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WO2019100842A1 - 一种检测核苷酸特异性和/或非特异性吸附的方法 - Google Patents

一种检测核苷酸特异性和/或非特异性吸附的方法 Download PDF

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Publication number
WO2019100842A1
WO2019100842A1 PCT/CN2018/107249 CN2018107249W WO2019100842A1 WO 2019100842 A1 WO2019100842 A1 WO 2019100842A1 CN 2018107249 W CN2018107249 W CN 2018107249W WO 2019100842 A1 WO2019100842 A1 WO 2019100842A1
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image
chip
detecting
nucleotide
preprocessed
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English (en)
French (fr)
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赵陆洋
黄天逊
吴增丁
颜钦
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Direct Genomics Co Ltd
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Direct Genomics Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/0002—Inspection of images, e.g. flaw detection
    • G06T7/0012—Biomedical image inspection
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/10—Image acquisition modality
    • G06T2207/10004—Still image; Photographic image
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/30—Subject of image; Context of image processing
    • G06T2207/30004—Biomedical image processing
    • G06T2207/30072—Microarray; Biochip, DNA array; Well plate

Definitions

  • the present invention relates to the field of nucleic acid detection, and in particular to a method for detecting nucleotide specific and/or non-specific adsorption.
  • nucleic acid detection on a chip for example, using a chip on which a probe is immobilized on a surface, and further detecting and detecting the captured nucleic acid, including sequence determination, information analysis, etc., probe/nucleic acid complex on the surface of the chip and/or thereon
  • the non-specific adsorption of the substrate to the reaction substrate affects the amount of valid data obtained and the accuracy of the test results.
  • Qualitative or quantitative detection of non-specific adsorption conditions can determine the quality of the chip and the prediction of the test results.
  • a substrate nucleotide (Terminator) on the chip, including non-specific adsorption of substrate nucleotides on the surface of the chip and chip surface.
  • Non-specific adsorption of nucleotides by the probe/probe complex can result in increased sequencing error rates, sequencing throughput, and/or reduced sequencing quality.
  • nucleotides include nucleotide analogs, and different nucleotide analogs may have different non-specific adsorption capacities on the chip surface, probe/nucleic acid complex. For example, performing nucleic acid sequence determination on a chip, the substrate, that is, the molecular size, structure of the nucleotide analog added in the biochemical reaction, and the charge property in the reaction system, etc., affecting the substrate and the chip and/or nucleic acid complex. Binding/adsorption.
  • nucleotide design including nucleotide design, preparation and production, chip production, and nucleic acid sequence detection using a chip.
  • Embodiments of the present invention are directed to at least one of the technical problems existing in the prior art or at least provide a useful commercial choice. To this end, the present invention provides a method of detecting nucleotide-specific and/or non-specific adsorption.
  • a method for detecting nucleotide-specific and/or non-specific adsorption comprising: reacting a nucleotide to be tested with a first template strand, the first template strand and the first chip a probe connection, the first chip comprising a surface of the substrate and a probe fixed at one end to the surface of the substrate, the first template strand carrying a first label, the nucleotide to be tested being capable of pairing with a corresponding base of the first template strand,
  • the other end of the probe extends one base
  • the nucleotide to be detected carries a second mark
  • the first mark can generate a first signal
  • the second mark can generate a second signal
  • the signal on the first chip is detected a first detection result
  • a method of detecting non-specific adsorption of a nucleotide comprising: reacting a nucleotide to be tested with a third template strand, the third template strand being linked to a probe on a third chip,
  • the third chip comprises a surface of the substrate and a probe fixed at one end to the surface of the substrate, and the third template chain carries a third mark, and the nucleotide to be tested cannot be paired with the corresponding base of the third template chain, and the probe cannot be The other end extends one base, the nucleotide to be tested carries a fourth mark, the third mark can generate a third signal, and the fourth mark can generate a fourth signal; the signal on the third chip is detected to obtain a third detection result;
  • the nucleotide to be tested is reacted with a fourth template strand, the fourth template strand is linked to the fourth chip, the fourth chip comprises a surface of the substrate without a probe, and the fourth template
  • a method for detecting nucleotide-specific and/or non-specific adsorption comprising: reacting a nucleotide to be tested with a fifth template strand, the fifth template strand and the fifth chip a probe connection, the fifth chip comprising a surface of the substrate and a probe fixed at one end to the surface of the substrate, the fifth template chain carrying a fifth label, the nucleotide to be tested being capable of pairing with the corresponding base of the fifth template strand,
  • the other end of the probe extends one base
  • the nucleotide to be tested carries a sixth mark
  • the fifth mark can generate a fifth signal
  • the sixth mark can generate a sixth signal
  • the signal on the fifth chip is detected.
  • a fifth detection result reacting the nucleotide to be tested with a sixth template chain, the sixth template chain being connected to a probe on the sixth chip, the sixth chip comprising a substrate surface and a probe having one end fixed to the surface of the substrate
  • the sixth template chain carries a fifth label, and the nucleotide to be tested cannot be paired with the corresponding base of the sixth template strand, and the other end of the probe cannot be extended by one base; the signal on the sixth chip is detected to obtain the first Six test results;
  • reacting the nucleotide to be tested with a seventh template chain the seventh template chain is connected to the seventh chip, the seventh chip comprises a surface of the substrate without the probe; detecting the signal on the seventh chip, obtaining the seventh detection result And detecting the specificity and/or non-specific adsorption of the nucleotide to be detected based on at least two of the fifth detection result, the sixth detection result, and the seventh detection result.
  • the above method for detecting nucleic acid-specific and/or non-specific adsorption can qualitatively or quantitatively detect non-specific adsorption conditions and/or specific adsorption conditions of a probe/template strand on a substrate surface and/or a substrate surface; By detecting the signal on the surface of the substrate by the detection system, it is possible to qualitatively or quantitatively distinguish between specific and non-specific adsorption, and obtain information such as quantification and distribution of non-specific or specific adsorption, which can be used for structural design and preparation of nucleoside analogs, and chip production.
  • FIG. 1 is a schematic diagram showing a pairing/unpairing relationship between a nucleotide to be tested and a corresponding base on a template strand in an embodiment of the present invention
  • FIG. 2 is a schematic flowchart diagram of an image processing method according to an embodiment of the present invention.
  • FIG. 3 is another schematic flowchart of an image processing method according to an embodiment of the present invention.
  • FIG. 4 is a schematic diagram showing the principle of probe immobilization, template strand hybridization, and nucleotide polymerization/adsorption on a substrate surface according to an embodiment of the present invention
  • FIG. 5 is a schematic diagram showing the principle of probe immobilization, template strand hybridization, and nucleotide polymerization/adsorption on the surface of another substrate in an embodiment of the present invention
  • FIG. 6 is a schematic diagram showing the principle of template strand hybridization and nucleotide adsorption on the surface of still another substrate in the embodiment of the present invention.
  • FIG. 7 is an image of a Cy3 fluorescent dot (left image) and an ATTO647N fluorescent dot (right image) in a field of view obtained by photographing an experimental group in an embodiment of the present invention
  • Fig. 9 is a view showing Cy3 fluorescence dots (left image) and ATTO647N fluorescent dots (right image) in one field of view obtained by the control group 2 in the embodiment of the present invention.
  • nucleotides include ribonucleotides, deoxyribonucleotides, and the like, including A, T, C, G, and U, and the like.
  • C is used for cytosine or cytosine analogs
  • G for guanine or guanine analogs
  • A for adenine or adenine analogs
  • T for thymine or thymine analogs
  • U is used to represent uracil or uracil analogs.
  • the "template strand” referred to in the embodiments of the present invention may be DNA and/or RNA, etc., and may also be referred to as a "hybrid strand” in some embodiments, for example, a target DNA strand in the process of detecting nucleic acid using a chip.
  • the "probe” referred to in the embodiments of the present invention may be DNA and/or RNA, etc., and may also be referred to as "primer”, “capture strand” or “fixed strand” in some embodiments.
  • the so-called “probes” can be randomly distributed or regularly distributed on the surface of the substrate, such as arrays, such as capture chips currently on the market, where the probes are generally distributed in an array on the surface of the chip.
  • the so-called “substrate” can be any solid support that can be used to immobilize a nucleic acid sequence, such as a nylon membrane, a glass slide, a plastic, a silicon wafer, a magnetic bead, etc., and the surface of the chip can be used interchangeably with the surface of the substrate unless otherwise specified.
  • the probes are typically attached/immobilized on the surface of the substrate by chemical bonds.
  • the surface of the substrate is a chemically modified surface with reactive groups that can be attached to the probe. Surface modification, fixation, etc. can be carried out using known methods or directly customized or purchased.
  • non-specific adsorption generally refers to the adsorption caused by the force of non-covalent bonds, the force of non-covalent bonds Including hydrophobic interaction, van der Waals force, electrostatic force, etc.; on the chip with probe on the surface, in the process of nucleic acid detection based on the principle of base complementation, generally, the non-specific adsorption of nucleic acid refers to the non-covalent attachment of nucleic acid to the chip. On the surface and / or probe.
  • the so-called “marker” may be any physical, chemical or biologically detectable label.
  • the “signal” referred to in the embodiments of the present invention is a signal generated by the “marker” in a specific situation, typically but not limited.
  • a sexual "marker” is an optically detectable label, such as a fluorescent dye, and correspondingly, the “signal” is a fluorescent signal.
  • the fluorescent dyes such as Cy3 and/or ATTO-647N, are capable of emitting a green fluorescent signal and a red fluorescent signal, respectively, under the excitation of 532 nm and 640 nm wavelengths.
  • test result can be any suitable qualitative or quantitative result, such as visual results, instrumental test results, and the like.
  • the non-specific adsorption of the nucleotide to be tested can be judged by visually measuring the signal on the surface of the substrate, such as the distribution, intensity, etc. of the fluorescent signal.
  • the signal on the surface of the substrate can be detected by means of an instrument/signal detection device to determine the non-specific adsorption of the nucleotide to be tested, for example, the instrument can be an optical detection device with an imaging system, etc. Including the light source, the objective lens, and the camera, correspondingly, the so-called "detection result” includes obtaining an image.
  • the so-called “bright spot” refers to the spot/spot on the image, and one spot/spot occupies at least one pixel.
  • the so-called “pixel” is the same as “pixel.”
  • the so-called “bright spot detection” corresponds to the detection of an optical signal of the nucleic acid or base or base cluster.
  • a method for detecting non-specific adsorption of a nucleotide comprises: S100: reacting a nucleotide to be tested with a first template strand, the first template strand being linked to a probe on the first chip
  • the first chip comprises a surface of the substrate and a probe fixed at one end to the surface of the substrate, the first template chain carries a first label, and the nucleotide to be tested can be paired with the corresponding base of the first template strand, thereby enabling the probe
  • the other end extends one base, the nucleotide to be tested carries a second mark, the first mark can generate a first signal, the second mark can generate a second signal;
  • S300 reacting the nucleotide to be tested with the second template chain, the second template chain is connected to the probe on the second chip, and the second chip comprises a surface of the substrate and a probe fixed to the surface of the substrate at
  • the non-specific adsorption and/or specific adsorption of the probe/template strand on the substrate surface and/or the substrate surface can be qualitatively or quantitatively detected; the detection system can detect the signal on the surface of the substrate, Qualitative or quantitative distinction between specific and non-specific adsorption, obtaining information on the quantification and distribution of non-specific or specific adsorption, can be used for nucleoside analog structure design and preparation, chip production, and all methods involving chip detection of nucleic acid processes or In applications, for example, evaluation of performance of an idle chip (without a probe) or a capture chip (with a probe), quality control of chip production, prediction of effects of chip capture nucleic acid, analysis and comparison, and the like.
  • the first signal and the second signal are detection-distinguishable signals, that is, the first signal and the second signal can be detected as two different signals, such as fluorescent signals of different colors.
  • S100 and S300 are performed in no order, and can be performed sequentially or simultaneously. Similarly, the same is true for S200 and S400. In some embodiments, S100 and S300 are parallel tests performed simultaneously.
  • the materials and surface characteristics of the substrates of the first chip and the second chip in S100 and S300 are substantially the same or identical, such as hydrophilicity, surface size, and the like.
  • the substrate surfaces of the first chip and the second chip are substrates of the same surface properties that are subjected to the same surface treatment.
