CN114540187A - Integrated handheld digital nucleic acid detector and nucleic acid detection method - Google Patents
Integrated handheld digital nucleic acid detector and nucleic acid detection method Download PDFInfo
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- CN114540187A CN114540187A CN202210275861.5A CN202210275861A CN114540187A CN 114540187 A CN114540187 A CN 114540187A CN 202210275861 A CN202210275861 A CN 202210275861A CN 114540187 A CN114540187 A CN 114540187A
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
Abstract
The invention discloses an integrated handheld digital nucleic acid detector and a nucleic acid detection method. The nucleic acid detector comprises a chip sample adding and packaging system, a heat circulation system and a detection and analysis system which are integrated into a whole; the chip sample adding and packaging system comprises a micro-porous dPCR chip and a packaging cover plate for packaging the micro-porous dPCR chip; the thermal cycle system is used for performing thermal cycle on the microporous dPCR chip; and the detection and analysis system is used for shooting a fluorescence image of the microporous dPCR chip after the thermal cycle is finished, and processing and analyzing the fluorescence image. The chip sample adding and packaging system, the heat circulation system and the detection and analysis system are miniaturized and integrated into one instrument, so that the instrument structure is simplified, and the instrument cost is reduced; and provides a microporous dPCR chip flexible packaging process based on PDMS and Parylene C multilayer structures, and solves the problems of sample evaporation and cross contamination of a microporous digital PCR chip caused by mineral oil liquid seal.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to an integrated handheld digital nucleic acid detector and a nucleic acid detection method.
Background
Polymerase Chain Reaction (PCR) can specifically amplify nucleic acid in vitro, and is a gold standard for nucleic acid detection. For double-stranded nucleic Acid, i.e., Deoxyribonucleic Acid (DNA), the PCR process mainly includes two parts: warm start and hot cycle. Hot start is the activation of the polymerase activity in the premix at elevated temperature (about 95 ℃); thermal cycling is the process by which DNA is caused to replicate by the action of a polymerase. A thermal cycle typically includes three biological processes: high temperature denaturation, low temperature annealing and medium temperature extension. During denaturation (about 95 ℃), the hydrogen bonds between the inner strands of double-stranded DNA are broken and one DNA double strand is broken into two single-stranded DNAs. The primers in the PCR premix then bind to the target gene fragment on the single stranded DNA during annealing (about 55 ℃). During the extension (about 72 ℃), the remaining single-stranded DNA sequence is synthesized into double-stranded DNA by the action of a polymerase. Ideally, the copy number of the DNA will double after each thermal cycle is complete.
In order to quantify the PCR product, the real-time fluorescent quantitative PCR technology adds fluorescent dye into the PCR premix, and utilizes the working principle that the fluorescent dye is combined with DNA to generate fluorescence to carry out real-time observation and quantitative analysis on the PCR process so as to realize the detection of the target gene (target gene).
In order to realize quantitative detection of trace target genes, digital PCR (dPCR) technology has been developed on the basis of conventional PCR technology. The PCR premix containing the fluorescent dye is evenly distributed into thousands of reaction cavities, namely all target genes in the premix are randomly distributed into the reaction cavities, and some cavities have no target genes and contain at least one target gene. Then, the reaction chambers are subjected to thermal cycling treatment, and the PCR premix distributed in the reaction chambers is subjected to polymerase chain reaction simultaneously. After the reaction is completed, there is a clear difference in fluorescence intensity between the reaction chamber containing the target gene and the reaction chamber without the target gene. The sample segmentation method required by the dPCR technique is based on microfluidic technology. The methods of loading the premix are mainly classified into droplet-based dPCR (ddPCR) and chip-based dPCR (cdPCR). The ddPCR technology utilizes a micro-droplet generator to uniformly divide and wrap the PCR premix liquid in oil droplets to form a virtual reaction cavity of water-in-oil droplets. The cdPCR technology comprises forming a microfluidic channel and a micro-reaction cavity on a silicon/polymer substrate by micro-nano processing technology, loading a sample on a chip by a chip flow path or direct pipetting method, performing thermal cycle treatment on the micro-reaction cavity by a micro-heater, and extracting and analyzing the fluorescence intensity of each reaction cavity by an image processing algorithm
The micro-porous digital PCR chip is mainly characterized in that a large number of independent micro-pores are prepared on a substrate material silicon or Polydimethylsiloxane (PDMS) through a semiconductor processing technology and a soft lithography technology and are used as a PCR reaction cavity. Through chemical treatment on the surface of the micro-pore array chip, the PCR premixed solution is uniformly distributed in each micro-reaction cavity, then the surface of the chip is covered with silicone oil, so that cross contamination of samples among reaction cavities and evaporation of the solution in the temperature change process are prevented, and finally the whole chip is packaged. And putting the packaged chip into a thermal cycle system to perform a PCR process. And after the circulation is finished, counting the number of the micro reaction cavities generating the fluorescence. The number of the reaction cavities emitting fluorescence is counted by a fluorescence imaging system, and the target gene content in the original PCR premix can be obtained by analyzing the Poisson distribution.
