CN113174327B - Stirring control method based on PCR amplification bin - Google Patents

Abstract

The invention relates to a stirring control method based on a PCR amplification bin, wherein the amplification bin comprises a reactant to be amplified, a freeze-dried ball and memory metal; when reactants to be amplified and the freeze-dried balls enter the amplification bin, recording a first length and a second length of the memory metal at a first temperature and a second temperature; a first perturbation function T1(T1i, L1i, Q1i) and a second perturbation function T2(T2i, L2i, Q2i) are established. The memory metal is arranged in the amplification bin, a plurality of reaction periods are arranged in the actual amplification reaction process, so that the amplification reaction is more sufficient, the reaction in the amplification bin can be disturbed by the aid of the deformation of the memory metal at the deformation temperature, the reactant to be amplified and the freeze-dried ball are reacted, and related chemical reactions are more uniform and more sufficient under the action of the disturbance.

Description

Stirring control method based on PCR amplification bin

Technical Field

The invention relates to the field of nucleic acid detection, in particular to a temperature control method based on a PCR amplification bin.

Background

In the detection process of gene chip, the decisive factor of temperature control in the amplification process of PCR (Polymerase Chain Reaction) is the key technology for determining whether the fluorescence signal can be seen.

Temperature control typically involves three stages of temperature control, high (92-96 deg.C), medium (69-73 deg.C) and low (52-56 deg.C). In a complete PCR amplification process, a gene chip is denatured at a high temperature stage, then annealed at a low temperature stage, and finally extended at a medium temperature stage, and one PCR amplification is completed after the three stages are completed. Multiple of the above-described processes are typically required to achieve the predetermined fluorescence detection criteria.

The amplification process is completed in an amplification chamber, which is arranged in a pipeline layer that is a part of the chip to be detected. PCR is carried out by using DNA to be changed into single strand at 95 deg.C, combining primer and single strand at low temperature (usually about 60 deg.C) according to base complementary pairing principle, adjusting temperature to optimum reaction temperature (about 72 deg.C) of DNA polymerase, synthesizing complementary strand by DNA polymerase along phosphoric acid to pentose (5'-3'), and completing PCR amplification.

Generally, in order to monitor the real-time temperature of the amplification reaction, a temperature sensor and a temperature control device are required to be arranged, the temperature control device is used for controlling the temperature of the nucleic acid in the amplification bin, the temperature sensor is used for detecting the real-time temperature of the amplification bin in real time, and the temperature control device is adjusted according to the real-time temperature, so that each stage of the PCR amplification is kept in a corresponding temperature range. However, the existing temperature monitoring method is not very sensitive to temperature control and adjustment, and can affect the PCR reaction to a certain extent, thereby affecting the final fluorescence detection result.

Disclosure of Invention

Therefore, the invention provides a stirring control method based on a PCR amplification bin, which realizes the temperature control in an amplification reaction period and the correction of the mixing degree of the reactant to be amplified and the freeze-dried ball according to the shape memory property of the memory metal and the disturbance formed in the amplification bin in the deformation process.

In order to achieve the above object, the present invention provides a method for controlling agitation based on a PCR amplification chamber, comprising: the amplification bin contains a reactant to be amplified, a freeze-dried ball and memory metal; recording a first length and a second length of the memory metal at a first temperature and a second temperature when the reactant to be amplified enters the amplification bin; establishing a first disturbance function T1(T1i, L1i, Q1i) and a second disturbance function T2(T2i, L2i, Q2i), wherein T1i and T2i respectively represent a first temperature and a second temperature in the ith period, and L1i and L2i respectively represent the length of the corresponding memory metal in the ith period; q1i and Q2i respectively represent the mixing degree of the reactants to be amplified and the freeze-dried spheres in the ith period; comparing the first perturbation function with the second perturbation function to obtain a first comparison function T12(T1i-T2i, L1i-L2i, Q1i-Q2 i); setting a first comparison standard function T120, if all parameters of the first comparison function are within the range of the first comparison standard function T120, continuing the subsequent amplification reaction, otherwise, adjusting the length parameters at the corresponding temperature; and in a plurality of reaction cycles, adjusting the current reaction temperature according to the first length and the second length of the memory metal so as to enable the reactant to be amplified to be carried out at a preset temperature, and obtaining the mixing degree of the reactant to be amplified and the freeze-dried ball which meets the requirement.