  • the substrate surfaces of the first chip and the second chip are different surface areas of the same substrate that are subjected to the same surface treatment.
  • the substrate surfaces of the first chip and the second chip in S100 and S300 are in various aspects such as surface size, probe type and nucleotide sequence, probe immobilization density, probe distribution, amount of nucleotide to be detected,
  • the reaction conditions and the like are basically the same.
  • the terms "substantially identical”, “substantially identical” and “consistent” or “identical” mean that the differences produced by different batch preparation, processing, and/or parallel tests are within the allowable tolerances.
  • the first template strand differs from the second template strand in that the nucleotide to be tested is capable of pairing with the corresponding base of the first template strand and not with the corresponding base of the second template strand, as shown in FIG. 1 shows a paired/unpaired relationship of an exemplary nucleotide to be tested and a corresponding base on a template strand in an embodiment of the present invention, in this example, the nucleotide C to be tested and the first template strand Corresponding base G pairing, but not paired with the corresponding base A, C, T or U on the second template strand, thus, reacting the nucleotide C to be tested with the first template strand can keep the probe away One end of the surface of the substrate extends one base C to form a CG pairing relationship; and the nucleotide C to be tested reacts with the second template strand to extend one end of the probe away from the surface of the substrate by one base.
  • the nucleotide to be tested is another nucleotide
  • the distribution or arrangement of the probes on the surface of the substrate is not limited.
  • the probes on the first chip and the second chip are randomly distributed and/or regularly distributed (for example, in an array) on the surface of the substrate. on.
  • the probes were identical on different chip surfaces or in different surface areas of the same chip.
  • the first marker and the second marker can be different fluorescent dyes.
  • detecting a signal on the first chip, obtaining the first detection result includes: taking a photo of the first chip surface by the imaging system to obtain a first image; and detecting the first image to obtain the first detection result.
  • Detecting a signal on the second chip, obtaining a second detection result includes: taking a photo of the second chip surface by the imaging system to obtain a second image; and detecting the second image to obtain a second detection result.
  • the imaging system may be an optical detection device or the like with an imaging system, including a light source, an objective lens, and a camera.
  • detecting the first image to obtain the first detection result further comprises: detecting the first image to determine the number Na1 of the positions of the first signal and the second signal simultaneously on the surface of the first chip and only There is a number Na3 of positions of the second signal.
  • Detecting the second image to obtain the second detection result further comprises: detecting the second image to determine the number Nb1 of locations where the first signal and the second signal are simultaneously present on the surface of the second chip, and the number of locations where only the second signal is present Nb3.
  • the image comprises a plurality of pixel points.
  • the detection of the first image is taken as an example, and it should be understood that the following image detection method is also applicable to the processing of the second image, and the parallel test of the detection example of the same nucleotide-specific/non-specific adsorption.
  • the same treatment can be used to obtain reliable and comparable detection results.
  • an image detecting method includes: an optional image pre-processing step S11, wherein the image pre-processing step S11 includes pre-processing the first image to obtain a first pre-processed image.
  • Image includes pre-processing the first image to obtain a first pre-processed image.
  • Bright spot detecting step S12 the bright spot detecting step S12 includes the steps of: S21, analyzing the first image to calculate a bright spot determination threshold, S22, analyzing the first image to obtain a candidate bright spot, and S23, determining whether the candidate bright spot is a bright spot according to the bright spot determination threshold.
  • the first image is processed by the image pre-processing step, and the calculation amount of the bright spot detecting step can be reduced.
  • whether the candidate bright spot is a bright spot is determined by the bright spot determining threshold, and the accuracy of determining the bright spot of the image can be improved.
  • the image pre-processing step S11 is a step taken to obtain a better detection effect.
  • the image may be directly subjected to a bright spot detection step.
  • the input first image to be detected may be a 16-bit tiff format image of 512*512 or 2048*2048, and the image of the tiff format may be a grayscale image. In this way, the processing of the image detecting method can be simplified.
  • the image pre-processing step S11 includes performing a background subtraction process on the first image to obtain a pre-processed first image. In this way, the noise of the first image can be further reduced, and the accuracy of the image detecting method is higher.
  • the image pre-processing step S11 includes: performing a simplification process on the first image subjected to the subtractive background processing to obtain a pre-processed first image. In this way, the amount of calculation of the subsequent image detecting method can be reduced.
  • the image pre-processing step S11 includes filtering the first image to obtain a pre-processed first image. In this way, filtering the first image can obtain the pre-processed first image under the condition that the image detail features are retained as much as possible, thereby improving the accuracy of the image detection method.
  • the image pre-processing step S11 includes performing a subtractive background process on the first image and then performing a filtering process to obtain the pre-processed first image. In this way, after the background is subtracted from the first image and then filtered, the noise of the first image can be further reduced, and the accuracy of the image detection method is higher.
  • the image pre-processing step S11 includes: performing a simplification process on the first image after performing the subtractive background processing and then performing the filtering process to obtain the pre-processed first image. In this way, the amount of calculation of the subsequent image detecting method can be reduced.
  • the image pre-processing step S11 includes performing a simplification process on the first image to obtain a pre-processed first image. In this way, the amount of calculation of the subsequent image detecting method can be reduced.
  • the step of determining whether the candidate bright spot is a bright spot according to the bright spot determination threshold includes: step S31, searching for the greater than (p*p-1) connectivity in the preprocessed first image. Pixel points and the found pixel points as the center of the candidate bright points, p*p and the bright points are in one-to-one correspondence, each value in p*p corresponds to one pixel point, p is a natural number and is an odd number greater than 1; S32.
  • I max can be understood as the center strongest intensity of the candidate bright spot.
  • I max is the strongest intensity of the center of the 3*3 window.
  • a BI is the ratio of the set value in the first image preprocessed in the 3*3 window, and ceof guass is the pixel and 2D of the 3*3 window. Correlation coefficient of Gaussian distribution.
  • the preprocessed first image is a simplified processed image.
  • the preprocessed first image may be a binarized image, that is, the set value in the binarized image may be that the pixel meets the set condition. The value corresponding to the time.
  • the binarized image may contain two values of 0 and 1 characterizing different attributes of the pixel, the set value is 1, and A BI is the ratio of 1 in the binarized image in the p*p window. .
  • the image includes a plurality of pixel points.
  • detecting the first image includes: highlighting the first image by using a k*k matrix.
  • the detecting comprises determining that the matrix whose central pixel value of the matrix is not less than any pixel value of the matrix non-center corresponds to a bright point, k is an odd number greater than 1, and the k*k matrix comprises k*k pixels.
  • the method is based on the difference between the brightness/intensity of the signal generated by the mark and the background brightness/intensity, enabling simple and rapid detection of the image, detection and acquisition of signal information.
  • the pixel value of the matrix center is greater than a first preset value, and any one of the matrix non-center pixel values is greater than the second pixel value.
  • the first preset value and the second preset value may be set according to experience or a certain amount of pixel/intensity data of a normal bright spot of a normal image, and the so-called "normal image” and "normal bright spot” may be normal to the naked eye. If the image looks clear, the background is clean, the size and intensity of the highlights are relatively uniform.
  • the first preset value and the second preset value are related to an average pixel value of the image. For example, the first preset value is set to be 1.4 times the average pixel value of the image, and the second preset value is 1.1 times the average pixel value of the image, which can eliminate interference and obtain a better bright spot detection result.
  • the first image is a color image
  • one pixel of the color image has three pixel values
  • the color image can be converted into a gray image, and then image detection is performed to reduce the calculation of the image detection process.
  • Quantity and complexity may be selected to be converted into a grayscale image using, but not limited to, a floating point algorithm, an integer method, a shift method, or an average value method.
  • the color image can also be directly detected.
  • the size comparison of the pixel values mentioned above can be regarded as a three-dimensional value or a comparison of the size of an array of three elements, and the relative sizes of the plurality of multi-dimensional values can be customized according to experience and needs, for example. When any two-dimensional value in the three-dimensional value a is larger than the value of the corresponding dimension of the three-dimensional value b, the three-dimensional value a is considered to be larger than the three-dimensional value b.
  • the first image is a grayscale image
  • the pixel value of the grayscale image is a one-dimensional value
  • the pixel value of the grayscale image is the same as the grayscale value
  • the surface of the substrate also referred to as the surface of the chip, is a surface-modified surface with an epoxy group: a probe (DNA capture strand, Capture DNA)
  • the probe is an amino-modified probe having an amino group at the end, and the probe is reacted with an epoxy group on the surface of the chip by -NH 3 , fixed on the surface of the substrate, and then the chip is passivated using a passivation solution. To block unreacted epoxy groups. Further, a hybrid template strand is added, which is a DNA strand of a specific sequence which is end-labeled with a Cy3 fluorescent dye and which is complementary to the base of the probe.
  • the nucleotide to be tested which is complementary to the first base to be tested of the hybrid template strand and labeled with ATTO-647N fluorescent dye is added, and then the surface is irradiated by a fluorescence microscope such as total internal reflection fluorescence microscopy (TIRF). Take a picture and get an image with signal points/spots.
  • TIRF total internal reflection fluorescence microscopy
  • the surface of the substrate is also referred to as a chip surface, and is a surface-modified epoxy group-bearing surface: the same reaction system and time, the same concentration, the same concentration
  • the probe by reacting -NH 3 with an epoxy group on the surface of the chip, bonds the probe to the surface of the chip by a chemical bond, and then passivates the chip with a passivation solution to block the unreacted epoxy group. Further, a hybrid template strand is added, which is a DNA strand with a Cy3 fluorescent dye label at the end and a complementary pair with the probe sequence, but the first base to be tested is not paired with the nucleotide to be added to be added.
  • the nucleotide to be tested (which is the same as the nucleotide to be tested in the example of Fig. 4) is added, labeled with ATTO-647N fluorescent dye, and then the surface is subjected to total internal reflection fluorescence microscopy (TIRF). The image is taken to obtain an image with fluorescent signal spots/spots.
  • TIRF total internal reflection fluorescence microscopy
  • a method for detecting non-specific adsorption of a nucleotide comprises: S1000: reacting a nucleotide to be tested with a third template strand, the third template strand being linked to a probe on a third chip
  • the third chip includes a surface of the substrate and a probe fixed to the surface of the substrate at one end, and the third template chain carries a third mark, and the nucleotide to be tested cannot be paired with the corresponding base of the third template chain, and the probe cannot be made.
  • the other end extends one base, the nucleotide to be tested carries a fourth mark, the third mark can generate a third signal, and the fourth mark can generate a fourth signal; S2000: detects the signal on the third chip, obtains a third The detection result; S3000: reacting the nucleotide to be tested with the fourth template chain, the fourth template chain is connected to the fourth chip, the fourth chip includes the surface of the substrate without the probe, and the fourth template chain has the third mark
  • the nucleotide to be tested can be paired with the corresponding base of the fourth template chain; S4000: detecting the signal on the fourth chip to obtain the fourth detection result; S5000: detecting the test based on the third detection result and the fourth detection result Nucleotide Specific adsorption.
  • the non-specific adsorption of the probe/template strand on the substrate surface and/or the surface of the substrate can be qualitatively or quantitatively detected; and the non-specific adsorption can be qualitatively or quantitatively distinguished by detecting the signal on the surface of the substrate by the detection system.
  • Type, information such as non-specific quantification, distribution, etc. can be used in chip production and in all methods or applications involving chip detection of nucleic acid processes, such as for no-load chips (without probes) or capture chips (with probes) Evaluation of performance, quality control of chip production, prediction, analysis and comparison of effects of chip capture nucleic acid.
  • the S1000 and S3000 are carried out in no order, and can be carried out sequentially or simultaneously. Similarly, the S2000 and S4000 are also carried out without order. In some embodiments, S1000 and S3000 are parallel tests for simultaneous ⁇ ⁇
  • the materials and surface characteristics of the substrates of the third chip and the fourth chip in S1000 and S3000 are substantially the same or identical, such as hydrophilicity, surface size, and the like.
  • the substrate surfaces of the third and fourth chips are substrates of the same surface properties that are subjected to the same surface treatment.
  • the substrate surfaces of the third and fourth chips are different surface areas of the same substrate that have been subjected to the same surface treatment.
