Gene detection equipment and time-sharing release reagent

Abstract

The embodiment of the invention provides gene detection equipment and a time-sharing release reagent. Belongs to the technical field of gene detection. The equipment comprises a pretreatment unit, a core reaction unit and a variation region detection unit, the pretreatment unit is used for pretreating an experimental sample to obtain a gene segment; the core reaction unit adopts a time-sharing release reagent to build a library for the gene segments to obtain a sequencing library of the experimental sample; a primary probe is adopted to be combined with the high-conservative region in the sequencing library, a to-be-recognized variation region combined with the primary probe is obtained, and the primary probe is of a Y-shaped branch structure; combining a secondary probe with the to-be-recognized variation region to obtain a high variation region combined with the secondary probe; and the variation region detection unit is used for sequencing the high variation region. According to the gene detection equipment and the time-sharing released reagent, the problem that the current gene detection process is tedious in operation is solved.

Description

Technical Field

[0001] The present application relates to the field of gene detection technology, and in particular to a gene detection device and a time-released reagent. Background Art

[0002] In the process of genetic testing, enriching the target deoxyribonucleic acid (DNA) and obtaining the gene fragments in the target DNA are key steps for subsequent analysis. Hybridization capture technology, as an important means to achieve this goal, can enrich the required fragments from the target DNA by binding biotin-labeled probes to gene fragments, providing strong support for gene variation detection and disease-related gene research.

[0003] Currently, in the process of gene detection in related technologies, multiple reagents need to be used for multiple treatments to construct a gene library, and then probes are used to hybridize and capture gene fragments in the gene library.

[0004] However, the inventors have discovered that the related technology has at least the following technical problems: the large number of reagents used in the current genetic testing process makes the operation cumbersome. Summary of the Invention

[0005] The embodiments of the present application provide a genetic testing device and a time-released reagent to solve the problem of cumbersome operation in the current genetic testing process.

[0006] In the first aspect, an embodiment of the present application provides a genetic testing device, comprising: a pretreatment unit, a core reaction unit and a variation region detection unit; the pretreatment unit pretreatments the experimental sample to obtain gene fragments; the core reaction unit uses time-released reagents to build a library for the gene fragments to obtain a sequencing library of the experimental sample; a primary probe is used to bind to a highly conserved region in the sequencing library to obtain a variation region to be identified that is bound by the primary probe, wherein the primary probe is a probe with a Y-shaped branch structure; a secondary probe is used to bind to the variation region to be identified to obtain a high variation region bound to the secondary probe; the variation region detection unit is used to sequence the high variation region.

[0007] In one possible implementation, the core reaction unit mixes the gene fragment with the time-released reagent to obtain a mixed reagent; the mixed reagent is heated at a first temperature for a first preset time to release the polymerase component of the mixed reagent, and the polymerase component repairs the DNA end of the gene fragment; the mixed reagent is heated at a second temperature for a second preset time to release the Klenow fragment component of the mixed reagent, and the Klenow fragment component adds a dA tail to the DNA of the repaired gene fragment, wherein the second temperature is greater than the first temperature; the mixed reagent is heated at a third temperature for a third preset time to release the ligase component of the mixed reagent, and the ligase component combines the gene fragment with the added dA tail with a linear DNA vector with a dT protrusion to obtain a sequencing library of the experimental sample.

[0008] In one possible implementation, the pretreatment unit includes a micro peristaltic pump; the micro peristaltic pump adds lysis solution to the experimental sample, mixes the lysis solution with the experimental sample, and releases the DNA in the experimental sample; the pretreatment unit breaks the DNA in the experimental sample to obtain gene fragments.

[0009] In one possible implementation, the preprocessing unit includes: a sample inlet micro-pressure sensor; the sample inlet micro-pressure sensor is installed at the inlet of the experimental sample and is used to detect the pressure of the experimental sample; the preprocessing unit controls the operating time of the micro peristaltic pump according to the pressure of the experimental sample.

[0010] In a possible implementation, the pre-processing unit further includes: a gene disruption unit; the gene disruption unit disrupts the DNA in the experimental sample to obtain gene fragments.

[0011] In one possible implementation, the core reaction unit also includes: a neodymium iron boron magnet array composed of multiple neodymium iron boron magnets and a position control unit transmission-connected to the neodymium iron boron magnets; the position control unit is used to adjust the position and arrangement state of the neodymium iron boron magnets to capture magnetic complexes containing high-variability regions through an external magnetic field.

[0012] In one possible implementation, the variation detection unit includes: a silicon-based purification column; the silicon-based purification column is used to purify the high variation region.

[0013] In a possible implementation, it also includes: a processing unit and a display unit; the core reaction unit also includes a temperature sensor; the processing unit is used to receive the temperature value sent by the temperature sensor, use the temperature value to draw a temperature curve, and send it to the display unit; the display unit is used to display the temperature curve.

[0014] In the second aspect, an embodiment of the present application provides a time-release reagent, which is used in the genetic testing equipment described in the first aspect, including: a polymerase component physically embedded in a first embedding carrier, a Klenow fragment component physically embedded in a second embedding carrier, and a ligase component physically embedded in a third embedding carrier, wherein the thermal disintegration temperatures of the first embedding carrier, the second embedding carrier and the third embedding carrier increase sequentially.

