
Substantially the same as in example 1, except that: the gene chip region comprises a base film layer and a bearing layer which are connected up and down, wherein the bearing layer is a nitrocellulose layer, the base film layer is connected with a solid phase carrier layer, the probe array is arranged on the bearing layer, two adjacent rings forming a concentric circle array are arranged on the bearing layer, the height of an inner ring is higher than that of an outer ring, the height difference of any two adjacent rings forming the concentric circle array on the bearing layer is equal, and the height difference of the innermost ring and the outermost ring forming the concentric circle array on the bearing layer is 1/3 of the radius of the concentric circle array. On the one hand, the design can enable the reaction solution to flow to the outer ring in sequence in the hybridization process, improve the hybridization efficiency, and on the other hand, the eluent can flow to the outer ring in the elution process, so that the elution is convenient and quick.
Example 3
Substantially the same as in example 2, except that: the height difference between the innermost circular ring and the outermost circular ring forming the concentric circular array on the bearing layer is 1/5 of the radius of the concentric circular array, and the bearing layer is a slide layer.
Example 4
Substantially the same as in example 2, except that: the height difference between the innermost circular ring and the outermost circular ring forming the concentric circular array on the bearing layer is 1/2 of the radius of the concentric circular array, and the bearing layer is a nylon layer.
Example 5
Substantially the same as in example 2, except that: in order to realize the measurement of a plurality of targets, the probe array consists of more than two groups of test units, and each group of test units comprises a detection probe, a positive quality control probe, a negative quality control probe and a blank reference point.
Example 6
Substantially the same as in example 5, except that: the detection probes, the positive quality control probes, the negative quality control probes and the blank reference points of the same group of test units are arranged on the same circular ring.
Example 7
Substantially the same as in example 5, except that: the detection probes, the positive quality control probes, the negative quality control probes and the blank reference points of the same group of test units are arranged on the same sector or on the same radial direction.
The probe arrays of the gene chips are concentric circular arrays, and the probe array points of two adjacent circular rings are staggered, so that the mutual interference between the two adjacent points in the probe synthesis process can be avoided, the final signal identification is influenced, and the detection accuracy is further improved; the structure is simple, the use is convenient, the experiment difficulty is reduced, and the accuracy of the detection result is improved; further, the circular rings forming the concentric circular array are gradually reduced from inside to outside, so that on one hand, the reaction liquid can flow outwards in sequence in the hybridization process, the hybridization efficiency is improved, and the eluent can flow outwards again in the other direction during elution, so that the elution is convenient and rapid.
Application examples
The chip used was an autonomously synthesized gene chip, and the chip of the experimental group was the chip of example 7, the substrate of which was aldehyde-formed, using a full-automatic spotter of Genomic Solution company in the united states. The gene chip of the control group adopts an aldehyde substrate of Boao biological limited company.
In the embodiment, five types of 3, 7, 14, 11 and 55 of the subgenera of the adenovirus B are selected in a GeneBank database, a gene chip is designed aiming at the conserved regions of the five viruses, and the detection results of the two chips are compared by detecting the viruses, and the detection method adopts a chemiluminescence biochip imager to carry out chip scanning analysis.
Step one: extraction of viral nucleic acid:
and selecting a DNA/RNA extraction kit, extracting nucleic acid from the adenovirus of the types 3, 7, 14, 11 and 55 according to the kit instruction, and storing the extracted sample nucleic acid in a refrigerator at the temperature of-70 ℃.
Step two: designing a primer:
selecting a nucleic acid sequence of common adenovirus in GenBank database, comparing the downloaded sequences by using alignment software, selecting a sequence region which is conserved in the types and specific among the types, and designing a specific primer by combining with molecular biology software, wherein the 11 type and 55 type viruses share a pair of primers, and the primers are as follows:
PCR amplification is carried out by taking virus nucleic acid as a template, and a vector is constructed and transformed into DH5 alpha competent cells.