  • the substrate surfaces of the third chip and the fourth chip in S1000 and S3000 are substantially identical except for whether or not the probe is immobilized, such as the surface size, the amount of the nucleotide to be detected, the reaction conditions, and the like.
  • the terms “substantially identical”, “substantially identical” and “consistent” or “identical” mean that the differences produced by different batch preparation, processing, and/or parallel tests are within the allowable tolerances.
  • the distribution or arrangement of the probes on the surface of the third chip is not limited. In the embodiment of the invention, the probes are randomly distributed or regularly distributed on the surface of the third chip (for example, in an array).
  • the third and fourth labels are different fluorescent dyes.
  • detecting a signal on the third chip, obtaining the third detection result includes: taking a photo of the third chip surface by the imaging system to obtain a third image; and detecting the third image to obtain the third detection result.
  • Detecting a signal on the fourth chip, obtaining a fourth detection result includes: taking a photo of the fourth chip surface by the imaging system to obtain a fourth image; and detecting the fourth image to obtain a fourth detection result.
  • the imaging system may be an optical detection device or the like with an imaging system, including a light source, an objective lens, and a camera.
  • detecting the third image to obtain the third detection result further comprises: detecting the third image to determine the number Nc1 of positions at which the third signal and the fourth signal appear simultaneously on the surface of the third chip, and only The number of positions Nc3 at which the fourth signal appears.
  • Detecting the fourth image to obtain the fourth detection result further includes: detecting the fourth image to determine the number Nd1 of positions at which the third signal and the fourth signal appear simultaneously on the surface of the fourth chip, and the number of positions where only the fourth signal appears Nd3.
  • the detection of the image can be referred to the above-described detection example of the first image.
  • the formula (Nc1+Nc3-Nd1-Nd3)/(Nc1+Nc3) can be used to determine the non-specific adsorption change ratio of the nucleotide to be measured after the substrate surface is fixed by the probe, thereby realizing the third detection result and the fourth detection result. Non-specific adsorption of the nucleotide to be tested is detected.
  • the surface of the substrate also referred to as the surface of the chip, is a surface-modified surface with an epoxy group: a probe (DNA capture strand, Capture DNA)
  • the probe is an amino-modified probe having an amino group at the end, and the probe is reacted with an epoxy group on the surface of the chip by -NH 3 , fixed on the surface of the substrate, and then the chip is passivated using a passivation solution. To block unreacted epoxy groups.
  • a hybrid template strand is added, which is a DNA strand with a Cy3 fluorescent dye label at the end and a complementary pair with the probe sequence, but the first base to be tested is not paired with the nucleotide to be added to be added.
  • the nucleotide to be tested labeled with ATTO-647N fluorescent dye is added to carry out hybridization.
  • the surface is photographed by a fluorescence microscope including a total internal reflection fluorescence microscope (TIRF) to obtain an image with a fluorescent signal.
  • TIRF total internal reflection fluorescence microscope
  • the tendency is to indicate that there is a non-specific adsorption of the template strand and the nucleotide at the same position, the number is Nc1; (2) if only the green fluorescent signal is observed, the position tends to indicate the position For the non-specific adsorption of the template strand, the number is Nc2; (3) If only the red fluorescent signal is observed, the tendency is to indicate that the position is a non-specific adsorption of nucleotides, the number is Nc3.
  • the surface of the substrate is also referred to as a chip surface, and is a surface-modified epoxy group-bearing surface.
  • a fixing solution containing no probe is added.
  • the reaction system and reaction time were the same as in the above-described example of Fig. 5, and then the chip was passivated using a passivation liquid.
  • a hybrid template strand is added, which is a DNA strand of a specific sequence tagged with a Cy3 fluorescent dye at the end.
  • the nucleotide to be tested labeled with ATTO-647N fluorescent dye is added, and then the surface is imaged by a fluorescence microscope including a total internal reflection fluorescence microscope (TIRF) to obtain an image with a fluorescent signal.
  • TIRF total internal reflection fluorescence microscope
  • the formula (Nc1+Nc3-Nd1-Nd3)/(Nc1+Nc3) can be further used to determine the proportion of non-specific adsorption changes of the nucleotide to be measured after the substrate surface passes through the immobilized probe.
  • a method for detecting nucleotide-specific and/or non-specific adsorption comprises: S10000: reacting a nucleotide to be tested with a fifth template strand, the fifth template strand and a fifth chip a probe connection, the fifth chip comprising a surface of the substrate and a probe fixed to the surface of the substrate at one end, the fifth template chain carrying a fifth label, the nucleotide to be tested being capable of pairing with the corresponding base of the fifth template strand So that the other end of the probe extends one base, the nucleotide to be tested carries a sixth mark, the fifth mark can generate a fifth signal, and the sixth mark can generate a sixth signal; S20000: detecting the fifth chip Signal, obtaining a fifth detection result; S30000: reacting the nucleotide to be tested with a sixth template chain, the sixth template chain being connected to the probe on the sixth chip, the sixth chip including the surface of the substrate and one end fixed thereto The probe
  • the specificity and/or non-specific adsorption of the probe/template strand on the substrate surface and/or the substrate surface can be qualitatively or quantitatively detected; the detection system can detect the signal on the surface of the substrate, and can be qualitative or Quantitatively distinguish between specific and/or non-specific types of adsorption, obtain specific and/or non-specific quantification, distribution, etc., can be used for nucleotide analog performance evaluation, chip production, and all methods involving chip detection of nucleic acid processes or In applications, for example, evaluation of performance of an idle chip (without a probe) or a capture chip (with a probe), quality control of chip production, prediction of effects of chip capture nucleic acid, analysis and comparison, and the like.
  • S10000, S30000, and S50000 are performed in no order, and may be performed sequentially or simultaneously. Similarly, the execution of S20000, S40000, and S60000 is also not limited in order. In some embodiments, S10000, S30000, and S50000 are parallel tests, performed simultaneously.
  • the materials, surface characteristics such as hydrophilicity, surface size, and the like of the substrates of the fifth chip, the sixth chip, and the seventh chip in S10000, S30000, and S50000 are substantially the same or identical.
  • the substrate surfaces of the fifth, sixth, and seventh chips are substrates of the same surface properties that are subjected to the same surface treatment.
  • the substrate surfaces of the fifth, sixth, and seventh chips are different surface areas of the same substrate that are subjected to the same surface treatment.
  • the substrate surfaces of the fifth chip and the sixth chip in S10000 and S30000 are in various aspects such as surface size, probe type and nucleotide sequence, probe immobilization density, probe distribution, amount of nucleotide to be detected,
  • the reaction conditions and the like are basically the same.
  • the seventh chip in S50000 is basically the same as the fifth chip and the sixth chip in S10000 and S30000, except for the absence of the fixed probe, such as the surface size, the amount of the nucleotide to be tested, and the reaction conditions. .
  • the terms “substantially identical”, “substantially identical” and “consistent” or “identical” mean that the differences produced by different batch preparation, processing, and/or parallel tests are within the allowable tolerances.
  • the distribution or arrangement of the probes on the surface of the fifth chip and the sixth chip is not limited.
  • the probes are randomly distributed or regularly distributed on the surfaces of the fifth chip and the sixth chip (for example, in an array). distributed). In parallel experiments, the probes were distributed in the same state on different chip surfaces.
  • the fifth and sixth indicia are different fluorescent dyes.
  • detecting a signal on the fifth chip, obtaining a fifth detection result includes: taking a photo of the fifth chip surface by the imaging system to obtain a fifth image; and detecting the fifth image to obtain a fifth detection result.
  • Detecting the signal on the sixth chip, obtaining the sixth detection result includes: taking a picture of the sixth chip surface by the imaging system to obtain a sixth image; and detecting the sixth image to obtain a sixth detection result.
  • Detecting the signal on the seventh chip, and obtaining the seventh detection result includes: taking a photo of the seventh chip surface by the imaging system to obtain a seventh image; and detecting the seventh image to obtain a seventh detection result.
  • the imaging system may be an optical detection device or the like with an imaging system, including a light source, an objective lens, and a camera.
  • detecting the fifth image to obtain the fifth detection result further comprises: detecting the fifth image to determine the number Ne1 of the positions of the fifth signal and the sixth signal simultaneously on the surface of the fifth chip and only There is a number Ne3 of positions of the sixth signal.
  • Detecting the sixth image to obtain the sixth detection result further includes: detecting the sixth image to determine the number Nf1 of positions at which the fifth signal and the sixth signal are simultaneously present on the surface of the sixth chip, and the number of positions where only the sixth signal exists Nf3.
  • Detecting the seventh image to obtain the seventh detection result further includes: detecting the seventh image to determine the number Ng1 of positions at which the fifth signal and the sixth signal appear simultaneously on the surface of the seventh chip, and the number of positions where only the sixth signal appears Ng3.
  • the detection of the image can be referred to the above-described detection example of the first image.
  • Detecting the specificity and/or non-specific adsorption of the nucleotide to be tested (a) determining the ratio of non-specific adsorption of the nucleotide to be detected on the surface of the substrate and the nucleic acid strand using the formula (Nf1+Nf3)/(Ne1+Ne3); (b) Using the formula Nf1/(Ne1+Ne3) to determine the ratio of non-specific adsorption of the nucleotide to be detected in the nucleic acid strand; (c) using the formula (Ne1+Ne3-Nf1-Nf3)/(Ne1+Ne3) to determine the test The effective ratio of nucleotide to template strand; (d) using the formula (Ng1+Ng3-Nf1-Nf3)
  • the surface of the substrate also referred to as the surface of the chip, is a surface-modified surface with an epoxy group: a probe (DNA capture strand, Capture DNA)
  • the probe is an amino-modified probe having an amino group at the end, and the probe is reacted with an epoxy group on the surface of the chip by -NH 3 , fixed on the surface of the substrate, and then the chip is passivated using a passivation solution. To block unreacted epoxy groups. Further, a hybrid template strand is added, which is a DNA strand of a specific sequence which is end-labeled with a Cy3 fluorescent dye and which is complementary to the base of the probe.
  • the nucleotide to be tested which is complementary to the first base to be tested of the hybrid template strand and labeled with ATTO-647N fluorescent dye is added, and then the surface is irradiated by a fluorescence microscope such as total internal reflection fluorescence microscopy (TIRF). Take a picture and get an image with signal points/spots.
  • TIRF total internal reflection fluorescence microscopy
  • the surface of the substrate is also referred to as a chip surface, and is a surface-modified epoxy group-bearing surface: the same reaction system and time, the same concentration, the same concentration
  • the probe by reacting -NH 3 with an epoxy group on the surface of the chip, bonds the probe to the surface of the chip by a chemical bond, and then passivates the chip with a passivation solution to block the unreacted epoxy group. Further, a hybrid template strand is added, which is a DNA strand with a Cy3 fluorescent dye label at the end and a complementary pair with the probe sequence, but the first base to be tested is not paired with the nucleotide to be added to be added.
  • the nucleotide to be tested (which is the same as the nucleotide to be tested in the example of Fig. 4) is added, labeled with ATTO-647N fluorescent dye, and then the surface is subjected to total internal reflection fluorescence microscopy (TIRF). The image is taken to obtain an image with fluorescent signal spots/spots.
  • TIRF total internal reflection fluorescence microscopy
  • the nucleotides at this position are non-specifically adsorbed with the template strand, the number is Nf1; (2) If only the green fluorescent signal is observed, the position is The non-specific adsorption of the template strand is in the number of Nf2; (3) If only the red fluorescent signal is observed, the position is the surface non-specific adsorption of the nucleotide, and the number is Nf3.
  • the surface of the substrate is also referred to as a chip surface, and is a surface-modified epoxy group-bearing surface.
  • a fixing solution containing no probe is added.
  • the reaction system and reaction time were the same as in the above-described example of Fig. 5, and then the chip was passivated using a passivation liquid.
  • a hybrid template strand is added, which is a DNA strand of a specific sequence tagged with a Cy3 fluorescent dye at the end.
  • the nucleotide to be tested labeled with ATTO-647N fluorescent dye is added, and then the surface is imaged by a fluorescence microscope including a total internal reflection fluorescence microscope (TIRF) to obtain an image with a fluorescent signal.