Further, comparing the first perturbation functions T1(T1i +1, L1i +1, Q1i +1) -T1(T1i, L1i, Q1i) in adjacent periods respectively to obtain a first perturbation function difference value; comparing the second perturbation functions T2(T2i +1, L2i +1, Q2i +1) -T2(T2i, L2i and Q2i) in adjacent periods to obtain a second perturbation function difference value; and establishing a first disturbance standard difference value and a second disturbance standard difference value, if the first disturbance function difference value is in accordance with the first disturbance standard difference value, if the second disturbance function difference value is in accordance with the second disturbance standard difference value, continuing the periodic amplification reaction, and otherwise, adjusting the length parameter of the memory metal at the corresponding temperature.

Further, the mixing degree of the reactants to be amplified in the amplification bin and the freeze-dried balls in the current time node is compared with the mixing degree of the reactants to be amplified and the freeze-dried balls in the first period; if the mixing degree of the reactant to be amplified and the freeze-dried ball is not increased, the mixing is sufficient.

Further, comparing the mixing degree of the reactant to be amplified and the freeze-dried balls in the last period in the current time node with the mixing degree of the reactant to be amplified and the freeze-dried balls in the penultimate period; if the mixing degree of the reactant to be amplified and the freeze-drying balls is not increased, the stirring and mixing are finished fully, and if the mixing degree is increased, the stirring is continued.

Further, a mixedness matrix (Q1i, Q2i) is established, wherein Q1i, Q2i respectively represent the first mixedness and the second mixedness in the ith period;

and comparing the mixing degree matrixes in the adjacent periods to obtain a mixing degree difference value, and if the mixing degree difference value is 0, fully mixing.

Further, it is characterized in that the original length of the memory metal is a half-ring-shaped ring length.

Further, the first temperature is an initial temperature, and the second temperature is a deformation temperature.

Further, the disturbance coefficient X in each period is | L1i-L2i |/L1i, a standard disturbance coefficient for each period is preset, and if X is greater than the standard disturbance coefficient X0, the first temperature, the second temperature and the third temperature are adjusted so that the disturbance coefficient approaches the standard disturbance coefficient.

Further, the difference of the perturbation coefficients in adjacent periods is compared, and if the difference is greater than 0.1 × X0, the influence coefficient of the fluorescence intensity in the corresponding period in the amplification reaction result is adjusted.

Further, the difference of the perturbation in any two periods is compared, and if the difference is greater than 0.09 × X0, the influence coefficient of the fluorescence intensity in the corresponding period in the amplification reaction result is adjusted.

Compared with the prior art, the method has the advantages that a plurality of reaction cycles are set in the actual amplification reaction process, so that the amplification reaction is more sufficient, the reactant to be amplified and the freeze-dried ball in the amplification bin can be disturbed by utilizing the deformation of the memory metal at different temperatures, and the related chemical reaction is more uniform and more sufficient under the action of disturbance.

Particularly, in the embodiment of the invention, the memory metal is arranged in the amplification bin, the three disturbance functions are used for establishing the change of the length and the fluorescence intensity of the memory metal in the reaction at different temperatures, the relation functions of the three are established, the difference relation between any two disturbance functions is compared, the comparison standard function at each reaction stage is established, whether the difference relation of the corresponding disturbance functions is in the range of the comparison standard function is determined, if the difference relation is in the range, the reaction activity in the whole reaction period is normal, the subsequent amplification reaction is continued, if the difference relation is beyond the range of the comparison standard function, the phenomenon that the temperature, the length of the memory metal or the mixing degree is not in the normal range appears in the three stages of the reaction, and the data in the period needs to be corrected.