Step three: designing a probe:
the gene sequence between the upstream and downstream primers is designed into a probe, a (T) 12 structure is added at the 3' end of the probe, and the terminal of the (T) 12 is marked with-NH 2, so that the space flexibility of the probe is improved. Designing an internal standard probe, and obtaining the following available probes after designing and screening the probe:
step four: preparation of the chip:
the probe lyophilized powder was dissolved by RNase-Free Water, and the probe spotting solution (6 XSSC, 0.1% SDS) was mixed with the same volume of each dissolved probe to give a final probe concentration of 50. Mu.M/L. The repeated sequence of (T) 20 is used as a mark column to prompt the relative position of the probe on the chip, and meanwhile, whether the probe is combined with the chip or not can be detected. The 5 '-end of the (T) 20 repetitive sequence is subjected to biotin labeling, and the 3' -end is subjected to NH2 modification. 10 μl of each of the probe mixed solution and the identification column is added into 384 well plates with coded serial numbers, 3 sites are designed for each probe by setting a sample application program on a sample application instrument, and 1 positive quality control probe, 1 negative quality control probe, 1 blank control probe and 1 identification column, namely a chip quality control probe are designed for each chip area, so that the preparation of the gene chip is completed. The arrangement of the probes is shown in FIG. 4, where the system numbers explain: (1) a sheet matrix control probe, (2) a positive quality control probe, (3)3 type adenovirus probe, (4)7 type adenovirus probe, (5) a 14 type adenovirus probe, (6) a 11 type adenovirus probe, (7) a 55 type adenovirus probe, (8) an internal standard probe, and (9) a negative quality control probe.
Step five: hybridization of chips:
performing PCR amplification by taking virus nucleic acid as a template, hybridizing an amplification product with a probe on a chip, and pre-denaturing for 10min at 95 ℃;95 ℃ for 30s,55 ℃ for 30s and 72 ℃ for 60s, and 35 cycles are total; final extension at 72℃for 5min.
The PCR reaction system is as follows:
the amplified products are denatured to be in a melting state, 5 mu l of the amplified products are evenly mixed with 5 mu l of preheated hybridization solution, 8 mu l of the amplified products are absorbed and added into a chip reaction zone, the chip is placed into a hybridization box, hybridization is carried out for 1h in a water bath at 45 ℃, and after the hybridization reaction is finished, the chip is washed and dried. Then 10 μl of the labeling solution was added, the mixture was washed with water bath at 37deg.C for 25min, followed by washing with 1xPBST and deionized water for 20s, and spin-dried on a chip centrifuge. And adding the chemiluminescent and chromogenic mixed solution into a chip reaction area, and putting the chip reaction area into an imager for scanning imaging and then analyzing results.
A scan of 5 adenovirus nucleic acid gene chips is shown in FIG. 5. The left graph shows the scanning result of the chip of the control group, and the right graph shows the scanning result of the chip of the application, so that the brightness of the chip of the application is obviously higher than that of the traditional chip under the conditions of identical detection samples, identical experimental steps and identical experimental conditions in the position areas of 3, 7, 14, 11 and 55 viruses. In the areas of the chip substrate control probe, the positive probe and the internal control probe, the chip has higher brightness, clear luminous points and no fuzzy halation around the points.
The reproducibility of the detection was evaluated for two gene chips:
the plasmids of each virus are taken for gene chip detection, no-template negative control is established, the same chip is provided with different reaction areas, the repeated 5 times are carried out among different batches of chips, the statistical results of the control group chips are shown in the following table 1, and the statistical results of the experimental chips are shown in the following table 2.
TABLE 1
TABLE 2
As can be seen from the data in the above table, for each probe of the 5 viruses, the intra-chip repeatability variation coefficient value was less than 15%, and the inter-chip repeatability variation coefficient value was also less than 15%, indicating that the detection method was better in repeatability. On the other hand, the repeatability coefficient value of the chip is obviously superior to that of the chip of the control group, which proves that the chip of the application has better detection effect than the chip of the control group.