  • TIRF total internal reflection fluorescence microscope
  • Example 1 Investigating the effects of non-specific adsorption during a nucleotide reaction
  • Chip glass with epoxy group on the surface (purchased from SCHOTT);
  • EKB-6P Fixed strand
  • Hybridization chain 1 (EKB-6T-2-Cy3): a specific sequence synthesized by a complementary pair with a fixed strand, with a short sequence of 33 bp of the sequence to be tested and a fluorophore Cy3 at the end.
  • the specific sequence is:
  • Hybridization chain 2 (EKB-6T-6-Cy3): a synthetically specific sequence that is not complementary to a fixed strand, with a 33 bp length to be tested (inconsistent with the EKB-6T-2-Cy3 sequence to be tested), terminus A short sequence of DNA with the fluorophore Cy3, the specific sequence is:
  • immobilized chain 1 (experimental group, control group 1): After washing and drying the glass, placed in a solution of 150 mM K 2 HPO 4 and containing a concentration of 1.0 M amino-modified EKB-6P nucleic acid sequence, 37 ° C conditions The reaction was carried out for 0.5 hour, washed successively with 3X SSC solution (containing 0.1% Triton), 3XSSC, 0.15 MK 2 HPO 4 solution, and then passivated with 1 M K 2 HPO 4 at 37 ° C for 17 hours.
  • 3X SSC solution containing 0.1% Triton
  • 3XSSC 0.15 MK 2 HPO 4 solution
  • Fixation of the fixed chain 2 (control group 2): After the glass was washed and dried, it was placed in a 150 mM K 2 HPO 4 solution, and reacted at 37 ° C for 0.5 hour, followed by 3XSSC solution (containing 0.1% Triton), 3XSSC, After washing with 0.15 MK 2 HPO 4 solution, 1 M K 2 HPO 4 was added and passivated at 37 ° C for 17 hours.
  • Chip (Flow Cell) Assembly The passivated chips are assembled into a four-channel flow cell.
  • Hybridization of hybridization chain 1 (experimental group, control group 2): The assembled four-channel flow cell was added to Rinse buffer (1XSSC + 150 mM HEPES + 0.1% SDS), reconstituted at 55 ° C for 0.5 hour, and then hybridized. A 3X SSC solution having a chain (EKB-6T-2-Cy3) concentration of 1 nM was reacted at 55 ° C for 0.5 hour. The channels were then washed sequentially using Rinse buffer (1XSSC + 150 mM HEPES + 0.1% SDS) and Buffer H (150 mM HEPES + 150 mM NaCl).
  • Hybridization of Hybrid Chain 2 (Control 1): The assembled four-channel flow cell was added to Rinse buffer (1XSSC + 150 mM HEPES + 0.1% SDS), reconstituted at 55 ° C for 0.5 hour, and then added to the hybrid strand (EKB). -6T-6-Cy3) A 3XSSC solution having a concentration of 1 nM was reacted at 55 ° C for 0.5 hour. The channels were then washed sequentially using Rinse buffer (1XSSC + 150 mM HEPES + 0.1% SDS) and Buffer H (150 mM HEPES + 150 mM NaCl).
  • Nucleotide reaction Add a reaction solution containing a concentration of 200 nM of nucleotide A to the channel, and react at 37 ° C for 90 s, then use Rinse buffer (1XSSC + 150 mM HEPES + 0.1% SDS) and Buffer H (150 mM HEPES + 150 mM). NaCl) Wash the channels sequentially and then add imaging buffer.
  • Photo detection Photographing the chip channel using TIRF.
  • the results of the experimental group are shown in Fig. 7.
  • the left image shows the Cy3 fluorescence point in one field of view.
  • the number of Cy3 fluorescence points in all the fields of the multiple channels is 19852.
  • the right picture shows the ATTO647N fluorescence points in the same field of view.
  • the ATTO647N fluorescence point of the field of view is 16744, and the coincidence points are 13358.
  • the results of the control group 1 are shown in Fig. 8.
  • the left image shows the Cy3 fluorescence point in one field of view.
  • the number of Cy3 fluorescence points in all the fields of the multiple channels is counted as 19971.
  • the right image shows the ATTO647N fluorescence points in the same field of view.
  • the ATTO647N fluorescence point of all fields of view was 619, and the coincidence points were 358.
  • the results of the control group 2 are shown in Fig. 9.
  • the left image shows the Cy3 fluorescence point in one field of view.
  • the number of Cy3 fluorescence points in all the fields of the multiple channels is 603.
  • the right image shows the ATTO647N fluorescence points in the same field of view.
  • the ATTO647N fluorescence point of all fields of view is 524.
  • the Cy3 fluorescence point is the impurity on the surface of the chip, and the brightness is weak.
  • the ATTO647N fluorescence point is the non-specific adsorption of nucleotides on the surface of the chip.
  • the ratio of the non-specific adsorption of the nucleotide to be detected on the surface of the substrate and the nucleic acid strand is 3.25%; the ratio of the non-specific adsorption of the nucleotide to be detected in the nucleic acid strand is 1.19%; the nucleotide to be tested and the template The effective proportion of the chain was 96.75%; the ratio of non-specific adsorption of the nucleotide to be measured after the surface of the substrate was fixed by the probe was -86.5%.

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Abstract

一种检测核苷酸特异性和/或非特异性吸附的方法，包括：使待测核苷酸与第一模板链反应，第一模板链与第一芯片上的探针连接，待测核苷酸能够与第一模板链的相应碱基配对而使探针延伸一个碱基，第一模板链和待测核苷酸带有产生不同信号的标记；检测第一芯片上的信号，获得第一检测结果；使待测核苷酸与第二模板链反应，第二模板链与第二芯片上的探针连接，第二模板链和待测核苷酸带有不同标记，待测核苷酸与第二模板链的相应碱基不能配对；检测第二芯片上的信号，获得第二检测结果；基于上述检测结果，检测待测核苷酸的特异性和/或非特异性吸附，对核苷酸与基底表面或其上探针的特异性和/或非特异性吸附进行检测，用于芯片生产质控。

Description

一种检测核苷酸特异性和/或非特异性吸附的方法 技术领域
本发明涉及核酸检测领域，具体涉及一种检测核苷酸特异性和/或非特异性吸附的方法。
背景技术
在芯片上进行核酸检测，例如利用表面固定有探针的芯片捕获核酸以及进一步对捕获得的核酸进行分析检测，包括序列测定、信息分析等，芯片表面和/或其上的探针/核酸复合物对反应底物(例如测序反应中加入的核苷类似物)的非特异性吸附，会影响获得的有效数据量以及检测结果的准确性。对非特异性吸附情况的定性或定量检测，能够判定芯片的质量以及用于检测结果预测等。