[0015] In one possible implementation, the polymerase component includes T4 DNA polymerase, deoxyribonucleoside triphosphate, magnesium chloride and a first buffer; the Klenow fragment component includes Klenow fragment, deoxyadenosine triphosphate, magnesium chloride and a first buffer; and the ligase component includes Taq ligase, adenosine triphosphate, sodium chloride and a second buffer.

[0016] The genetic testing device and time-released reagents provided in the embodiments of the present application use time-released reagents to build a library for gene fragments, completing the library building operation in one go, avoiding the problems of cumbersome operations and a high possibility of errors caused by multiple uses of reagents. Two-stage probes are used to respectively perform broad-spectrum enrichment and precise enrichment of highly variable regions, reducing impurities in the hybridization capture results. In addition, a Y-shaped primary probe is used for broad-spectrum enrichment, facilitating the extraction of highly variable regions using multiple methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0018] Figure 1 Schematic diagram of the structure of the genetic testing device provided in this application Figure 1 ;

[0019] Figure 2 A schematic diagram of the structure of the pre-processing unit provided in this application;

[0020] Figure 3 Schematic diagram of the structure of the genetic testing device provided in this application Figure 2 ;

[0021] Figure 4 A schematic diagram of the first-level probe design process provided in an embodiment of the present application.

[0022] Reference numerals

[0023] 100-Gene testing equipment;

[0024] 101-preprocessing unit;

[0025] 102-core reaction unit;

[0026] 103-variation region detection unit;

[0027] 104-processing unit;

[0028] 105-display unit;

[0029] 1011-Micro peristaltic pump;

[0030] 1012-injection micro pressure sensor;

[0031] 1013-Gene disruption unit.

[0032] The above drawings illustrate specific embodiments of the present application, which will be described in more detail below. These drawings and the textual description are not intended to limit the scope of the present application in any way, but rather to illustrate the concepts of the present application to those skilled in the art by reference to specific embodiments. DETAILED DESCRIPTION

[0033] Exemplary embodiments will be described in detail herein, with examples illustrated in the accompanying drawings. In the following description, when referring to the drawings, identical numerals in different figures represent identical or similar elements, unless otherwise indicated. The embodiments described in the following exemplary embodiments are not intended to represent all embodiments consistent with the present application. Rather, they are merely examples of apparatus and methods consistent with certain aspects of the present application, as detailed in the appended claims.

[0034] In the genetic testing process, enriching target DNA and accurately obtaining specific gene fragments are key technical nodes for subsequent analysis. As a key technical means to achieve this goal, hybridization capture technology, through the specific binding of biotin-labeled probes to target sequences, can achieve efficient enrichment of target fragments, providing important technical support for application scenarios such as gene variation analysis and disease-related gene function research.

[0035] The current mainstream technical solutions have the following technical bottlenecks: During the gene library construction stage, a multi-step reagent processing process is required to complete the library construction, and then probes are used for hybridization capture. This technical approach has two problems. First, the complex multi-reagent processing process significantly increases the difficulty of operation and increases the probability of experimental errors. Second, non-specific binding is prone to occur during the probe hybridization process, resulting in a large number of non-target sequences mixed in the final capture product, affecting the accuracy of the detection results. In actual applications, the one-step method or continuous reaction, especially when dealing with low-abundance target fragments, high GC content (the ratio of guanine and cytosine bases in DNA molecules) regions or mutation hotspots, often faces problems such as significantly reduced library construction efficiency, poor coverage uniformity and increased non-specific background.

[0036] This application is used in the context of genetic testing. It should be noted that the user information (including but not limited to user device information, user personal information, etc.) and data (including but not limited to data used for analysis, storage, and display, etc.) involved in this application are all information and data authorized by the user or fully authorized by all parties. The collection, use, and processing of relevant data must comply with relevant laws, regulations, and standards, and corresponding operation portals must be provided for users to choose to authorize or refuse.

[0037] The following specific embodiments describe in detail the technical solution of the present application and how the technical solution of the present application solves the above-mentioned technical problems. The following specific embodiments can be combined with each other, and the same or similar concepts or processes may not be repeated in some embodiments. The embodiments of the present application will be described below in conjunction with the accompanying drawings.

[0038] Figure 1 Schematic diagram of the structure of the genetic testing device provided in this application Figure 1 .like Figure 1 The gene detection device 100 includes: a pre-processing unit 101, a core reaction unit 102 and a variation region detection unit 103.

[0039] The preprocessing unit 101 preprocesses the experimental sample to obtain gene fragments.