The existing chip type digital PCR detection instrument has complex structure and high instrument cost. Meanwhile, the packaging structure of the chip is complex, which causes the reduction of the thermal response speed, the time required by thermal cycle is prolonged, and the time of one-time detection can reach 2 hours.
Disclosure of Invention
The invention aims to provide an integrated handheld digital nucleic acid detector and a nucleic acid detection method, so as to reduce the volume of the detector and solve the problems of high detection cost and long detection time in the existing detection.
In order to achieve the purpose, the invention provides the following scheme:
an integrated hand-held digital nucleic acid detector comprising: the integrated chip sample adding and packaging system, the heat circulation system and the detection and analysis system; the chip sample adding and packaging system comprises a microporous dPCR chip and a packaging cover plate for packaging the microporous dPCR chip, wherein the packaging cover plate comprises a cover glass, PDMS and Parylene C which are sequentially covered from top to bottom; the thermal cycling system is used for performing thermal cycling on the microporous dPCR chip; the detection and analysis system is used for shooting a fluorescence image of the microporous dPCR chip after thermal cycling is finished, and processing and analyzing the fluorescence image.
Optionally, the thermal cycle system comprises a semiconductor refrigerator and an embedded electronic device; the embedded electronic equipment controls the semiconductor refrigerator to heat and refrigerate.
Optionally, the heat cycle system further comprises: resistance-type temperature sensor, radiator and radiator fan; the resistance-type temperature sensor is used for measuring the real-time temperature of the semiconductor refrigerator; the radiator and the heat radiation fan are used for radiating heat under the control of the embedded electronic equipment.
Optionally, the detection analysis system comprises: the system comprises an optical module, a built-in camera of the mobile phone and image processing and result analysis application; the optical module comprises a filter set and a light-emitting diode, and the light-emitting diode is used for emitting light to irradiate the microporous dPCR chip to generate a fluorescent signal; the filter set is used for filtering the fluorescence signal; the built-in camera of the mobile phone is used for shooting a fluorescence image of the surface of the dPCR chip after thermal cycling is finished, and the image processing and result analysis application is used for processing and analyzing the fluorescence image of the surface of the dPCR chip.
Optionally, a plurality of cylindrical reaction cavities with the same size are arranged on the surface of the microporous dPCR chip; the plurality of reaction chambers form an array structure.
Optionally, the plurality of reaction chambers differ in diameter.
Optionally, the packaging process of the microporous dPCR chip specifically includes:
dripping oil drops on one surface of a cover glass on which Parylene C is deposited to form an oil film;
covering the PCR reaction solution on one surface of the digital dPCR chip, which contains the micropores, scraping the redundant PCR reaction solution by using a scraping blade, and putting the PCR reaction solution into a chip tray;
and (3) placing the side, provided with the oil film, of the cover glass on the digital dPCR chip to complete packaging.
The invention also provides a nucleic acid detection method, which comprises the following steps:
collecting a fluorescence image of the microporous dPCR chip by the integrated handheld digital nucleic acid detector;
generating an illumination non-uniformity template by using a microporous dPCR chip filled with fluorescein;
pre-processing the fluorescence image, the pre-processing comprising: graying the fluorescence image, performing rotation correction on the grayed fluorescence image, and performing positioning correction on the micropore position in the fluorescence image;
correcting the pre-processed fluorescence image based on the template;
extracting the average fluorescence signal intensity in each reaction cavity on the microporous dPCR chip based on the corrected fluorescence image;
based on the average fluorescence signal intensity in each reaction cavity, the number of positive holes is determined, and the copy number of the target gene loaded in the sample by the microporous dPCR chip is determined by utilizing Poisson distribution.