例如在目前的核酸测序中，特别是利用芯片的基因测序中，在芯片上存在底物核苷酸(Terminator)的非特异性吸附，包括芯片表面对底物核苷酸的非特异性吸附和芯片表面上的探针/探针复合物对核苷酸的非特异性吸附。这些非特异性吸附会造成测序结果错误率增加、测序通量和/或测序质量下降等。
另外，核苷酸包括核苷酸类似物，不同的核苷酸类似物对芯片表面、探针/核酸复合物的非特异性吸附能力可能不同。例如，在芯片上进行核酸序列测定，底物即生化反应中加入的核苷酸类似物的分子大小、结构以及在反应体系中的电荷性质等，影响底物与芯片和/或核酸复合物的结合/吸附。
因此，检测这些非特异性吸附情况，对于核苷酸包括核苷酸类似物的结构设计、制备生产，芯片生产，利用芯片进行核酸序列检测等领域具有重要意义。
发明内容
本发明实施例旨在至少解决现有技术中存在的技术问题之一或者至少提供一种有用的商业选择。为此，本发明提供一种检测核苷酸特异性和/或非特异性吸附的方法。
根据第一方面，提供一种检测核苷酸特异性和/或非特异性吸附的方法，该方法包括：使待测核苷酸与第一模板链反应，该第一模板链与第一芯片上的探针连接，该第一芯片包括基底表面以及一端固定在该基底表面的探针，第一模板链带有第一标 记，待测核苷酸能够与第一模板链的相应碱基配对，从而使探针的另一端延伸一个碱基，待测核苷酸带有第二标记，第一标记能够产生第一信号，第二标记能够产生第二信号；检测第一芯片上的信号，获得第一检测结果；使待测核苷酸与第二模板链反应，该第二模板链与第二芯片上的探针连接，该第二芯片包括基底表面以及一端固定在该基底表面的探针，第二模板链带有第一标记，待测核苷酸与第二模板链的相应碱基不能配对，无法使探针的另一端延伸一个碱基；检测第二芯片上的信号，获得第二检测结果；基于第一检测结果和第二检测结果，检测待测核苷酸的特异性和/或非特异性吸附。
根据第二方面，提供一种检测核苷酸非特异性吸附的方法，该方法包括：使待测核苷酸与第三模板链反应，该第三模板链与第三芯片上的探针连接，该第三芯片包括基底表面以及一端固定在该基底表面的探针，第三模板链带有第三标记，待测核苷酸与第三模板链的相应碱基不能配对，无法使探针的另一端延伸一个碱基，待测核苷酸带有第四标记，第三标记能够产生第三信号，第四标记能够产生第四信号；检测第三芯片上的信号，获得第三检测结果；使待测核苷酸与第四模板链反应，该第四模板链与第四芯片连接，第四芯片包括不带探针的基底表面，第四模板链带有第三标记，待测核苷酸能够与第四模板链的相应碱基配对；检测第四芯片上的信号，获得第四检测结果；基于第三检测结果和第四检测结果，检测待测核苷酸的非特异性吸附。
根据第三方面，提供一种检测核苷酸特异性和/或非特异性吸附的方法，该方法包括：使待测核苷酸与第五模板链反应，该第五模板链与第五芯片上的探针连接，该第五芯片包括基底表面以及一端固定在该基底表面的探针，第五模板链带有第五标记，待测核苷酸能够与第五模板链的相应碱基配对，从而使探针的另一端延伸一个碱基，待测核苷酸带有第六标记，第五标记能够产生第五信号，第六标记能够产生第六信号；检测第五芯片上的信号，获得第五检测结果；使待测核苷酸与第六模板链反应，该第六模板链与第六芯片上的探针连接，该第六芯片包括基底表面以及一端固定在该基底表面的探针，第六模板链带有第五标记，待测核苷酸与第六模板链的相应碱基不能配对，无法使探针的另一端延伸一个碱基；检测第六芯片上的信号，获得第六检测结果；使待测核苷酸与第七模板链反应，该第七模板链与第七芯片连接，该第七芯片包括不带探针的基底表面；检测第七芯片上的信号，获得第七检测结果；基于第五检测结果、第六检测结果和第七检测结果中的至少两个检测结果，检 测待测核苷酸的特异性和/或非特异性吸附。
上述检测核酸特异性和/或非特异性吸附的方法，能够定性或定量检测基底表面和/或基底表面上的探针/模板链对核苷酸的非特异吸附情况和/或特异性吸附情况；通过检测系统检测基底表面的信号，能够定性或定量区分特异性和非特异性吸附，获得非特异性或特异性吸附的定量、分布情况等信息，能够用于核苷类似物结构设计及制备，芯片生产以及所有涉及芯片检测核酸过程的方法或应用中，例如用于核酸序列测定中的反应底物的结构设计、空载芯片(不带探针)或者捕获芯片(带探针)性能的评价、芯片生产的质控，用于芯片检测核酸的效果的预测、分析比较等。
附图说明
图1为本发明实施方式中的待测核苷酸与模板链上相应碱基的配对/不配对关系示意图；
图2为本发明实施方式中的图像处理方法的流程示意图；
图3为本发明实施方式中的图像处理方法的另一流程示意图；
图4为本发明实施方式中一个基底表面上探针固定、模板链杂交和核苷酸聚合/吸附的原理示意图；
图5为本发明实施方式中另一个基底表面上探针固定、模板链杂交和核苷酸聚合/吸附的原理示意图；
图6为本发明实施方式中又一个基底表面上模板链杂交和核苷酸吸附的原理示意图；
图7为本发明实施例中实验组拍照获得的一个视野中的Cy3荧光点(左图)和ATTO647N荧光点(右图)图像；
图8为本发明实施例中对照组1拍照获得的一个视野中的Cy3荧光点(左图)和ATTO647N荧光点(右图)图像；
图9为本发明实施例中对照组2拍照获得的一个视野中的Cy3荧光点(左图)和ATTO647N荧光点(右图)图像。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。在以下的实施方式中，很多细节描述是为了使得本发明能被更好的理解。然而，本领域技术人员不需创造性劳动可以认识到，其中部分特征在不同情况下是可以省略的，或者可以由其他元件、材料、方法所替代。本领域技术人员能够根据说明书中的描述以及本领域的一般技术知识实现相关操作。
另外，说明书中所描述的特点、操作或者特征可以以任意适当的方式结合形成各种实施方式。同时，方法描述中的各步骤或者动作也可以按照本领域技术人员所能显而易见的方式进行顺序调换或调整。因此，说明书和附图中的各种顺序只是为了清楚描述某一个实施例，并不意味着是必须的顺序，除非另有说明其中某个顺序是必须遵循的。
为便于理解，以下结合附图通过举例的方式对本发明实施例进行详细说明。应当理解的是，如下举例中的对具体操作及操作细节的描述为示意，非对发明方案的限定。
在本发明的描述中，需要理解的是，术语“第一”、“第二”、“第三”、“第四”、“第五”、“第六”、“第七”等仅用于描述目的，而不能理解为指示或暗示相对重要性、相对顺序或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”、“第三”、“第四”、“第五”、“第六”、“第七”等的特征可以明示或者隐含地包括一个或者更多个所述特征。在本发明的描述中，“多个”的含义是两个或两个以上，除非另有明确具体的限定。
所称的“核苷酸”包括核糖核苷酸、脱氧核糖核苷酸及其类似物，包括A、T、C、G和U及其类似物。示例中如无特殊说明，利用C表示胞嘧啶或者胞嘧啶类似物，利用G表示鸟嘌呤或者鸟嘌呤类似物，利用A表示腺嘌呤或者腺嘌呤类似物，利用T表示胸腺嘧啶或者胸腺嘧啶类似物，利用U表示尿嘧啶或者尿嘧啶类似物。
本发明实施方式所称的“模板链”可以是DNA和/或RNA等，在一些实施方式中也可称为“杂交链”，例如，在利用芯片检测核酸过程中的目标DNA链。
本发明实施方式所称的“探针”可以是DNA和/或RNA等，在一些实施方式中也可称为“引物”、“捕获链”或“固定链”。所称的“探针”可以在基底表面上随机分布或者规则分布，如阵列分布，例如在目前市面上的捕获芯片，探针在芯片表面上一般呈阵列分 布。
所称的“基底”可以是任何可用于固定核酸序列的固体支持物，例如尼龙膜、玻璃片、塑料、硅片、磁珠等，如无特殊说明，芯片表面与基底表面可互换使用。固相探针，探针一般是通过化学键连接/固定在基底表面上，一般地，基底表面为经过化学修饰的表面，带有反应基团，可与探针连接。表面修饰、固定等可利用已知方法进行或者直接定制或者购买。
所称的“非特异性吸附”和“特异性吸附”/“特异性结合”是相对的，“非特异性吸附”一般是指非共价键的作用力导致的吸附，非共价键的作用力包括疏水作用力、范德华力、静电作用力等；在表面带有探针的芯片上、基于碱基互补原则的核酸检测过程中，一般地，核酸非特异性吸附指核酸非共价的连接在芯片表面和/或探针上。
所称的“标记”可以是任何物理、化学或生物可检测标记，相应的，本发明实施方式所称的“信号”，是所述“标记”在特定情形下产生的信号，典型但非限定性的“标记”是光学可检测标记，例如荧光染料，相应的，所述“信号”是荧光信号。在本发明一些实施例中，标记为荧光染料，例如为Cy3和/或ATTO-647N，分别能够在532nm和640nm波长的激发光作用下发出绿色荧光信号和红色荧光信号。
所称的“检测结果”可以是任何合适的定性或定量结果，例如目测结果、仪器检测结果等。在一些不需要精确定量的应用场景下，可以通过目测基底表面上的信号，例如荧光信号的分布、强度等来判断待测核苷酸的非特异性吸附情况。在一些需要精确定量的应用场景下，可以借助仪器/信号检测装置检测基底表面上的信号来判断待测核苷酸的非特异性吸附情况，例如仪器可以是带有成像系统的光学检测装置等，包括光源、物镜以及相机，相应的，所称的“检测结果”包括获得图像。
所称的“亮点”，指图像上的光点/光斑，一个光点/光斑占有至少一个像素点。所称“像素点”同“像素”。利用光学检测系统对带有荧光标记的核酸进行检测时，所称的“亮点检测”对应于对该核酸或者碱基或碱基簇的光学信号的检测。
在本发明的一个实施例中，检测核苷酸非特异性吸附的方法，包括：S100：使待测核苷酸与第一模板链反应，该第一模板链与第一芯片上的探针连接，该第一芯片包括基底表面以及一端固定在该基底表面的探针，第一模板链带有第一标记，待测核苷酸能够与第一模板链的相应碱基配对，从而使探针的另一端延伸一个碱基，待测核苷酸带有第二标记，第一标记能够产生第一信号，第二标记能够产生第二信号；S200：检测第一芯片上的信号，获得第一检测结果；S300：使待测核苷酸与第二模 板链反应，该第二模板链与第二芯片上的探针连接，该第二芯片包括基底表面以及一端固定在该基底表面的探针，第二模板链带有第一标记，待测核苷酸与第二模板链的相应碱基不能配对，无法使探针的另一端延伸一个碱基；S400：检测第二芯片上的信号，获得第二检测结果；S500：基于第一检测结果和第二检测结果，检测待测核苷酸的特异性和/或非特异性吸附。
利用该方法，能够定性或定量检测基底表面和/或基底表面上的探针/模板链对核苷酸的非特异吸附情况和/或特异性吸附情况；通过检测系统检测基底表面的信号，能够定性或定量区分特异性和非特异性吸附，获得非特异性或特异性吸附的定量、分布情况等信息，能够用于核苷类似物结构设计及制备、芯片生产以及所有涉及芯片检测核酸过程的方法或应用中，例如用于空载芯片(不带探针)或者捕获芯片(带探针)性能的评价、芯片生产的质控，用于芯片捕获核酸的效果的预测、分析比较等。
上述方法中，第一信号和第二信号是检测上可区分的信号，即能够检测到第一信号和第二信号是两个不同的信号，例如不同颜色的荧光信号等。
S100和S300的进行无顺序要求，可先后进行，也可同时进行。类似地，S200和S400也是如此。在一些实施例中，S100和S300为平行试验，同时进行。
在一平行试验示例中，S100和S300中的第一芯片和第二芯片的基底的材质、表面特性如亲疏水、表面大小等是基本相同或一致的。在一个例子中，第一芯片和第二芯片的基底表面是经过相同表面处理的、相同材料性能的基底。在另一个例子中，第一芯片和第二芯片的基底表面是经过相同表面处理的同一基底的不同表面区域。例如，S100和S300中的第一芯片和第二芯片的基底表面在各方面如表面大小、探针种类和核苷酸序列、探针固定密度、探针分布、待测核苷酸的量、反应条件等基本一致。所称的“基本一致”、“基本相同”同“一致”或“相同”，指不同批次制备、处理和/或平行试验产生的差异在允许的偏差范围内。
第一模板链与第二模板链的差别在于，待测核苷酸能够与第一模板链的相应碱基配对，而与第二模板链的相应碱基不能配对，如图1所示。图1示出了本发明实施方式中一个示例性的待测核苷酸与模板链上相应碱基的配对/不配对关系，在该示例中，待测核苷酸C与第一模板链上的相应碱基G配对，而与第二模板链上的相应碱基A、C、T或U不配对，因此，使待测核苷酸C与第一模板链反应，能够使探针的远离基底表面的一端延伸一个碱基C形成CG配对关系；使待测核苷酸C与第二模板链反应，无法使探针的远离基底表面的一端延伸一个碱基。在待测核苷酸是其 它核苷酸的情况下，原理类似。所称的“配对”指碱基的互补结合，如A和T配对、C和G配对等，属于特异性结合。
对探针在基底表面上的分布或排布方式不作限制，在本发明实施例中，第一芯片和第二芯片上的探针随机分布和/或规则分布(例如呈阵列分布)在基底表面上。在平行实验中，探针在不同的芯片表面上或者在同一芯片的不同表面区域的分布状态相同。