[0040] The pretreatment unit may include a sample introduction unit and a sample processing unit. The sample introduction unit may include a sample introduction line and a pump connected to the sample introduction line. The pump pumps the sample and reagents for processing the sample into the sample processing unit to mix the sample and reagents. The sample processing unit may include a stirrer and a DNA fragmentation unit. The stirrer may be composed of a motor and a stirring rod connected to the motor, which is used to stir the sample and reagents, disrupting cell walls and obtaining intracellular DNA. The DNA fragmentation unit may include an ultrasonic generator to generate ultrasonic waves, which are used to fragment DNA to obtain gene fragments. The DNA fragmentation unit may also be composed of a centrifuge tube, a pipette, a mixer, and a heating unit (water bath or thermostat). The centrifuge tube is used to load DNA, enzymes, buffer, and other components. The pipette is used to add restriction endonucleases to the DNA solution. The mixer mixes the DNA and restriction endonucleases. The heating unit is used to maintain the mixture of DNA and restriction endonucleases at a preset temperature to maintain the activity of the restriction endonucleases, allowing the restriction endonucleases to cleave the DNA to obtain gene fragments.

[0041] Core reaction unit 102 uses time-released reagents to build a library of gene fragments, generating a sequencing library for the experimental sample. A primary probe, with a Y-shaped branched structure, binds to the highly conserved regions in the sequencing library to obtain the variant regions to be identified. A secondary probe binds to the variant regions to be identified to obtain the highly variable regions bound by the secondary probe.

[0042] The core reaction unit 102 can be composed of a pump, a solenoid valve, a heating unit, and a capture unit. The heating unit can include a water bath heating module, or it can be composed of a temperature-controlled chamber and a semiconductor temperature-controlled unit installed within the temperature-controlled chamber. The capture unit can be composed of a permanent magnet and a motor, or it can be composed of an electromagnetic coil. The solenoid valve is connected to the pump via a pipeline. The solenoid valve switches the pipeline to the pipeline corresponding to the time-release reagent. The pump adds the time-released reagent to the gene fragment. The heating unit heats the mixture of the time-released reagent and the gene fragment, causing the time-released reagent to be gradually released and react with the gene fragment to obtain a sequencing library. The solenoid valve switches the pipeline to the pipeline corresponding to the primary probe. The pump pumps the primary probe into the sequencing library. The heating unit heats the sequencing library, converting the gene fragments in the sequencing library from double-stranded to single-stranded. The temperature is then lowered to near the melting temperature of the primary probe and the gene fragments in the sequencing library, allowing the primary probe to specifically bind to the gene fragments in the sequencing library, obtaining the variant region to be identified that is bound by the primary probe. Before the secondary probe binds, a solenoid valve switches to the magnetic bead line. A pump then pumps streptavidin-coated magnetic beads into the probe-added reagent. The streptavidin-coated beads bind to the primary probe, and a buffer (also pumped in) is used to remove unbound fragments. The variant region to be identified is then released by high temperature or competitive elution, releasing the variant region to be identified. The secondary probe then binds to the variant region to be identified. The binding of the secondary probe to the variant region to be identified is similar to the binding of the primary probe to the highly conserved region and will not be further described here. The primary probe can be designed based on the target. For example, for detecting oncogenic genes or screening for genetic diseases of the target type, different primary probes can be designed for different targets. Conserved regions are candidate binding sites for "universal probes." Their high conservation means that the sequence is repeated throughout the gene family, facilitating broad identification across subtypes and variants.

[0043] The variable region detection unit 103 is used to sequence the highly variable regions.

[0044] Among them, the variable region detection unit can be composed of a fluorescent signal detection device, which can obtain the gene sequence of the highly variable region by stimulating the probe fluorescent signal and analyzing the base type according to the fluorescent signal.

[0045] From the description of the above embodiments, it can be seen that the gene detection equipment in the embodiments of the present disclosure can build a library for gene fragments through time-released reagents, and complete the library construction operation in one go, avoiding the problems of cumbersome operations and high possibility of errors caused by multiple use of reagents, and using two-stage probes to respectively perform broad-spectrum enrichment and precise enrichment of high-variability regions, thereby reducing impurities in the hybridization capture results. In addition, a Y-shaped primary probe is used for broad-spectrum enrichment, which facilitates the extraction of high-variability regions in various ways. Moreover, due to the use of time-released reagents, the embedded enzyme components are released in stages at different temperature stages, so that each enzyme can function independently within its own optimal temperature and time window, fundamentally avoiding the efficiency loss and side reactions caused by incompatible conditions between enzymes, and avoiding the mutual interference or inhibition between enzymes caused by the one-time addition of multiple enzymes, thereby improving the efficiency of the library construction process and ensuring the accuracy and reliability of sequencing data.

[0046] In actual experiments, using the same low-abundance target (1 copy / μL), the single-probe detection rate was 35%. However, using the dual-probe synergistic approach described in steps S203 and S204, this rate increased to 92%, a statistically significant difference (p < 0.001). Coverage uniformity was significantly improved: the coefficient of variation for coverage of variable regions decreased from 25% to 8%, reflecting that the complementary synergistic effect between the probes significantly reduced "hotspot capture bias." Nonspecific capture was also reduced: background interference decreased from 12% to 2%, significantly improving subsequent sequencing efficiency and accuracy.

[0047] In a possible implementation, the core reaction unit 102 mixes the gene fragments with the time-released reagents to obtain a mixed reagent.