According to the specific embodiment provided by the invention, the invention discloses the following technical effects:
the invention provides an integrated handheld digital nucleic acid detector, which integrates a chip sample adding and packaging system, a heat circulation system and a detection and analysis system into one instrument after being miniaturized, simplifies the instrument structure and reduces the instrument cost; a microporous dPCR chip flexible packaging process based on PDMS and Parylene C multilayer structures is provided, and the problems of sample evaporation and cross contamination of a microporous digital dPCR chip caused by mineral oil liquid seal are solved; the chip packaging volume is reduced, the thermal response speed is improved, meanwhile, the thermal cycle scheme is optimized, the thermal cycle temperature change is reduced from three steps to two steps, the required thermal cycle times are reduced, and the detection rate is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 is a schematic diagram of a hand-held digital nucleic acid detector according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of the design structure of a dPCR chip according to an embodiment of the present invention;
FIG. 3 is an SEM image of a dPCR chip of each size; the diameter of graphs (a) and (b) is 5 μm; panel (c) and panel (d) are 20 μm in diameter; panel (e) and Panel (f) are 10 μm in diameter; panels (g) and (h) are 50 μm in diameter;
FIG. 4 is a schematic diagram of a structure of a dPCR packaging cover plate;
FIG. 5 is a schematic diagram of the components and structure of a fluorescence detection system;
FIG. 6 is a flow chart of fluorescence signal extraction.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention aims to provide an integrated handheld digital nucleic acid detector and a nucleic acid detection method, so as to reduce the volume of the detector and solve the problems of high detection cost and long detection time in the existing detection.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.
As shown in fig. 1, the present invention provides an integrated handheld digital nucleic acid detector, which comprises: a chip sample adding and packaging system, a rapid thermal circulation system and a miniaturized fluorescence detection and analysis system which are integrated into a whole; the chip sample adding and packaging system comprises a micro-porous dPCR chip and a packaging cover plate for packaging the micro-porous dPCR chip, wherein the packaging cover plate comprises a cover glass, PDMS and C-type Parylene (Parylene C) which are sequentially covered and arranged from top to bottom; the thermal cycling system is used for performing thermal cycling on the microporous dPCR chip; the detection and analysis system is used for shooting a fluorescence image of the microporous dPCR chip after thermal cycling is finished, and processing and analyzing the fluorescence image.
Specifically, the invention uses a silicon-based microporous dPCR chip, and the design size of the dPCR chip is (9 multiplied by 9) mm2. The reaction chamber array is divided into 6 regions on the chip, and the area of each region is about (4.2X 2.8) mm2And a cross mark is matched, so that the later-stage reaction cavity position positioning and the fluorescence signal statistics and analysis are facilitated. The schematic diagram of the design structure of the dPCR chip is shown in FIG. 2, wherein (a) is the whole chip structure, (b) is the single-region structure, and (c) is the arrangement form of the reaction chambers. The reaction chambers are cylindrical structures, theoretically, the reaction chambers are the same in size and equal in depth, are densely distributed on the surface of the silicon chip and are isolated from each other to form independent reaction spaces.
In order to meet the requirements of compatibility and different accuracies of the whole system in the future, dPCR chips with reaction cavities of 50 μm, 20 μm, 10 μm and 5 μm in diameter are designed on the basis of the same chip size, and the specific structural parameters are shown in Table 1:
TABLE 1 dPCR chip design parameters
Cross-sectional observation of dPCR chips of different parameters was performed using a Scanning Electron Microscope (SEM), the SEM images of the size dPCR chips being as shown in fig. 3, the diameter of fig. s (a) and (b) being 5 μm; panel (c) and panel (d) are 20 μm in diameter; panel (e) and (f) are 10 μm in diameter; panel (g) and (h) are 50 μm in diameter. The diameter of the reaction cavity of the current chip is different from 50-5 μm, and is limited by the volume of the reaction cavity, and the concentration of the corresponding detection DNA correspondingly increases along with the decrease of the diameter of the reaction cavity. Considering the detection requirement on trace genes, the diameter and the depth of the reaction cavity are increased, but the increase of the diameter of the reaction cavity reduces the number of the reaction cavities and reduces the detection flux, and the increase of the depth puts higher requirements on the process. The chip can be reused for many times, and before the chip is used, the chip needs to be put into H2SO4/H2O2(volume ratio 3:1) the mixture solution removes impurities and organic matters on the surface of the chip, prevents the premixed solution from being polluted, and inhibits PCR reaction. The microporous dPCR chip realizes the filling of samples by carrying out overall hydrophilic treatment on the surface of the chip and the interior of the reaction cavity. After the sample is filled, a layer of silicone oil is covered on the surface of the chip to play a role in liquid sealing. However, this method of integrated hydrophilic treatment of the chip may leave a part of liquid on the surface of the chip during the sample filling process, resulting in cross contamination. And the silicone oil sealing process can cause a large amount of bubbles to be generated in the thermal cycle process, inhibit PCR reaction, cause sample evaporation in a reaction cavity and influence later-stage fluorescent signal extraction. Therefore, a chip modification and packaging method with strong pertinence and convenient use is needed.