第一标记和第二标记可以为不同的荧光染料。在本发明的实施例中，检测第一芯片上的信号，获得第一检测结果包括：通过成像系统对第一芯片表面进行拍照，获得第一图像；以及检测第一图像，以获得第一检测结果。检测第二芯片上的信号，获得第二检测结果包括：通过成像系统对第二芯片表面进行拍照，获得第二图像；以及检测第二图像，以获得第二检测结果。成像系统可以是带有成像系统的光学检测装置等，包括光源、物镜以及相机。
在本发明的实施例中，检测第一图像，以获得第一检测结果进一步包括：检测第一图像，以确定第一芯片表面上同时存在第一信号和第二信号的位置的数量Na1以及只存在第二信号的位置的数量Na3。检测第二图像，以获得第二检测结果进一步包括：检测第二图像，以确定第二芯片表面上同时存在第一信号和第二信号的位置的数量Nb1以及只存在第二信号的位置的数量Nb3。
进一步，进行以下(a)，(b)和(c)中的至少之一，来实现基于第一检测结果和第二检测结果，检测待测核苷酸的特异性和/或非特异性吸附：(a)利用公式(Nb1+Nb3)/(Na1+Na3)确定待测核苷酸非特异性吸附在所述基底表面和第一模板链的比例；(b)利用公式Nb1/(Na1+Na3)确定待测核苷酸非特异性吸附在第一模板链或者第二模板链的比例；(c)利用公式(Na1+Na3-Nb1-Nb3)/(Na1+Na3)确定待测核苷酸与第一模板链的有效正配比例，即核苷酸特异性结合到第一模板链的比例。
以下结合附图说明示例图像的检测方法。本发明实施例中，图像包含多个像素点。以下示例描述中，以第一图像的检测为例进行描述，应当理解以下图像检测方法同样也适用于第二图像的处理，并且在同一核苷酸特异性/非特异性吸附的检测示例的平行试验中，如同一核苷酸特异性/非特异性吸附的检测方案的试验组和对照组的图像，均可采用相同的处理方式，以获得可信的、可对比的检测结果。
如图2所示，在本发明的一个的实施例中，图像检测方法，包括：可选的图像预处理步骤S11，图像预处理步骤S11包括预处理第一图像以获得预处理后的第一图像；亮点检测步骤S12，亮点检测步骤S12包括步骤：S21，分析第一图像以计算亮点判定阈 值，S22，分析第一图像以获取候选亮点，S23，根据亮点判定阈值判断候选亮点是否为亮点。
上述图像检测方法，通过图像预处理步骤对第一图像进行处理，可减少亮点检测步骤的计算量，同时，通过亮点判断阈值判断候选亮点是否为亮点，可提高判断图像亮点的准确性。需要说明的是，图像预处理步骤S11是为了获得更好的检测效果所采用的步骤，在其它实施方式中，可以直接对图像进行亮点检测步骤。
具体地，在一个例子中，输入的待检测的第一图像可为512*512或2048*2048的16位tiff格式的图像，tiff格式的图像可为灰度图像。如此，可简化图像检测方法的处理过程。
在某些实施方式中，图像预处理步骤S11包括：对第一图像进行减背景处理，以获得预处理的第一图像。如此，能够进一步减少第一图像的噪声，使图像检测方法的准确性更高。
在某些实施方式中，图像预处理步骤S11包括：对进行减背景处理后的第一图像进行简化处理，以获得预处理的第一图像。如此，可减少后续图像检测方法的计算量。
在某些实施方式中，图像预处理步骤S11包括：对第一图像进行滤波处理，以获得预处理后的第一图像。如此，对第一图像进行滤波可在尽量保留图像细节特征的条件下获取预处理的第一图像，进而可提高图像检测方法的准确性。
在某些实施方式中，图像预处理步骤S11包括：对第一图像进行减背景处理后再进行滤波处理，以获得预处理后的第一图像。如此，对第一图像进行减背景后再进行滤波，能够进一步减少第一图像的噪声，使图像检测方法的准确性更高。
在某些实施方式中，图像预处理步骤S11包括：对进行减背景处理后再进行滤波处理后的第一图像进行简化处理，以获得预处理的第一图像。如此，可减少后续图像检测方法的计算量。
在某些实施方式中，图像预处理步骤S11包括：对第一图像进行简化处理以获得预处理的第一图像。如此，可减少后续图像检测方法的计算量。
在某些实施方式中，请参图3，根据亮点判定阈值判断候选亮点是否为亮点的步骤，包括：步骤S31，在预处理后的第一图像中查找大于(p*p-1)连通的像素点并将查找到的像素点作为候选亮点的中心，p*p与亮点是一一对应的，p*p中的每个值对应一个像素点，p为自然数且为大于1的奇数；步骤S32，判断候选亮点的中心是否满足条件：I max*A BI*ceof guass＞T，其中，I max为p*p窗口的中心最强强度，A BI为p*p窗口中预处 理后的第一图像中为设定值所占的比率，ceof guass为p*p窗口的像素和二维高斯分布的相关系数，T为亮点判定阈值。若满足上述条件，S33，判断候选亮点的中心对应的亮点为待处理图像所包含的亮点；若不满足上述条件，S34，弃去候选亮点的中心对应的亮点。如此，实现了亮点的检测。
具体地，I max可理解为候选亮点的中心最强强度。在一个例子中，p＝3，查找大于8连通的像素点。将查找到的像素点作为候选亮点的像素点。I max为3*3窗口的中心最强强度，A BI为3*3窗口中预处理后的第一图像中为设定值所占的比率，ceof guass为3*3窗口的像素和二维高斯分布的相关系数。
预处理后的第一图像为简化处理后的图像，例如预处理后的第一图像可为二值化图像，也就是说，二值化图像中的设定值可为像素点满足设定条件时所对应的值。在另一个例子中，二值化图像可包含表征像素点不同属性的0和1二个数值，设定值为1，A BI为p*p窗口中二值化图像中为1所占的比率。
在本发明的另一个实施例中，对于拍照得的图像的检测，图像均包含多个像素点，以第一图像为例，检测第一图像包括：利用k*k矩阵对第一图像进行亮点检测，包括判定该矩阵的中心像素值不小于该矩阵非中心任一像素值的矩阵对应一个亮点，k为大于1的奇数，k*k矩阵包含k*k个像素点。该方法基于标记所产生的信号的亮度/强度与背景亮度/强度的差异，能够简单快速的检测图像，检测到以及获得信号的信息。
在某些实施例中，所述矩阵中心的像素值大于第一预设值，所述矩阵非中心任一像素值大于第二像素值。
第一预设值和第二预设值可以根据经验或者一定量的正常图像的正常亮点的像素/强度数据来设定，所称的“正常图像”、“正常亮点”可以是肉眼看起来正常，如图像看起来清晰、背景较干净，亮点大小及强度较均匀等。在一个实施例中，第一预设值和第二预设值与该图像的平均像素值相关。例如，设定第一预设值为该图像的平均像素值的1.4倍，第二预设值为该图像的平均像素值的1.1倍，能够排除干扰、获得较佳的亮点检测结果。
具体地，在一个示例中，第一图像是彩色图像，彩色图像的一个像素点具有三个像素值，可以将该彩色图像转化为灰度图像，再进行图像检测，以降低图像检测过程的计算量和复杂度。可选择但不限于利用浮点算法、整数方法、移位方法或平均值法等将非灰度图像转换成灰度图像。当然，也可以直接检测彩色图像，上述涉及的像素值的大小 比较可看成是三维值或者具有三个元素的数组的大小比较，可根据经验及需要自定义多个多维值的相对大小，例如当三维值a中的任两维数值比三维值b的对应维度的数值大，可认为三维值a大于三维值b。
在另一个示例中，第一图像是灰度图像，灰度图像的像素值为一维数值，灰度图像的像素值同灰度值。
在一个例子中，如图4所示，在一基底表面上，该基底表面也称为芯片表面，为经过表面修饰后带有环氧基团的表面：加入探针(DNA捕获链，Capture DNA)，探针为经过氨基化修饰的探针、末端带有氨基基团，探针通过-NH 3与芯片表面的环氧基团反应，固定在基底表面，然后使用钝化液钝化芯片，以封闭未反应的环氧基团。再加入杂交模板链，该模板链为末端带有Cy3荧光染料标记并与探针碱基互补配对的特定序列的DNA链。
杂交完成后，加入与杂交模板链的第一个待测碱基互补配对且带有ATTO-647N荧光染料标记的待测核苷酸，然后通过荧光显微镜例如全内反射荧光显微镜(TIRF)对表面进行拍照，可得到带有信号点/斑的图像。比较可能的结果，(1)如果同一个位置观察到两种荧光信号，倾向说明该位置的核苷酸与模板链发生正配反应或者少量的链上吸附，数量为Na1；(2)如果只观察到绿色荧光信号，倾向说明该位置为模板链的非特异性吸附，数量为Na2；(3)如果只观察到红色荧光信号，倾向说明该位置为核苷酸的非特异性吸附或者模板链的Cy3荧光染料发生猝灭但是反应正常，数量为Na3。
如图5所示，在一基底表面上，该基底表面也称为芯片表面，为经过表面修饰后带有环氧基团的表面：利用相同的反应体系及时间，将同一种、同样浓度的探针，通过-NH 3与芯片表面的环氧基团反应，将探针通过化学键连接到芯片表面，然后使用钝化液钝化芯片，以封闭未反应的环氧基团。再加入杂交模板链，该模板链为末端带有Cy3荧光染料标记且杂交部分与探针序列互补配对，但第一个待测碱基与将要加入的待测核苷酸不配对的DNA链。
杂交完成后，加入与待测核苷酸(其与图4的示例中的待测核苷酸相同)，带有ATTO-647N荧光染料标记，然后通过全内反射荧光显微镜(TIRF)对表面进行成像拍照，可得到带有荧光信号点/斑的图像。比较可能的结果，(1)如果同一个位置观察到两种荧光信号，倾向说明该位置的核苷酸与模板链发生非特异性吸附，数量为Nb1；(2)如果只观察到绿色荧光信号，倾向说明该位置为模板链的非特异性吸 附，数量为Nb2；(3)如果只观察到红色荧光信号，倾向说明该位置为核苷酸的表面非特异性吸附，数量为Nb3。
基于上述信息，可进一步量化区分特异性吸附和非特异性吸附，评估芯片表面和/或探针对待测核苷酸的非特异性吸附等，例如利用公式(Nb1+Nb3)/(Na1+Na3)确定待测核苷酸非特异性吸附在基底表面和核酸链的比例；利用公式Nb1/(Na1+Na3)确定待测核苷酸非特异性吸附在核酸链的比例；利用公式(Na1+Na3-Nb1-Nb3)/(Na1+Na3)确定待测核苷酸与模板链的有效正配比例。
在本发明的另一个实施例中，检测核苷酸非特异性吸附的方法包括：S1000：使待测核苷酸与第三模板链反应，该第三模板链与第三芯片上的探针连接，该第三芯片包括基底表面以及一端固定在该基底表面的探针，第三模板链带有第三标记，待测核苷酸与第三模板链的相应碱基不能配对，无法使探针的另一端延伸一个碱基，待测核苷酸带有第四标记，第三标记能够产生第三信号，第四标记能够产生第四信号；S2000：检测第三芯片上的信号，获得第三检测结果；S3000：使待测核苷酸与第四模板链反应，该第四模板链与第四芯片连接，第四芯片包括不带探针的基底表面，第四模板链带有第三标记，待测核苷酸能够与第四模板链的相应碱基配对；S4000：检测第四芯片上的信号，获得第四检测结果；S5000：基于第三检测结果和第四检测结果，检测待测核苷酸的非特异性吸附。
利用该方法，能够定性或定量检测基底表面和/或基底表面上的探针/模板链对核苷酸的非特异吸附情况；通过检测系统检测基底表面的信号，能够定性或定量区分非特异性吸附类型，获得非特异性定量、分布情况等信息，能够用于芯片生产以及所有涉及芯片检测核酸过程的方法或应用中，例如用于空载芯片(不带探针)或者捕获芯片(带探针)性能的评价、芯片生产的质控，用于芯片捕获核酸的效果的预测、分析比较等。
S1000和S3000的进行无顺序要求，可先后进行，也可同时进行。同样的，S2000和S4000的进行也无顺序限制。在一些实施例中，S1000和S3000是平行试验，为同时进行。
在一平行试验示例中，S1000和S3000中的第三芯片和第四芯片的基底的材质、表面特性如亲疏水、表面大小等是基本相同或一致的。在一个例子中，第三芯片和第四芯片的基底表面是经过相同表面处理的、相同材料性能的基底。在另一个例子中，第三芯片和第四芯片的基底表面是经过相同表面处理的同一基底的不同表面区域。例如，S1000和S3000中的第三芯片和第四芯片的基底表面除了是否固定探针以外，其它方面如表面 大小、待测核苷酸的量、反应条件等基本一致。所称的“基本一致”、“基本相同”同“一致”或“相同”，指不同批次制备、处理和/或平行试验产生的差异在允许的偏差范围内。
对探针在第三芯片表面上的分布或排布方式不作限制，在本发明实施例中，探针在第三芯片表面上随机分布或规则分布(例如呈阵列分布)。
在一个示例中，第三标记和第四标记为不同的荧光染料。在本发明的实施例中，检测第三芯片上的信号，获得第三检测结果包括：通过成像系统对第三芯片表面进行拍照，获得第三图像；以及检测第三图像，以获得第三检测结果。检测第四芯片上的信号，获得第四检测结果包括：通过成像系统对第四芯片表面进行拍照，获得第四图像；以及检测第四图像，以获得第四检测结果。成像系统可以是带有成像系统的光学检测装置等，包括光源、物镜以及相机。
在本发明的实施例中，检测第三图像，以获得第三检测结果进一步包括：检测第三图像，以确定第三芯片表面上同时出现第三信号和第四信号的位置的数量Nc1以及只出现第四信号的位置的数量Nc3。检测第四图像，以获得第四检测结果进一步包括：检测第四图像，以确定第四芯片表面上同时出现第三信号和第四信号的位置的数量Nd1以及只出现第四信号的位置的数量Nd3。图像的检测可参考上述对第一图像的检测示例。