In particular, by determining the magnitude of the fluorescence intensity, it is determined whether to terminate the amplification reaction experiment, and the amplification reaction is performed in an effective time, and if the experiment is completed in time when the amplification reaction is completed, or if other experimenters are replaced in a waiting time, the progress of the amplification reaction can be verified at any time.

In particular, if the disturbance coefficient in each period is too large, the disturbance formed in the amplification chamber is large, which may destroy the reactant to be amplified in the amplification chamber and affect the fluorescence intensity of the final amplification reaction, so that the reaction temperature in each stage needs to be adjusted to reduce the deformation amount of the memory metal, and thus it is necessary to ensure that the disturbance coefficient approaches the standard disturbance coefficient.

Drawings

FIG. 1 is a schematic structural diagram of a PCR amplification chamber-based agitation control method according to an embodiment of the present invention;

FIG. 2 is a schematic view of a first usage state of the enlarged structure at I in FIG. 1;

FIG. 3 is a second schematic view of the enlarged structure at I of FIG. 1;

FIG. 4 is a schematic view of a third usage state of the enlarged structure shown at I in FIG. 1;

FIG. 5 is a schematic structural diagram of a chip device according to an embodiment of the present invention;

FIG. 6 is an exploded view of a chip device according to an embodiment of the present invention;

FIG. 7 is a schematic structural view of a pressing structure in an embodiment of the present invention;

fig. 8 is a schematic structural diagram of a pipeline layer in an embodiment of the invention.

Detailed Description

In order that the objects and advantages of the invention will be more clearly understood, the invention is further described below with reference to examples; it should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only for explaining the technical principle of the present invention, and do not limit the scope of the present invention.

It should be noted that in the description of the present invention, the terms of direction or positional relationship indicated by the terms "upper", "lower", "left", "right", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, which are only for convenience of description, and do not indicate or imply that the device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.

Furthermore, it should be noted that, in the description of the present invention, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

Referring to fig. 1, the agitation control method based on the PCR amplification chamber according to the embodiment of the present invention is applied to the amplification reaction process, and it will be understood by those skilled in the art that after the sample is extracted and purified, the material to be amplified is placed in the amplification chamber, and the amplification reaction is about to proceed. As shown in fig. 1 to 4, a memory metal 100 is provided in the amplification chamber 27 provided on the pipe layer 101. The amplification bin 27 contains a reactant to be amplified and a freeze-dried ball, when the amplification reaction is about to start, the reactant to be amplified enters the amplification bin from the pipeline, the freeze-dried ball is arranged in the amplification bin, and the reactant to be amplified is required to be fully mixed with the freeze-dried ball for subsequent reaction. Recording a first length and a second length of the memory metal 100 at a first temperature and a second temperature during the amplification reaction; establishing a first disturbance function T1(T1i, L1i, Q1i) and a second disturbance function T2(T2i, L2i, Q2i), wherein T1i and T2i respectively represent a first temperature and a second temperature in the ith period, and L1i and L2i respectively represent the length of the corresponding memory metal in the ith period; q1i and Q2i respectively represent the mixing degree of the reactants to be amplified and the freeze-dried spheres in the ith period; sequentially comparing the first perturbation function with the second perturbation function to obtain a first comparison function T12(T1i-T2i, L1i-L2i, Q1i-Q2 i); setting a first comparison standard function T120, if all parameters of the first comparison function are within the range of the first comparison standard function T120, continuing the amplification reaction, otherwise, adjusting the length parameters at the corresponding temperature. And in a plurality of reaction cycles, adjusting the current reaction temperature according to the first length and the second length of the memory metal so as to enable the reactant to be amplified to be carried out at a preset temperature, and obtaining the mixing degree of the reactant to be amplified and the freeze-dried ball which meets the requirement.