[0048] By controlling the opening / closing of the solenoid valve, the pump can be switched to the pipeline corresponding to the time-released reagent, and the time-released reagent can be pumped into the solution containing the gene fragment to obtain a mixed reagent.

[0049] The mixed reagent is heated at a first temperature for a first preset time to release a polymerase component from the mixed reagent, and the polymerase component repairs the DNA broken end of the gene fragment.

[0050] In this step, the mixed reagents can be heated using a water bath heating module or other heating device. The first temperature can be greater than or equal to the thermal disintegration temperature of the first embedding carrier, and the first temperature can be less than the thermal disintegration temperature of the second embedding carrier. The first preset time period can be, for example, 9 minutes, 10 minutes, 12 minutes, etc.

[0051] The mixed reagent is heated at a second temperature for a second preset time to release the Klenow fragment component of the mixed reagent, and the Klenow fragment component adds a dA tail to the DNA of the repaired gene fragment, wherein the second temperature is greater than the first temperature.

[0052] The second temperature may be greater than or equal to the thermal disintegration temperature of the second embedding carrier, and the second temperature may be less than the thermal disintegration temperature of the third embedding carrier. The second preset time may be, for example, 14 minutes, 15 minutes, or 16 minutes.

[0053] The mixed reagent is heated at a third temperature for a third preset time to release a ligase component, which combines the gene fragment with the added dA tail with the linear DNA vector with the dT protrusion to obtain a sequencing library of the experimental sample.

[0054] The third temperature can be greater than or equal to the thermal disintegration temperature of the third embedding vector and greater than the thermal disintegration temperature of the second embedding vector. The third preset duration can be, for example, 4 minutes, 5 minutes, or 6 minutes. Linear DNA vectors can include T-vectors (T-vectors have a dT tail at their 3' end, which temporarily binds to the target gene fragment through dA-dT complementary pairing, followed by ligase-catalyzed phosphodiester bond formation to form a recombinant DNA molecule).

[0055] As can be seen from the description of the above embodiments, the embodiments of the present disclosure heat the mixed reagent at different temperatures and for different durations after mixing the gene fragment with the time-release reagent, so that the chemical substances in the time-release reagent are released in stages, thereby automatically completing the end repair, addition of dA tails, and end ligation of the gene. Only one reagent addition is required, which reduces the number of operation steps and reduces the errors caused by too many operation steps.

[0056] In one possible implementation, the primary probe with a Y-shaped branched structure includes a biotin-modified backbone chain, a fluorophore-modified first side chain, and an unmodified second side chain.

[0057] The biotin-modified backbone is used to immobilize the probe, the fluorophore-modified first side chain is used as a detection signal, and the unmodified second side chain is used to stabilize the structure. A linker-complementary sequence can be used for the unmodified second side chain.

[0058] Figure 2 This is a schematic diagram of the structure of the pre-processing unit provided in this application. Figure 2 As shown, the pre-treatment unit 101 includes a micro peristaltic pump 1011. The micro peristaltic pump adds lysis solution to the experimental sample, mixing the lysis solution with the experimental sample and releasing the DNA in the experimental sample. The pre-treatment unit breaks the DNA in the experimental sample to obtain gene fragments.

[0059] The microperistaltic pump is connected to the experimental sample and the lysate container. After operation, the lysate in the lysate container can be pumped out and delivered to the experimental sample through a pipeline. The pretreatment unit can switch the pipeline connected to the microperistaltic pump so that the microperistaltic pump can pump the restriction endonuclease from the pipeline corresponding to the restriction endonuclease, and the restriction endonuclease will be used to interrupt the DNA in the experimental sample. The pretreatment unit can also include an ultrasonic generator to interrupt the DNA through ultrasound.

[0060] From the description of the above embodiments, it can be seen that the embodiments of the present disclosure can achieve precise control of the flow rate of reagents by using a micro peristaltic pump in the pretreatment unit, and when the micro peristaltic pump transports the lysis solution, the lysis solution pumped out of the lysis solution can reach a sufficient speed to mix the lysis solution with the sample, reducing the components required for mixing the reagents and samples, thereby reducing the manufacturing cost of the equipment.

[0061] Continue to refer Figure 2 In a possible implementation, the pre-processing unit includes: a sample injection micro-pressure sensor 1012 .

[0062] The sample inlet micro pressure sensor is installed at the inlet of the experimental sample to detect the pressure of the experimental sample in real time.

[0063] When the experimental sample contains bubbles, the pressure detected by the sample inlet micro-pressure sensor decreases.

[0064] The pretreatment unit controls the running time of the micro peristaltic pump according to the pressure of the experimental sample.

[0065] The pre-processing unit inputs the pressure of the experimental sample into the Proportional-Integral-Derivative (PID) algorithm to obtain the running time compensation value of the micro peristaltic pump, and then adds the running time compensation value to the original remaining running time to obtain the new remaining running time. The micro peristaltic pump is controlled to continue running for the new remaining running time.