可利用公式(Nc1+Nc3-Nd1-Nd3)/(Nc1+Nc3)确定基底表面经过固定探针后对待测核苷酸非特异性吸附变化比例，来实现基于第三检测结果和第四检测结果，检测待测核苷酸的非特异性吸附。
在一个例子中，如图5所示，在一基底表面上，该基底表面也称为芯片表面，为经过表面修饰后带有环氧基团的表面：加入探针(DNA捕获链，Capture DNA)，探针为经过氨基化修饰的探针、末端带有氨基基团，探针通过-NH 3与芯片表面的环氧基团反应，固定在基底表面，然后使用钝化液钝化芯片，以封闭未反应的环氧基团。再加入杂交模板链，该模板链为末端带有Cy3荧光染料标记且杂交部分与探针序列互补配对，但第一个待测碱基与将要加入的待测核苷酸不配对的DNA链。
待杂交反应完成后，加入带有ATTO-647N荧光染料标记的待测核苷酸，进行杂交。杂交完成后，通过荧光显微镜包括全内反射荧光显微镜(TIRF)对表面进行拍照，可得到带有荧光信号的图像。(1)如果同一个位置观察到两种荧光信号，倾向说明该位置同时存在模板链和核苷酸的非特异性吸附，数量为Nc1；(2)如果只观察到 绿色荧光信号，倾向说明该位置为模板链的非特异性吸附，数量为Nc2；(3)如果只观察到红色荧光信号，倾向说明该位置为核苷酸的非特异性吸附，数量为Nc3。
如图6所示，在一基底表面上，该基底表面也称为芯片表面，为经过表面修饰后带有环氧基团的表面，与上述基底表面相同，加入不包含探针的固定液，反应体系及反应时间与上述图5示例相同，然后使用钝化液钝化芯片。再加入杂交模板链，该模板链为末端带有Cy3荧光染料标记的特定序列的DNA链。
待反应完成后，加入带有ATTO-647N荧光染料标记的待测核苷酸，然后通过荧光显微镜包括全内反射荧光显微镜(TIRF)对表面进行成像拍照，可得到带有荧光信号的图像。(1)如果同一个位置观察到两种荧光信号，则说明该位置同时存在模板链和核苷酸的非特异性吸附，数量为Nd1；(2)如果只观察到绿色荧光信号，则说明该位置为模板链的非特异性吸附，数量为Nd2；(3)如果只观察到红色荧光信号，则说明该位置为核苷酸的非特异性吸附，数量为Nd3。
可进一步利用公式(Nc1+Nc3-Nd1-Nd3)/(Nc1+Nc3)确定基底表面经过固定探针后对待测核苷酸非特异性吸附变化比例。
在本发明的又一个实施例中，检测核苷酸特异性和/或非特异性吸附的方法包括：S10000：使待测核苷酸与第五模板链反应，该第五模板链与第五芯片上的探针连接，该第五芯片包括基底表面以及一端固定在该基底表面的探针，第五模板链带有第五标记，待测核苷酸能够与第五模板链的相应碱基配对，从而使探针的另一端延伸一个碱基，待测核苷酸带有第六标记，第五标记能够产生第五信号，第六标记能够产生第六信号；S20000：检测第五芯片上的信号，获得第五检测结果；S30000：使待测核苷酸与第六模板链反应，该第六模板链与第六芯片上的探针连接，该第六芯片包括基底表面以及一端固定在该基底表面的探针，第六模板链带有第五标记，待测核苷酸与第六模板链的相应碱基不能配对，无法使探针的另一端延伸一个碱基；S40000：检测第六芯片上的信号，获得第六检测结果；S50000：使待测核苷酸与第七模板链反应，该第七模板链与第七芯片连接，该第七芯片包括不带探针的基底表面；S60000：检测第七芯片上的信号，获得第七检测结果；S70000：基于第五检测结果、第六检测结果和第七检测结果中的至少两个检测结果，检测待测核苷酸的特异性和/或非特异性吸附。
利用该方法，能够定性或定量检测基底表面和/或基底表面上的探针/模板链对核苷酸的特异性和/或非特异吸附情况；通过检测系统检测基底表面的信号，能够定性或定 量区分特异性和/或非特异性吸附类型，获得特异性和/或非特异性定量、分布情况等信息，能够用于核苷酸类似物性能评估、芯片生产以及所有涉及芯片检测核酸过程的方法或应用中，例如用于空载芯片(不带探针)或者捕获芯片(带探针)性能的评价、芯片生产的质控，用于芯片捕获核酸的效果的预测、分析比较等。
S10000、S30000和S50000的进行无顺序要求，可先后进行，也可同时进行。同样的，S20000、S40000和S60000的进行也无顺序限制。在一些实施例中，S10000、S30000和S50000是平行试验，为同时进行。
在一平行试验示例中，S10000、S30000和S50000中的第五芯片、第六芯片和第七芯片的基底的材质、表面特性如亲疏水、表面大小等是基本相同或一致的。在一个例子中，第五芯片、第六芯片和第七芯片的基底表面是经过相同表面处理的、相同材料性能的基底。在另一个例子中，第五芯片、第六芯片和第七芯片的基底表面是经过相同表面处理的同一基底的不同表面区域。例如，S10000和S30000中的第五芯片和第六芯片的基底表面在各方面如表面大小、探针种类和核苷酸序列、探针固定密度、探针分布、待测核苷酸的量、反应条件等基本一致。同时，S50000中的第七芯片与S10000和S30000中的第五芯片和第六芯片相比，除了没有固定探针以外，其它方面如表面大小、待测核苷酸的量、反应条件等基本一致。所称的“基本一致”、“基本相同”同“一致”或“相同”，指不同批次制备、处理和/或平行试验产生的差异在允许的偏差范围内。
对探针在第五芯片和第六芯片表面上的分布或排布方式不作限制，在本发明实施例中，探针在第五芯片和第六芯片表面上随机分布或规则分布(例如呈阵列分布)。在平行实验中，探针在不同的芯片表面上的分布状态相同。
在一个示例中，第五标记和第六标记为不同的荧光染料。在本发明的实施例中，检测第五芯片上的信号，获得第五检测结果包括：通过成像系统对第五芯片表面进行拍照，获得第五图像；以及检测第五图像，以获得第五检测结果。检测第六芯片上的信号，获得第六检测结果包括：通过成像系统对第六芯片表面进行拍照，获得第六图像；以及检测第六图像，以获得第六检测结果。检测第七芯片上的信号，获得第七检测结果包括：通过成像系统对第七芯片表面进行拍照，获得第七图像；以及检测第七图像，以获得第七检测结果。成像系统可以是带有成像系统的光学检测装置等，包括光源、物镜以及相机。
在本发明的实施例中，检测第五图像，以获得第五检测结果进一步包括：检测第五图像，以确定第五芯片表面上同时存在第五信号和第六信号的位置的数量Ne1以及只 存在第六信号的位置的数量Ne3。检测第六图像，以获得第六检测结果进一步包括：检测第六图像，以确定第六芯片表面上同时存在第五信号和第六信号的位置的数量Nf1以及只存在第六信号的位置的数量Nf3。检测第七图像，以获得第七检测结果进一步包括：检测第七图像，以确定第七芯片表面上同时出现第五信号和第六信号的位置的数量Ng1以及只出现第六信号的位置的数量Ng3。图像的检测可参考上述对第一图像的检测示例。
进一步，进行以下(a)，(b)，(c)和(d)中的至少之一，来实现基于第五检测结果、第六检测结果和第七检测结果中的至少两个检测结果，检测待测核苷酸的特异性和/或非特异性吸附：(a)利用公式(Nf1+Nf3)/(Ne1+Ne3)确定待测核苷酸非特异性吸附在基底表面和核酸链的比例；(b)利用公式Nf1/(Ne1+Ne3)确定待测核苷酸非特异性吸附在核酸链的比例；(c)利用公式(Ne1+Ne3-Nf1-Nf3)/(Ne1+Ne3)确定待测核苷酸与模板链的有效正配比例；(d)利用公式(Ng1+Ng3-Nf1-Nf3)/(Ng1+Ng3)确定基底表面经过固定探针后对待测核苷酸非特异性吸附变化比例。
在一个例子中，如图4所示，在一基底表面上，该基底表面也称为芯片表面，为经过表面修饰后带有环氧基团的表面：加入探针(DNA捕获链，Capture DNA)，探针为经过氨基化修饰的探针、末端带有氨基基团，探针通过-NH 3与芯片表面的环氧基团反应，固定在基底表面，然后使用钝化液钝化芯片，以封闭未反应的环氧基团。再加入杂交模板链，该模板链为末端带有Cy3荧光染料标记并与探针碱基互补配对的特定序列的DNA链。
杂交完成后，加入与杂交模板链的第一个待测碱基互补配对且带有ATTO-647N荧光染料标记的待测核苷酸，然后通过荧光显微镜例如全内反射荧光显微镜(TIRF)对表面进行拍照，可得到带有信号点/斑的图像。(1)如果同一个位置观察到两种荧光信号，则说明该位置的核苷酸与模板链发生正配反应或者少量的链上吸附，数量为Ne1；(2)如果只观察到绿色荧光信号，则说明该位置为模板链的非特异性吸附，数量为Ne2；(3)如果只观察到红色荧光信号，则说明该位置为核苷酸的非特异性吸附或者模板链的Cy3荧光染料发生猝灭但是反应正常，数量为Ne3。
如图5所示，在一基底表面上，该基底表面也称为芯片表面，为经过表面修饰后带有环氧基团的表面：利用相同的反应体系及时间，将同一种、同样浓度的探针，通过-NH 3与芯片表面的环氧基团反应，将探针通过化学键连接到芯片表面，然后使用钝化液钝化芯片，以封闭未反应的环氧基团。再加入杂交模板链，该模板链为末端 带有Cy3荧光染料标记且杂交部分与探针序列互补配对，但第一个待测碱基与将要加入的待测核苷酸不配对的DNA链。
杂交完成后，加入与待测核苷酸(其与图4的示例中的待测核苷酸相同)，带有ATTO-647N荧光染料标记，然后通过全内反射荧光显微镜(TIRF)对表面进行成像拍照，可得到带有荧光信号点/斑的图像。(1)如果同一个位置观察到两种荧光信号，则说明该位置的核苷酸与模板链发生非特异性吸附，数量为Nf1；(2)如果只观察到绿色荧光信号，则说明该位置为模板链的非特异性吸附，数量为Nf2；(3)如果只观察到红色荧光信号，则说明该位置为核苷酸的表面非特异性吸附，数量为Nf3。
如图6所示，在一基底表面上，该基底表面也称为芯片表面，为经过表面修饰后带有环氧基团的表面，与上述基底表面相同，加入不包含探针的固定液，反应体系及反应时间与上述图5示例相同，然后使用钝化液钝化芯片。再加入杂交模板链，该模板链为末端带有Cy3荧光染料标记的特定序列的DNA链。
待反应完成后，加入带有ATTO-647N荧光染料标记的待测核苷酸，然后通过荧光显微镜包括全内反射荧光显微镜(TIRF)对表面进行成像拍照，可得到带有荧光信号的图像。(1)如果同一个位置观察到两种荧光信号，则说明该位置同时存在模板链和核苷酸的非特异性吸附，数量为Ng1；(2)如果只观察到绿色荧光信号，则说明该位置为模板链的非特异性吸附，数量为Ng2；(3)如果只观察到红色荧光信号，则说明该位置为核苷酸的非特异性吸附，数量为Ng3。
基于上述信息，可进行量化区分特异性吸附和非特异性吸附，评估芯片表面和/或探针对待测核苷酸的特异性吸附和非特异性吸附等，例如利用公式(Nf1+Nf3)/(Ne1+Ne3)确定待测核苷酸非特异性吸附在基底表面和核酸链的比例；利用公式Nf1/(Ne1+Ne3)确定待测核苷酸非特异性吸附在核酸链的比例；利用公式(Ne1+Ne3-Nf1-Nf3)/(Ne1+Ne3)确定待测核苷酸与模板链的有效正配比例；利用公式(Ng1+Ng3-Nf1-Nf3)/(Ng1+Ng3)确定基底表面经过固定探针后对待测核苷酸非特异性吸附变化比例。
以下通过实施例详细说明本发明的技术方案，应当理解，实施例仅是示例性的，不能理解为对本发明保护范围的限制。涉及的材料、试剂以及序列等，如无特殊说明，可通过自己配制合成或者市售途径获取。
实施例1：考察核苷酸反应过程中的非特异性吸附的影响
芯片：表面带有环氧基团的玻璃(购自肖特)；
固定链(EKB-6P)：人工合成的特定序列，末端带有-NH 3的DNA短序列，序列具体为：
TTTTTTTTTTTTACTTTGCCTCCTTCTGCATGGTATTCTTTCTCTTCCGCACCCAG-3’(SEQ ID NO：1)；
杂交链1(EKB-6T-2-Cy3)：人工合成的特定序列，与固定链互补配对，带有长度为33bp待测序列，末端带有荧光基团Cy3的DNA短序列，具体序列为：
AGGCAACATGGATCTAGCATGATCACATGACATCTGGGTGCGGAAGAGAAAGAATACCATGCAGAAGGAGGCAAAGTA-3’(SEQ ID NO：2)；
杂交链2(EKB-6T-6-Cy3)：人工合成的特定序列，与固定链不互补配对，带有长度为33bp待测序列(与EKB-6T-2-Cy3待测序列不一致)，末端带有荧光基团Cy3的DNA短序列，具体序列为：
CCTCAGATGAATATTGAATCACATCACACGATACTGGGTGCGGAAGAGAAAGAATACCATGCAGAAGGAGGCAAAGTA-3’(SEQ ID NO：3)。
实验过程：
固定链的固定1(实验组，对照组1)：将玻璃清洗吹干后，置于150mM K 2HPO 4并含有浓度为1.0M的氨基修饰的EKB-6P核酸序列的溶液中，37℃条件下反应0.5小时，依次用3XSSC溶液(含0.1％的Triton)，3XSSC，0.15M K 2HPO 4溶液清洗后，加入1M K 2HPO 4在37℃条件下钝化17小时。
固定链的固定2(对照组2)：将玻璃清洗吹干后，置于150mM K 2HPO 4溶液中，37℃条件下反应0.5小时，依次用3XSSC溶液(含0.1％的Triton)，3XSSC，0.15M K 2HPO 4溶液清洗后，加入1M K 2HPO 4在37℃条件下钝化17小时。