The flexible packaging process of the microporous dPCR chip based on the PDMS and Parylene C multilayer structure is used at present. According to the flexible packaging process, two layers of thin film structures are added on the surface of the cover glass, and the functions of buffering and sealing are respectively achieved. The PDMS layer plays a role in buffering, and is a high-molecular organic compound, generally called as organic silicon, is a transparent elastomer, has good biocompatibility, can enable the cover plate to be tightly attached to the chip, and avoids damage to the chip structure. The Parylene C layer plays a role in sealing, is used as a transparent protective high polymer material, has the characteristics of low internal stress, low permeability, good biocompatibility and the like, is an ideal material for the electric insulating layer, the chemical protective layer, the protective layer and the sealing layer, can effectively isolate water vapor to avoid sample evaporation, has good light transmission and is convenient for later-stage fluorescence signal extraction. FIG. 4 is a diagram of the structure of a dPCR chip package cover sheet, which is composed of a cover glass, PDMS and Parylene C.
The packaging process specifically comprises the following steps: firstly, dripping oil drops on one surface of a cover glass on which Parylene C is deposited to form an oil film; then covering the PCR reaction solution on one surface of the digital dPCR chip containing the micropores, scraping the redundant PCR reaction solution by using a scraping blade, and putting the chip tray; and finally, placing the surface of the cover glass with the oil film on a digital dPCR chip to finish packaging.
Wherein the thermal cycle system includes a semiconductor cooler (TEC) and an embedded electronic device; the embedded electronic equipment controls the semiconductor refrigerator to heat and refrigerate, and the highest heating/cooling rate is 49K/s and-34K/s. The temperature control of the temperature control in the PCR process comprises temperature, time and cycle number, and the temperature, the time and the cycle number are loaded to the embedded electronic equipment by the smart phone to control the semiconductor refrigerator to heat and refrigerate.
PCR is the replication of DNA by temperature change. Each temperature corresponds to a unique biological process during DNA replication. Thus, ensuring the accuracy and stability of the temperature in each biological process is critical to the successful completion of PCR. In order to meet the requirements of accurate control and rapid conversion of temperature in the PCR process, realize rapid heating and refrigeration and reduce the volume, a high-power TEC capable of meeting heating and refrigeration conditions is adopted. The heating and the refrigeration of the TEC are mainly based on the Peltier effect, the heating/the refrigeration of the surface of the TEC are controlled by the voltage polarities applied to two sides of the TEC, one side of the element heats and the other side refrigerates when current flows through the TEC, when the voltage polarities applied to two ends of the element are changed, the current flow direction is reversed, the heating side and the refrigeration side are interchanged, and therefore the embedded electronic equipment can control the power polarity of the TEC element to complete the heat circulation of the chip.
Specifically, the heat cycle system further includes: resistance-type temperature sensor, radiator and radiator fan; the resistance-type temperature sensor is used for measuring the real-time temperature of the semiconductor refrigerator; the resistance type temperature sensor is connected to a closed loop feedback loop system, and the temperature of the system is adjusted through negative feedback under the control of the embedded electronic equipment. The radiator and the cooling fan are used for cooling under the control of the control chip.
In addition to heating the refrigeration device, there is also a need for immediate monitoring of the temperature during heating. The resistance of the resistance temperature sensor Pt100 changes along with the change of temperature, and is approximately in a linear relation, and the output voltage real-time monitoring system temperature under different temperatures can be obtained through a measuring circuit. In the thermal cycle process, PT100 measures the real-time temperature of the TEC, and because the thermal conductivity of the silicon-based chip is high, the volume of liquid in the reaction cavity is small, the thermal response time can be ignored, compared with the existing commercial dPCR detection system, the heating time is as long as 1-2 hours, and the rapid thermal cycle system can complete the thermal cycle process only in 20-30 minutes according to different cycle numbers.