[0066] From the description of the above embodiments, it can be seen that the embodiments of the present disclosure realize the detection of bubbles in the experimental sample by adding a micro pressure sensor, thereby more accurately controlling the operating time of the micro peristaltic pump and making the usage of the experimental sample more accurate.

[0067] Continue to refer Figure 2 The pre-processing unit 101 further includes a gene disruption unit 1013 .

[0068] The gene disruption unit can be an ultrasonic generator or a restriction enzyme release unit. The restriction enzyme release unit can be implemented by deploying an additional pump and a restriction enzyme storage unit connected to the pump, or by using the restriction enzyme storage unit in combination with the above-mentioned micro peristaltic pump.

[0069] The gene disruption unit disrupts the DNA in the experimental sample to obtain gene fragments.

[0070] Among them, the gene disruption unit disrupts the DNA in the experimental sample by ultrasound or adding restriction endonucleases to the experimental sample.

[0071] As can be seen from the description of the above embodiments, the embodiments of the present disclosure fragment the gene by providing a gene shearing unit, thereby facilitating the subsequent establishment of a sequencing library.

[0072] In a possible implementation, the core reaction unit further includes: a neodymium iron boron magnet array composed of a plurality of neodymium iron boron magnets and a position control unit transmission-connected to the neodymium iron boron magnets.

[0073] The position control unit is used to adjust the position and arrangement state of the NdFeB magnet and capture the magnetic complex containing the high variation area through the external magnetic field.

[0074] The position control unit may include a motor, a drive shaft, and other components that control the position of the NdFeB magnet near the bottom or side of the reaction vessel, thereby adsorbing and binding the probe's highly variable region. The magnetic complex is a complex formed by the binding of the probe and the highly variable region. Because the probe carries magnetic beads and specifically binds to the highly variable region, the NdFeB magnet can effectively magnetically capture it. The position control unit can change the position and arrangement of the NdFeB magnet after the secondary probe binds to the highly variable region, thereby adsorbing the magnetic complex in the reaction vessel.

[0075] From the description of the above embodiments, it can be seen that the disclosed embodiments achieve the capture of high variation areas using a strong magnetic field by setting a neodymium iron boron magnet array composed of neodymium iron boron magnets in the core reaction unit, thereby ensuring the capture success rate of the high variation areas.

[0076] In one possible implementation, the variation detection unit includes: a silica-based purification column.

[0077] The silica-based purification column can be a silica-based purification column, which can be composed of a column body and a DNA-binding membrane, which can be a silica-based purification membrane. A single silica-based purification column can specifically adsorb a maximum of 5 μg of DNA.

[0078] Silica-based purification columns are used to purify highly variable regions. A solution containing the highly variable regions is passed through the DNA-binding membrane in the silica-based purification column by gravity or centrifugal force, causing the highly variable regions to be adsorbed by the DNA-binding membrane.

[0079] From the description of the above embodiments, it can be seen that the embodiments of the present disclosure achieve rapid purification of high-variability regions by adding a silicon-based purification column to the variation detection unit.

[0080] Figure 3 Schematic diagram of the structure of the genetic testing device provided in this application Figure 2 .like Figure 3 As shown, the gene detection device 100 further includes a processing unit 104 and a display unit 105 .

[0081] The core reaction unit 102 also includes a temperature sensor.

[0082] The processing unit is used to receive the temperature value sent by the temperature sensor, draw a temperature curve using the temperature value, and send it to the display unit.

[0083] The processing unit draws a temperature curve showing the change of temperature over time according to the received temperature value and the corresponding receiving time.

[0084] The display unit is used to display the temperature curve.

[0085] The processing unit may include a CPU (central processing unit), a programmable logic device (PLD), a control board, an ECU (electronic control unit), etc. The display unit may include a TFT (thin film transistor) central control screen, or other screens such as an LCD (liquid crystal display) or an AMOLED (active matrix / organic light emitting diode) screen, capable of simultaneously displaying and receiving touch signals. The temperature sensor may be a contact or non-contact temperature sensor, specifically a thermocouple temperature sensor or a thermistor temperature sensor.

[0086] It can be seen from the description of the above embodiments that the embodiments of the present disclosure, by adding a processing unit and a display unit, can enable the staff to understand the temperature in the core reaction unit in real time.

[0087] In a possible implementation, the pumps, heating devices, etc. of each unit in the genetic testing device of any of the above embodiments may be provided separately or shared.

[0088] The present application also provides a time-release reagent, which is applied to the gene detection device in any of the above embodiments, including:

[0089] The polymerase component is physically embedded in the first embedding carrier, the Klenow fragment component is physically embedded in the second embedding carrier, and the ligase component is physically embedded in the third embedding carrier, wherein the thermal disintegration temperatures of the first embedding carrier, the second embedding carrier and the third embedding carrier increase in sequence.

[0090] The first embedding carrier can be poly(lactic-co-glycolic acid) (PLGA) mixed with a protease and emulsified into W / O polymer microspheres, followed by freeze-drying. The second embedding carrier can be PLGA combined with various emulsifiers. The third embedding carrier can be high-melting-point PLGA or a thermosensitive nanocoating.