芯片(Flow Cell，流通池)组装：将钝化完成的芯片组装成四通道的流通池。
杂交链的杂交1(实验组，对照组2)：将组装好的四通道流通池加入Rinse buffer(1XSSC+150mM HEPES+0.1％SDS)，在55℃条件下复溶0.5小时，然后再加入杂交链(EKB-6T-2-Cy3)浓度为1nM的3XSSC溶液，在55℃条件下反应0.5小时。然后依次使用Rinse buffer(1XSSC+150mM HEPES+0.1％SDS)和Buffer H(150mM HEPES+150mM NaCl)冲洗通道。
杂交链的杂交2(对照组1)：将组装好的四通道流通池加入Rinse buffer (1XSSC+150mM HEPES+0.1％SDS)，在55℃条件下复溶0.5小时，然后再加入杂交链(EKB-6T-6-Cy3)浓度为1nM的3XSSC溶液，在55℃条件下反应0.5小时。然后依次使用Rinse buffer(1XSSC+150mM HEPES+0.1％SDS)和Buffer H(150mM HEPES+150mM NaCl)冲洗通道。
核苷酸反应：加入含有浓度为200nM的核苷酸A的反应溶液到通道，37℃条件下反应90s，然后使用Rinse buffer(1XSSC+150mM HEPES+0.1％SDS)和Buffer H(150mM HEPES+150mM NaCl)依次冲洗通道，然后加入成像缓冲液。
拍照检测：使用TIRF对芯片通道的拍照。
实验组结果如图7所示，其中左图显示一个视野中的Cy3荧光点，统计多个通道全部视野的Cy3荧光点数为19852，右图显示相同视野中的ATTO647N荧光点，统计多个通道全部视野的ATTO647N荧光点数为16744，二者重合点数为13358。
对照组1结果如图8所示，其中左图显示一个视野中的Cy3荧光点，统计多个通道全部视野的Cy3荧光点数为数19971，右图显示相同视野中的ATTO647N荧光点，统计多个通道全部视野的ATTO647N荧光点数为619，二者重合点数为358。
对照组2结果如图9所示，其中左图显示一个视野中的Cy3荧光点，统计多个通道全部视野的Cy3荧光点数为603，右图显示相同视野中的ATTO647N荧光点，统计多个通道全部视野的ATTO647N荧光点数为524，其中Cy3荧光点为芯片表面的杂质，亮度较弱，ATTO647N荧光点为核苷酸在芯片表面的非特异性吸附。
依据上述数据可以确定：待测核苷酸非特异性吸附在基底表面和核酸链的比例为3.25％；待测核苷酸非特异性吸附在核酸链的比例为1.19％；待测核苷酸与模板链的有效正配比例为96.75％；基底表面经过固定探针后对待测核苷酸非特异性吸附变化比例-86.5％。
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (10)

  1. 一种检测核苷酸特异性和/或非特异性吸附的方法，其特征在于，所述方法包括：
    使待测核苷酸与第一模板链反应，所述第一模板链与第一芯片上的探针连接，所述第一芯片包括基底表面以及一端固定在该基底表面的探针，所述第一模板链带有第一标记，所述待测核苷酸能够与所述第一模板链的相应碱基配对，从而使所述探针的另一端延伸一个碱基，所述待测核苷酸带有第二标记，所述第一标记能够产生第一信号，所述第二标记能够产生第二信号；
    检测所述第一芯片上的信号，获得第一检测结果；
    使所述待测核苷酸与第二模板链反应，所述第二模板链与第二芯片上的探针连接，所述第二芯片包括基底表面以及一端固定在该基底表面的探针，所述第二模板链带有所述第一标记，所述待测核苷酸与所述第二模板链的相应碱基不能配对，无法使所述探针的另一端延伸一个碱基；
    检测所述第二芯片上的信号，获得第二检测结果；
    基于所述第一检测结果和所述第二检测结果，检测所述待测核苷酸的特异性和/或非特异性吸附。
  2. 一种检测核苷酸非特异性吸附的方法，其特征在于，所述方法包括：
    使待测核苷酸与第三模板链反应，所述第三模板链与第三芯片上的探针连接，所述第三芯片包括基底表面以及一端固定在该基底表面的探针，所述第三模板链带有第三标记，所述待测核苷酸与所述第三模板链的相应碱基不能配对，无法使所述探针的另一端延伸一个碱基，所述待测核苷酸带有第四标记，所述第三标记能够产生第三信号，所述第四标记能够产生第四信号；
    检测所述第三芯片上的信号，获得第三检测结果；
    使所述待测核苷酸与第四模板链反应，所述第四模板链与第四芯片连接，所述第四芯片包括不带探针的基底表面，所述第四模板链带有所述第三标记，所述待测核苷酸能够与所述第四模板链的相应碱基配对；
    检测所述第四芯片上的信号，获得第四检测结果；
    基于所述第三检测结果和所述第四检测结果，检测所述待测核苷酸的非特异性吸附。
  3. 根据权利要求1或2所述的方法，其特征在于，所述第一芯片和第二芯片上的探针随机分布和/或规则分布在基底表面上；和/或
    所述第三芯片上的探针随机分布和/或规则分布在基底表面上。
  4. 根据权利要求1或2所述的方法，其特征在于，
    (i)所述检测所述第一芯片上的信号，获得第一检测结果包括：通过成像系统对所述第一芯片表面进行拍照，获得第一图像；以及检测所述第一图像，以获得所述第一检测结果；
    所述检测所述第二芯片上的信号，获得第二检测结果包括：通过所述成像系统对所述第二芯片表面进行拍照，获得第二图像；以及检测所述第二图像，以获得所述第二检测结果；和/或
    (ii)所述检测所述第三芯片上的信号，获得第三检测结果包括：通过成像系统对所述第三芯片表面进行拍照，获得第三图像；以及检测所述第三图像，以获得所述第三检测结果；
    所述检测所述第四芯片上的信号，获得第四检测结果包括：通过所述成像系统对所述第四芯片表面进行拍照，获得第四图像；以及检测所述第四图像，以获得所述第四检测结果。
  5. 根据权利要求4所述的方法，其特征在于，
    (i)中检测所述第一图像，以获得所述第一检测结果包括：检测所述第一图像，以确定所述第一芯片表面上同时存在第一信号和第二信号的位置的数量Na1以及只存在第二信号的位置的数量Na3；
    检测所述第二图像，以获得所述第二检测结果包括：检测所述第二图像，以确定所述第二芯片表面上同时存在第一信号和第二信号的位置的数量Nb1以及只存在第二信号的位置的数量Nb3；和/或
    (ii)中检测所述第三图像，以获得所述第三检测结果包括：检测所述第三图像，以确定所述第三芯片表面上同时出现第三信号和第四信号的位置的数量Nc1以及只出现第四信号的位置的数量Nc3；
    检测所述第四图像，以获得所述第四检测结果包括：检测所述第四图像，以确定 所述第四芯片表面上同时出现第三信号和第四信号的位置的数量Nd1以及只出现第四信号的位置的数量Nd3。
  6. 根据权利要求5所述的方法，其特征在于，
    进行(i)后，所述基于所述第一检测结果和所述第二检测结果，检测所述待测核苷酸的特异性和/或非特异性吸附包括进行以下(a)，(b)和(c)中的至少之一：
    (a)利用公式(Nb1+Nb3)/(Na1+Na3)确定所述待测核苷酸非特异性吸附在所述基底表面和第一模板链的比例；
    (b)利用公式Nb1/(Na1+Na3)确定所述待测核苷酸非特异性吸附在第一模板链上的比例；
    (c)利用公式(Na1+Na3-Nb1-Nb3)/(Na1+Na3)确定所述待测核苷酸特异性结合到所述第一模板链的比例；和/或
    进行(ii)后，所述基于所述第三检测结果和所述第四检测结果，检测所述待测核苷酸的非特异性吸附包括：利用公式(Nc1+Nc3-Nd1-Nd3)/(Nc1+Nc3)确定基底表面经过固定所述探针后对所述待测核苷酸非特异性吸附变化比例。
  7. 根据权利要求4或5所述的方法，其特征在于，
    (i)中，所述第一图像和/或所述第二图像包含多个像素点，检测所述第一图像和/或检测所述第二图像包括：
    利用k*k矩阵对所述第一图像和/或所述第二图像进行亮点检测，包括判定所述矩阵的中心像素值不小于该矩阵非中心任一像素值的矩阵对应一个亮点，k为大于1的奇数，k*k矩阵包含k*k个像素点；
    任选的，所述矩阵的中心像素值大于第一预设值，所述矩阵非中心任一像素值大于第二预设值；
    任选的，所述第一预设值和所述第二预设值与该图像的平均像素值相关；和/或
    (ii)中，所述第三图像和/或所述第四图像包含多个像素，检测所述第三图像和/或检测所述第四图像包括：
    利用k*k矩阵对所述第三图像和/或所述第四图像进行亮点检测，包括判定所述矩阵中心的像素值不小于该矩阵非中心任一像素值的矩阵对应一个亮点，k为大于1的奇数，k*k矩阵包含k*k个像素点；
    任选的，所述矩阵中心的像素值大于第一预设值，所述矩阵非中心任一像素值大于第二预设值；
    任选的，所述第一预设值和所述第二预设值与该图像的平均像素值相关。
  8. 根据权利要求4或5所述的方法，其特征在于，
    (i)中，所述第一图像和/或所述第二图像包含多个像素点，检测所述第一图像和/或检测所述第二图像包括：
    亮点检测步骤，所述亮点检测步骤包括：分析所述第一图像和/或分析所述第二图像以计算亮点判定阈值，分析所述第一图像和/或所述第二图像以获取候选亮点，根据所述亮点判定阈值判断所述候选亮点是否为所述亮点，和/或
    (ii)中，所述第三图像和/或所述第四图像包含多个像素点，检测所述第三图像和/或检测所述第四图像包括：
    亮点检测步骤，所述亮点检测步骤包括：分析所述第三图像和/或分析所述第四图像以计算亮点判定阈值，分析所述第三图像和/或所述第四图像以获取候选亮点，根据所述亮点判定阈值判断所述候选亮点是否为所述亮点。
  9. 根据权利要求8所述的方法，其特征在于，(i)和/或(ii)的亮点检测步骤之前还包括：
    图像预处理步骤，所述图像预处理步骤包括预处理所述第一图像、第二图像、第三图像和/或第四图像，以获得预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像；
    所述图像预处理步骤包括进行以下(a)、(b)、(c)、(d)、(e)和(f)至少之一：
    (a)对所述第一图像、第二图像、第三图像和/或第四图像进行减背景处理，以获得预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像；
    (b)对进行减背景处理后的第一图像、减背景处理后的第二图像、减背景处理后的第三图像和/或减背景处理后的第四图像进行简化处理，以获得预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像；
    (c)对所述第一图像、第二图像、第三图像和/或所述第二图像进行滤波处理，以获得预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像；
    (d)对所述第一图像、第二图像、第三图像和/或第四图像进行减背景处理后再进行滤波处理，以获得预处理后的第一图像、预处理后的第二图像、预处理后的第三图像 和/或预处理后的第四图像；
    (e)对进行减背景处理后再进行滤波处理的第一图像、进行减背景处理后再进行滤波处理的第二图像、进行减背景处理后再进行滤波处理的第三图像和/或进行减背景处理后再进行滤波处理的第四图像进行简化处理，以获得预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像；
    (f)对所述第一图像、第二图像、第三图像和/或第四图像进行简化处理，以获得预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像。
  10. 根据权利要求9所述的(b)、(e)和(f)任一方法，其特征在于，所述根据所述亮点判定阈值判断所述候选亮点是否为所述亮点，包括：在所述预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像中查找大于(p*p-1)连通的像素点并将查找到的所述像素点作为所述候选亮点的中心，p为自然数且为大于1的奇数；
    判断所述候选亮点的中心是否满足条件：I max*A BI*ceof guass＞T，其中，I max为p*p窗口的中心最强强度，A BI为p*p窗口中所述预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像中为设定值所占的比率，ceof guass为p*p窗口的像素和二维高斯分布的相关系数，T为所述亮点判定阈值，
    若满足上述条件，判定所述候选亮点为一个亮点。
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