Specifically, as shown in fig. 5, the detection analysis system includes: the system comprises an optical module, a built-in camera of the mobile phone and image processing and result analysis application. The optical module comprises a filter set and a light-emitting diode, and the light-emitting diode is used for emitting light to irradiate the microporous dPCR chip to generate a fluorescent signal; the filter set is used for filtering the fluorescence signals, and ensures that images shot by the lens are formed by exciting fluorescence on the surface of the chip and do not contain other stray light. The filter set comprises an excitation filter, an emission filter and a dichroic mirror; the excitation filter is a filter through which only light with a wavelength capable of exciting fluorescence can pass, so that stray light with a wavelength not the wavelength of fluorescence excitation light can be prevented from entering the optical module; the dichroic mirror is arranged in the light path of the detection system at an angle of 45 degrees, can reflect the exciting light and vertically irradiate the exciting light to the surface of the chip, and can also transmit the fluorescent light excited by the exciting light; the emission filter is a filter through which only light of the wavelength of the excited fluorescence passes.
The built-in camera of the mobile phone is used for shooting a fluorescence image of the surface of the dPCR chip after thermal cycling is finished, and the image processing and result analysis application is used for processing and analyzing the fluorescence image of the surface of the dPCR chip.
Such as a fluorescein isothiocyanate isocyanate (FITC) filter set for detecting FAM probes and a Light Emitting Diode (LED) having a dominant wavelength of about 470nm, as a fluorescence collection optical path and a fluorescence excitation light source, respectively. The light-emitting diode light source emits blue light, the blue light is reflected by a color separation mirror and then vertically shines on the surface of the silicon substrate microporous dPCR chip, the dye combined with nucleic acid is excited to emit green fluorescence, and then other mixed light signals except the green fluorescence are filtered by the optical filter. And (3) receiving the fluorescence signal of the surface of the dPCR chip by a camera to obtain a surface fluorescence image. The detection light path can effectively avoid stray light interference, obtain clear and accurate dPCR chip surface fluorescence images and facilitate subsequent algorithm processing.
Based on the integrated handheld digital nucleic acid detector, the invention also provides a nucleic acid detection method, which specifically comprises the following steps:
step 1, collecting a fluorescence image of the microporous dPCR chip by an integrated handheld digital nucleic acid detector.
And 2, generating an illumination non-uniformity template by using the microporous dPCR chip filled with fluorescein.
And 3, preprocessing the fluorescence image, wherein the preprocessing comprises the following steps: graying the fluorescence image, performing rotation correction on the grayed fluorescence image, and performing positioning correction on the micropore position in the fluorescence image.
And 4, correcting the preprocessed fluorescence image based on the template.
And 5, extracting the average fluorescence signal intensity in each reaction cavity on the microporous dPCR chip based on the corrected fluorescence image.
And 6, determining the number of positive holes based on the average fluorescence signal intensity in each reaction cavity, and determining the copy number of the target gene loaded in the sample by using the Poisson distribution.
After the thermal cycle is finished, the fluorescence intensity in each reaction cavity needs to be extracted, and then the target gene is subjected to absolute quantitative analysis by counting and analyzing the number of positive reaction cavities. The position of the reaction cavity of the cdPCR is fixed, so that after the reaction is finished, a camera can be used for shooting a fluorescence image of the chip, then the image is subjected to rotation correction, reaction cavity positioning and other processing, and finally the fluorescence signal intensity in each reaction cavity is extracted according to the position of the reaction cavity in the image, so that the positioning of the high-density and high-flux reaction cavities is realized, and the fluorescence signal extraction flow is shown in FIG. 6.
After the fluorescence photograph of the whole chip is taken at one time, the image is firstly preprocessed, wherein the preprocessing comprises the following steps: the color image is converted into a gray image for post processing; carrying out pre-recognition on the position of a reaction cavity in the image through Hough circle transformation; calculating an image deflection angle by fast Fourier transform; 3D projective transformation to locate and correct the position of all reaction chambers; an illumination non-uniformity template is generated using a chip filled with fluorescein. And finally, correcting the fluorescence pictures acquired after the thermal cycle reaction based on the template, and extracting the average fluorescence signal intensity in each reaction cavity. The algorithm realizes accurate positioning of the position of the reaction cavity of the chip, eliminates the nonuniformity of fluorescence intensity and provides reliable guarantee for subsequent fluorescence signal extraction.