Specifically, as shown in FIG. 2, the original length of the memory metal is a semi-annular length, and the shape shown in FIGS. 3 and 4 appears during the deformation process, the annular length changes, and the overall shape also changes slightly, and the change of the memory metal can strengthen the disturbance in the amplification chamber, so that the mixing between the lyophilized pellet and the reactant to be amplified is more sufficient. Furthermore, the temperature of the amplification chamber is increased after the amplification chamber is filled, the temperature is increased continuously, and the memory metal is slowly deformed along with the increase of the temperature until the deformation temperature is reached, so that the deformation is finished. Since the reaction materials required for the different reaction stages all require different temperatures of enzyme activity, the reactants to be amplified are mixed more thoroughly and uniformly with the lyophilized spheres when deformed. The mixing degree in the amplification reaction bin can be effectively improved through temperature control, so that the temperature conditions in each stage are appropriate.

Specifically, under a high-temperature environment, the template DNA is dissociated to form a template DNA single strand; under the low-temperature environment, the primer is matched and combined with the complementary sequence of the template DNA single strand; under the condition of medium temperature, a DNA template-primer combination takes dNTP as a reaction raw material and a target sequence as a template under the action of TaqDNA polymerase, a new semi-reserved replication chain complementary with a template DNA chain is synthesized according to the base pairing and semi-reserved replication principles, fluorescent dye can be embedded in the template DNA in advance, and thus a reaction cycle is completed.

Specifically, a first disturbance function T1(T1i, L1i, Q1i) and a second disturbance function T2(T2i, L2i, Q2i) are established, wherein T1i and T2i respectively represent a first temperature and a second temperature in the ith period, and L1i and L2i respectively represent the length of the corresponding memory metal in the ith period; q1i, Q2i represent the degree of mixing of the reactants to be amplified with the lyophilized pellet at the first temperature and the second temperature, respectively, during the i-th cycle; the first temperature is a starting temperature; the second temperature is a deformation temperature; sequentially comparing the first perturbation function with the second perturbation function to obtain a first comparison function T12(T1i-T2i, L1i-L2i, Q1i-Q2 i); setting a first comparison standard function T120, if all parameters of the first comparison function are within the range of the first comparison standard function T120, continuing the amplification reaction, otherwise, adjusting the length parameters at the corresponding temperature. The embodiment of the invention establishes the change of the length and the fluorescence intensity of the memory metal in the reaction at different temperatures through the two disturbance functions, establishes the relation function between the two disturbance functions, determines whether the difference relation of the corresponding disturbance functions is in the range of the comparison standard function through comparing the difference relation between the two disturbance functions and establishing the comparison standard function at each reaction stage, if so, indicates that the reaction activity in the whole reaction period is normal, continues the subsequent amplification reaction, if the difference relation exceeds the range of the comparison standard function, indicates that the phenomenon that the temperature, the length of the memory metal or the mixing degree of the reactant to be amplified and the freeze-dried ball is not in the normal range occurs in the two stages of the reaction, and the data in the period needs to be corrected.

Specifically, the memory metal can present different lengths at a specific temperature, and the memory metal has a specific memory property, so that the specific temperature can be memorized, and the specific length and shape can be presented at the specific temperature, therefore, the length change of the memory metal at each temperature in the amplification reaction can be utilized to form a certain disturbance in the amplification bin, the reaction of the reactant to be amplified in the amplification bin is more sufficient through the disturbance of the memory metal, and the fluorescence intensity obtained by the corresponding image acquisition device is more sufficient under the disturbance action, so that the corresponding fluorescence intensity meets the standard. Specifically, the time from the initial temperature to the deformation temperature may be divided into periods, and parameters such as deformation amount and temperature may be recorded in each period, so as to determine the degree of mixing according to the above data.