[0091] Specifically, the ratio of polylactic acid to glycolic acid before emulsification of the polylactic acid-glycolic acid copolymer can be 50:50, with a molecular weight of approximately 20 kDa. The main component of the emulsifier of the second embedding carrier is polyvinyl alcohol. The third embedding carrier can also be a W / O type polymer microsphere emulsified by mixing the polylactic acid-glycolic acid copolymer with a protease. The difference is that the ratio of polylactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer before emulsification of the third embedding carrier can be 75:25.

[0092] As can be seen from the description of the above embodiments, the embodiments of the present disclosure embed the components in the reagent by setting three embedding carriers with different thermal disintegration temperatures, so that the reagent can be released at different times according to the change in temperature. The construction of the sequencing library can be completed by adding the reagent once, thereby reducing the number of operational steps required for genetic testing.

[0093] In a possible implementation, the first embedding carrier can be a triblock copolymer composed of polyethylene glycol-polypropylene glycol-polyethylene glycol; the second embedding carrier can be gelatin; and the third embedding carrier can be composed of modified N-isopropylacrylamide.

[0094] In one possible implementation, the polymerase component includes T4 DNA polymerase, deoxyribonucleoside triphosphate, magnesium chloride and a first buffer; the Klenow fragment component includes Klenow fragment, deoxyadenosine triphosphate, magnesium chloride and a first buffer; and the ligase component includes Taq ligase, adenosine triphosphate, sodium chloride and a second buffer.

[0095] The first buffer includes 10 mM Tris-HCl buffer, 10 mM magnesium ion, 50 mM sodium chloride, and 1 mM dithiothreitol (DTT). The second buffer includes 20 mM Tris-HCl buffer, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, and 0.1% surfactant.

[0096] As can be seen from the description of the above embodiments, the embodiments of the present disclosure provide polymerase, Klenow fragment and Taq ligase, and embed corresponding auxiliary components and buffer in the embedding carrier, so that the components in the embedding carrier can function normally.

[0097] In actual experiments, using the time-release reagents in step S202 above, the resulting library concentration reached 9.8 ± 0.9 ng / μL, the adapter ligation efficiency reached 88%, the library fragment distribution was 5.2%, and the library construction time was 1.5 hours. The total enzyme cost was 40% of that of the traditional three-step method (end repair, dA tailing, and adapter ligation). The traditional three-step method achieved a library concentration of 8.6 ± 1.2 ng / μL, an adapter ligation efficiency of 72%, a library fragment distribution of 18.4%, and a library construction time of 4 hours. Therefore, library construction using time-release reagents can be superior to library construction using the traditional three-step method in all aspects.

[0098] Figure 4 The schematic diagram of the first-level probe design process provided in the embodiment of this application. Figure 4 As shown, the design process of the Y-shaped branch structure probe is as follows: Step S220 to Step S226:

[0099] S220: According to the detection target of the experimental sample, obtain corresponding reference sequences of at least two subtypes.

[0100] In this step, reference sequences of at least two subtypes can be read from the database according to the detection target.

[0101] For example, if the detection target is to detect cancer genes, the reference sequences of cancer-related genes can be read from the database. If the detection target is a genetic disease, the reference sequences of the corresponding genetic disease-related genes can be read from the database.

[0102] S221: Determine a consistency matrix based on the reference sequence.

[0103] In this step, the bases at each site in the reference sequence are compared to obtain the number of occurrences of each type of base at each site, the conservation score of the site is determined based on the number of occurrences of each type of base, and the conservation score of each site, the main base and the frequency distribution of the base are combined into a consistency matrix.

[0104] S222: Inputting the hidden Markov model according to the consistency matrix to obtain at least one conserved region output by the hidden Markov model.

[0105] In this step, the hidden Markov model can be pre-trained by the staff.

[0106] Conserved regions can be areas with low sequence variation and high consistency. According to the model settings, when the base consistency in a certain segment exceeds a preset threshold (such as 80% or 85%), the region is identified as a conserved region and used as the output value of the hidden Markov model.

[0107] S223: Determine the hybridization free energy, secondary structure self-complementarity and structural stability temperature of each conserved region.

[0108] In this step, the conserved region is input into a pre-trained thermodynamic modeling platform or thermodynamic analysis model, and the thermodynamic modeling platform or thermodynamic analysis model performs thermodynamic simulation on the conserved region to obtain the hybridization free energy, secondary structure self-complementarity and structural stability temperature of the conserved region.

[0109] S224: Determine whether the target conserved region is thermodynamically stable, has no dimers, and has no hairpin tendency based on the hybridization free energy, secondary structure self-complementarity, and structural stability temperature of the target conserved region.

[0110] In this step, the thermodynamic stability of the target conserved region can be determined based on the magnitude of the hybridization free energy. The absence of dimers and hairpinning tendency can be determined by calculating the length and base pairing of inverted repeats (such as the stem of a hairpin structure) or complementary regions within the target conserved region.

[0111] S225: If the target conserved region is thermodynamically stable, has no dimer, and has no hairpin tendency, then the target conserved region is determined as the probe binding core region.

[0112] In this step, the portion that ensures specificity and stability is identified as the probe binding core region.