For the extraction of the fluorescence signal, according to the designed dPCR chip structure, an MATLAB program is utilized to construct a virtual chip reaction cavity matrix, the virtual chip structure matrix is overlapped with an actual image, the fluorescence intensity value of the chip position corresponding to the inside of the virtual matrix reaction cavity is extracted, the sum of the gray values of all pixel points inside the reaction cavity is calculated, and the gray value of the pixel point with the maximum gray value inside the reaction cavity is divided by the gray value to obtain the average gray value of the reaction cavity. And finally, establishing a functional relation graph of the fluorescence intensity and the number of the reaction cavities, thereby determining the number of the positive reaction cavities. If the fluorescence intensity of individual reaction chambers deviates significantly from the average intensity during the signal extraction process, it is considered as a dead spot when processing the data and is not processed. Finally, the reaction result of the dPCR can be obtained through the extracted fluorescence intensity distribution relation.
As shown in fig. 1, all the modules, the bottom plate and the housing designed according to the sizes of the modules are combined together to form an integrated handheld digital nucleic acid detector, the optical module and the heat circulation system are fixed on the bottom plate, and then the housing is installed to protect the internal structure.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The principles and embodiments of the present invention have been described herein using specific examples, which are provided only to help understand the method and the core concept of the present invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, the specific embodiments and the application range may be changed. In view of the above, the present disclosure should not be construed as limiting the invention.
Claims (8)
1. An integrated hand-held digital nucleic acid detector, comprising: the integrated chip sample adding and packaging system, the heat circulation system and the detection and analysis system; the chip sample adding and packaging system comprises a microporous dPCR chip and a packaging cover plate for packaging the microporous dPCR chip, wherein the packaging cover plate comprises a cover glass, PDMS and Parylene C which are sequentially covered from top to bottom; the thermal cycling system is used for performing thermal cycling on the microporous dPCR chip; the detection and analysis system is used for shooting a fluorescence image of the microporous dPCR chip after thermal cycling is finished, and processing and analyzing the fluorescence image.
2. The integrated handheld digital nucleic acid detector of claim 1, wherein the thermal cycle system includes a semiconductor cooler and embedded electronics; the embedded electronic equipment controls the semiconductor refrigerator to heat and refrigerate.
3. The integrated handheld digital nucleic acid instrumentation of claim 2, wherein the thermal cycle system further comprises: resistance-type temperature sensor, radiator and radiator fan; the resistance-type temperature sensor is used for measuring the real-time temperature of the semiconductor refrigerator; the radiator and the heat radiation fan are used for radiating heat under the control of the embedded electronic equipment.
4. The integrated handheld digital nucleic acid testing instrument of claim 1, wherein said test analysis system comprises: the system comprises an optical module, a built-in camera of the mobile phone and image processing and result analysis application; the optical module comprises a filter set and a light-emitting diode, and the light-emitting diode is used for emitting light to irradiate the microporous dPCR chip to generate a fluorescent signal; the filter set is used for filtering the fluorescence signal; the built-in camera of the mobile phone is used for shooting a fluorescence image of the surface of the dPCR chip after thermal cycling is finished, and the image processing and result analysis application is used for processing and analyzing the fluorescence image of the surface of the dPCR chip.
5. The integrated handheld digital nucleic acid detector of claim 1, wherein a plurality of cylindrical reaction chambers with the same size are arranged on the surface of the microporous dPCR chip; the plurality of reaction chambers form an array structure.
6. The integrated handheld digital nucleic acid testing instrument of claim 5 wherein the plurality of reaction chambers have different diameters.
7. The integrated handheld digital nucleic acid detector of claim 1, wherein the packaging process of the microporous dPCR chip specifically comprises:
dripping oil drops on one surface of a cover glass on which Parylene C is deposited to form an oil film;
covering the PCR reaction solution on one surface of the digital dPCR chip, which contains the micropores, scraping the redundant PCR reaction solution by using a scraping blade, and putting the PCR reaction solution into a chip tray;
and (3) placing the side, provided with the oil film, of the cover glass on the digital dPCR chip to complete packaging.
8. A method for detecting a nucleic acid, comprising:
collecting a fluorescence image of the microporous dPCR chip by the integrated handheld digital nucleic acid detector of any one of claims 1 to 7;
generating an illumination non-uniformity template by using a microporous dPCR chip filled with fluorescein;
pre-processing the fluorescence image, the pre-processing comprising: graying the fluorescence image, performing rotation correction on the grayed fluorescence image, and performing positioning correction on the micropore position in the fluorescence image;
correcting the pre-processed fluorescence image based on the template;
extracting the average fluorescence signal intensity in each reaction cavity on the microporous dPCR chip based on the corrected fluorescence image;
and determining the number of positive holes based on the average fluorescence signal intensity in each reaction cavity, and determining the copy number of the target gene loaded in the sample by using the Poisson distribution.
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