Claims (6)
1. A high-throughput detection gene chip is characterized in that: high throughput detection gene chip: the gene chip region is provided with a probe array, the probe array is a concentric circle array, and probe points on two adjacent circles forming the concentric circle array are staggered;
the gene chip region comprises a base film layer and a bearing layer which are connected up and down, wherein the base film layer is connected with the solid phase carrier layer, the probe array is arranged on the bearing layer, and the height difference exists between two adjacent circular rings forming a concentric circular array on the bearing layer;
the probe array consists of more than two groups of test units, wherein each group of test units comprises a detection probe, a positive quality control probe, a negative quality control probe and a blank reference point;
the detection probes, the positive quality control probes, the negative quality control probes and the blank reference points of the same group of test units are arranged on the same sector or on the same radial direction;
the gene chip for high-throughput detection is used for detecting adenovirus type 3, adenovirus type 7, adenovirus type 14, adenovirus type 11 and adenovirus type 55;
in detection, the 11 type adenovirus and the 55 type adenovirus share a pair of primers, and the primers are designed as follows:
the probe design is as follows:
2. the high throughput assay gene chip of claim 1, wherein: the height difference of any two adjacent circular rings forming the concentric circular array on the bearing layer is equal.
3. The high throughput assay gene chip of claim 1 or 2, wherein: two adjacent rings forming a concentric circular array on the bearing layer, wherein the height of the inner ring is higher than that of the outer ring.
4. The high throughput assay gene chip of claim 3, wherein: the height difference between the innermost circular ring and the outermost circular ring on the bearing layer forming the concentric circular array is 1/5-1/2 of the radius of the concentric circular array.
5. The high throughput assay gene chip of claim 1 or 2, wherein: 2-10 gene chip areas are arranged on the same solid phase carrier layer; the bearing layer is at least one of a slide layer or a plastic layer.
6. The high throughput assay gene chip of claim 5, wherein: the bearing layer is at least one of a nitrocellulose layer, a nylon layer or a polystyrene layer.
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| CN1338519A (en) * | 2000-08-18 | 2002-03-06 | 莫志宏 | In-situ biochip and its preparing process |
| CN1608137A (en) * | 2001-10-15 | 2005-04-20 | 生物芯片技术有限公司 | Multiplexed analysis of polymorphic loci by concurrent interrogation and enzyme-mediated detection |
| JP2006201119A (en) * | 2005-01-24 | 2006-08-03 | Olympus Corp | Solid-phase carrier |
| CN205133582U (en) * | 2015-10-28 | 2016-04-06 | 北京泱深生物信息技术有限公司 | Genetic chip that layering set up |
| CN209443109U (en) * | 2018-12-03 | 2019-09-27 | 南京吉量生物科技有限公司 | A kind of genetic chip of high-throughput detection |
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| JP2005283306A (en) * | 2004-03-29 | 2005-10-13 | Lintec Corp | Member for manufacturing probe array, and manufacturing method for probe array |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1338519A (en) * | 2000-08-18 | 2002-03-06 | 莫志宏 | In-situ biochip and its preparing process |
| CN1608137A (en) * | 2001-10-15 | 2005-04-20 | 生物芯片技术有限公司 | Multiplexed analysis of polymorphic loci by concurrent interrogation and enzyme-mediated detection |
| JP2006201119A (en) * | 2005-01-24 | 2006-08-03 | Olympus Corp | Solid-phase carrier |
| CN205133582U (en) * | 2015-10-28 | 2016-04-06 | 北京泱深生物信息技术有限公司 | Genetic chip that layering set up |
| CN209443109U (en) * | 2018-12-03 | 2019-09-27 | 南京吉量生物科技有限公司 | A kind of genetic chip of high-throughput detection |
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