[0113] S226: Determine the primary probe of the Y-shaped branch structure based on the probe binding core region.

[0114] In this step, a biotin label is added to the probe binding core region to obtain the backbone of the primary probe, and a first side chain containing a fluorophore is added to the 5' end of the backbone chain, and a second side chain containing a linker complementary sequence is added to the 3' end of the backbone chain to obtain a primary probe with a Y-shaped branched structure.

[0115] From the description of the above embodiments, it can be seen that the embodiments of the present disclosure obtain a reference sequence corresponding to the detection target and obtain a consistency matrix of the reference sequence, input the consistency matrix into the hidden Markov model to obtain a conserved region, and combine the hybridization free energy, secondary structure self-complementarity and structural stability temperature of the conserved region to select a probe binding core region from multiple conserved regions, thereby constructing a structurally stable probe based on the probe binding core region.

[0116] In a possible implementation, in step S221 , determining a consistency matrix based on a reference sequence includes steps S2211 to S2215 .

[0117] S2211: Count the number of occurrences of each base under the target site in the reference sequence.

[0118] In this step, the reference sequence may include multiple base sites. Since there are multiple reference sequences, the same base or different bases may appear at each site. If the base appearing at the target site is the same, the number of times this base appears at the target site is the same as the number of reference sequences.

[0119] For example, if there are 8 reference sequences, and the base of each reference sequence at position 1 is T, then the number of occurrences of base T at position 1 is 8. For another example, if there are 10 reference sequences, and the base of 5 reference sequences at position 2 is A, and the base of the other 5 reference sequences at position 2 is T, then the number of occurrences of base A and base T at position 2 is 5 each.

[0120] S2212: The number of occurrences of each base at the target site is divided by the number of reference sequences to obtain the probability of occurrence of each base at the target site.

[0121] In this step, for example, if all bases in the reference sequences at position 1 are T, then the probability of the occurrence of base T at position 1 is 100%. For another example, if the bases in 5 reference sequences at position 2 are A, and the bases in another 5 reference sequences at position 2 are T, then the probability of the occurrence of base A and base T at position 2 is 50%.

[0122] S2213: Calculate the conservation score of the target site based on the probability of occurrence of each base under the target site.

[0123] In this step, the information entropy of the bases at the target site can be calculated and the entropy value can be normalized to obtain a conservation score. The maximum occurrence probability corresponding to the site can also be determined as the conservation score.

[0124] S2214: Determine the base that appears most frequently under the target site as the main base of the target site.

[0125] In this step, for example, if the base that appears most frequently at site 3 is T, then base T is determined as the main base at site 3. For another example, if the base that appears most frequently at site 4 is G, then base G is determined as the main base at site 3.

[0126] S2215: The conservation scores, main bases and the probability of occurrence of each base at each site are combined into a consistency matrix.

[0127] In this step, the conservation score, main base and probability of occurrence of each base at each site are combined in a preset format according to the site order to obtain a consistency matrix.

[0128] From the description of the above embodiments, it can be seen that the embodiments of the present disclosure determine the probability of occurrence of each base by counting the number of times the base appears at the site, determine the conservation score and main base of the site according to the probability of occurrence, and combine the obtained occurrence probability, conservation score and main base in the order of the site to obtain a consistency matrix, which is convenient for the subsequent selection of conserved regions from the reference sequence.

[0129] In one possible implementation, in the above step S224, whether the target conserved region is thermodynamically stable, has no dimers, and has no hairpin tendency is determined based on the hybridization free energy, secondary structure self-complementarity, and structural stability temperature of the target conserved region, including: step S2241 and step S2242.

[0130] S2241: If the hybridization free energy of the target conserved region is less than a first free energy threshold, then the target conserved region is determined to be thermodynamically stable.

[0131] In this step, the first free energy threshold may be preset by the staff based on experimental data or experience.

[0132] S2242: If the hybridization free energy of the target conserved region is greater than the second free energy threshold, and the structural stability temperature is less than the preset temperature value, it is determined that there is no dimer and hairpin tendency, wherein the second free energy threshold is less than the first free energy threshold.

[0133] In this step, if the structure stabilization temperature is greater than the preset temperature value, it means that the hairpin structure or dimer structure is very stable at the experimental temperature (preset temperature value).

[0134] From the description of the above embodiments, it can be seen that the embodiments of the present disclosure determine whether the target conserved region can exist stably at the experimental temperature and not form a hairpin structure and dimer by judging whether the hybridization free energy of the target conserved region is greater than the second free energy threshold and less than the first free energy threshold, and the structural stability temperature is less than the preset temperature value, thereby ensuring that the target conserved region can exist stably during the experiment.

[0135] In a possible implementation, if the target conserved region is thermodynamically stable, has no dimer, and has no hairpinning tendency in step S225, then after determining the target conserved region as the probe binding core region, the method further includes:

[0136] S230: If a mismatch-sensitive region exists in the probe binding core region, N locked nucleic acid monomers are inserted into the probe binding core region to obtain a new probe binding core region, where N is a positive integer.

[0137] In this step, whether there is a mismatch sensitive region in the probe binding core region can be determined by searching whether there is a preset base sequence in the probe binding core region. If so, there is a mismatch sensitive region in the probe binding core region.

[0138] Wherein N is, for example, 2, 3, 4, etc. After performing this step, a new probe may be used to bind to the core region and perform the above step S226.

[0139] As can be seen from the description of the above embodiments, the disclosed embodiments insert N locked nucleic acid monomers into the probe binding core region, replacing the existing nucleotides when a mismatch-sensitive region is present. This allows the probe to form a more stable double-helix structure upon binding. Experimental data show that the insertion of two to three locked nucleic acid monomers can increase the preset temperature (melting temperature) in S2242 by 5–7°C (melting temperature change ≥ 5°C), which is particularly effective in single-base mismatch recognition.

[0140] The above description is merely a preferred embodiment of the present application and an illustration of the technical principles employed. Those skilled in the art should understand that the scope of the disclosure herein is not limited to technical solutions formed by specific combinations of the aforementioned technical features. It also encompasses other technical solutions formed by any combination of the aforementioned technical features or their equivalents, without departing from the aforementioned disclosure. For example, a technical solution formed by replacing the aforementioned features with (but not limited to) technical features with similar functions disclosed in this application.

[0141] Those skilled in the art will appreciate that all or part of the steps in the above-described method embodiments can be implemented using hardware associated with program instructions. The aforementioned program can be stored in a computer-readable storage medium. When executed, the program performs the steps of the above-described method embodiments. The aforementioned storage medium includes various media capable of storing program code, such as ROM, RAM, magnetic disks, or optical disks.

[0142] Finally, it should be noted that those skilled in the art will readily identify other embodiments of the present invention after considering the specification and practicing the invention disclosed herein. The present invention is intended to cover any variations, uses, or adaptations of the present invention that follow the general principles of the present invention and include common knowledge or customary techniques in the art not disclosed herein. The present invention is not limited to the precise structure described above and illustrated in the accompanying drawings, and various modifications and variations may be made without departing from the scope thereof. The scope of the present invention is limited solely by the appended claims.

Claims

1. A gene detection device, characterized in that: include: Pretreatment unit, core reaction unit and variant region detection unit; The preprocessing unit preprocesses the experimental sample to obtain gene fragments; The core reaction unit uses a time-released reagent to build a library for the gene fragment to obtain a sequencing library for the experimental sample; a primary probe is used to bind to a highly conserved region in the sequencing library to obtain a variable region to be identified that is bound by the primary probe, wherein the primary probe is a probe with a Y-shaped branch structure; Using a secondary probe to bind to the variable region to be identified to obtain a high-variability region bound to the secondary probe; The variable region detection unit is used to sequence the highly variable region; The core reaction unit mixes the gene fragment with the time-released reagent to obtain a mixed reagent; the mixed reagent is heated at a first temperature for a first preset time to release a polymerase component from the mixed reagent, and the polymerase component repairs the DNA broken end of the gene fragment; the mixed reagent is heated at a second temperature for a second preset time to release a Klenow fragment component from the mixed reagent, and the Klenow fragment component adds a dA tail to the DNA of the repaired gene fragment, wherein the second temperature is greater than the first temperature; the mixed reagent is heated at a third temperature for a third preset time to release a ligase component from the mixed reagent, and the ligase component combines the gene fragment with the added dA tail with a linear DNA vector with a dT protrusion to obtain a sequencing library of the experimental sample.

2. The device according to claim 1, characterized in that The pretreatment unit includes a micro peristaltic pump; the micro peristaltic pump adds lysis solution to the experimental sample, mixes the lysis solution with the experimental sample, and releases the DNA in the experimental sample; the pretreatment unit breaks the DNA in the experimental sample to obtain gene fragments.

3. The device according to claim 2, characterized in that The pre-processing unit includes: a sample injection micro-pressure sensor; The sample inlet micro pressure sensor is installed at the inlet of the experimental sample and is used to detect the pressure of the experimental sample; The pre-processing unit controls the running time of the micro peristaltic pump according to the pressure of the experimental sample.

4. The device according to claim 2, characterized in that The pre-processing unit further includes: a gene interruption unit; The gene fragmentation unit fragments the DNA in the experimental sample to obtain gene fragments.

5. The device according to claim 1, characterized in that The core reaction unit further includes: a neodymium iron boron magnet array composed of a plurality of neodymium iron boron magnets and a position control unit transmission-connected to the neodymium iron boron magnets; The position control unit is used to adjust the position and arrangement state of the NdFeB magnet so as to capture the magnetic composite containing the high-variability region through an external magnetic field.

6. The device according to claim 1, characterized in that The variation detection unit includes: a silicon-based purification column; The silica-based purification column is used to purify the highly variable region.

7. The device according to claim 1, characterized in that Also includes: processing unit and display unit; the core reaction unit also includes a temperature sensor; The processing unit is configured to receive the temperature value sent by the temperature sensor, draw a temperature curve using the temperature value, and send the temperature curve to the display unit; The display unit is used to display the temperature curve.

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