Specifically, in the actual amplification reaction process, each amplification reaction at least comprises one reaction period, wherein one reaction period comprises the complete process of high-temperature deformation, low-temperature annealing and medium-temperature extension, and in the high-temperature stage, the first perturbation functions T1(T1i +1, L1i +1, Q1i +1) -T1(T1i, L1i and Q1i) in adjacent periods are compared to obtain a first perturbation function difference value; comparing the second perturbation functions T2(T2i +1, L2i +1, Q2i +1) -T2(T2i, L2i and Q2i) in adjacent periods to obtain a second perturbation function difference value; and establishing a first disturbance standard difference value and a second disturbance standard difference value, if the first disturbance function difference value is in accordance with the first disturbance standard difference value, if the second disturbance function difference value is in accordance with the second disturbance standard difference value, continuing the periodic amplification reaction, and otherwise, adjusting the length parameter of the memory metal at the corresponding temperature. The embodiment of the invention provides a stirring control method based on a PCR amplification bin, which is characterized in that corresponding first disturbance function difference values are obtained by comparing first disturbance functions in adjacent periods; and comparing the second disturbance functions in the adjacent periods to obtain corresponding second disturbance function difference values, comparing the first disturbance function difference value and the second disturbance function difference value with the first disturbance standard difference value and the second disturbance standard difference value respectively, if the corresponding first disturbance function difference value is within the range of the first disturbance standard difference value and the second disturbance function difference value is within the range of the second disturbance standard difference value, continuing the amplification reaction of the next period, and if not, adjusting the length parameter of the memory metal at the corresponding temperature.

Specifically, the mixing degree of the reactants to be amplified in the amplification bin and the freeze-dried balls in the current time node is compared with the mixing degree of the reactants to be amplified in the amplification bin and the freeze-dried balls in the first period; if the mixing degree of the reactant to be amplified and the freeze-dried ball is not increased, the reactant to be amplified and the freeze-dried ball are fully mixed, and the amplification reaction is continued. In the actual experimental operation process, in the amplification reaction, the mixing degree of the reactant to be amplified and the freeze-dried spheres in the sequential reaction period should be continuously increased, and whether the mixing degree of the reactant to be amplified and the freeze-dried spheres is uniformly mixed with the freeze-dried spheres is judged by judging whether the mixing degree of the reactant to be amplified and the freeze-dried spheres is continuously increased or not, so that the subsequent corresponding amplification reaction is facilitated.

Specifically, in a single cycle, as the temperature rises, the memory metal deforms with the passage of time, and in the deformation process, the memory metal stirs the reactant to be amplified, the lyophilized pellet and the frozen-dried pellet so as to be uniformly mixed, but after the deformation is finished, the stirring is finished, so that in practical application, a stirring cycle exists in each cycle, and in the stirring cycle, the starting time begins and the finishing time is reached after the deformation of the memory metal is finished. At the initial moment, the memory metal is not deformed, no stirring occurs, and as the time goes on, the memory metal begins to deform, the stirring also begins slowly, and as the stirring time goes on, the mixing degree of the reactant to be amplified and the freeze-dried ball also increases, and the mixing degree is more uniform and higher. The degree of mixing peaked after the experiment was completed. And in a single period, according to the judgment of the mixing degree, if the mixing degree of the reactant to be amplified and the freeze-drying balls is continuously increased, the reactant to be amplified is still mixed at present and does not reach a uniform state, and only after the mixing degree of the reactant to be amplified and the freeze-drying balls tends to be stable, the reactant to be amplified and the freeze-drying balls are fully mixed and are uniform. Therefore, in the embodiment of the present invention, the current mixing state can be determined according to the mixing degree of the reagent to be amplified for detecting nucleic acid and the freeze-dried ball, so that the progress of the experiment can be conveniently mastered. Whether the reactants to be amplified are uniformly mixed or not is judged by judging the mixing degree of the reactants to be amplified and the freeze-dried balls, so that the experiment progress is conveniently controlled.

Specifically, a mixing degree matrix (Q1i, Q2i) is established, wherein Q1i and Q2i respectively represent a first mixing degree and a second mixing degree in the ith period;

and comparing the mixing degree matrixes in the adjacent time periods to obtain a mixing degree difference value, and if the mixing degree difference value is 0, fully mixing.

Specifically, the mixing degree of the reactant to be amplified and the freeze-dried spheres determines the actual effect of the amplification reaction, so that in each period, the disturbance coefficient X of each period is determined to be | L1i-L2i |/L1i, the standard disturbance coefficient of each period is preset, and if X is greater than the standard disturbance coefficient X0, the deformation temperature is adjusted so that the disturbance coefficient approaches the standard disturbance coefficient. If the disturbance coefficient in each period is too large, the disturbance formed in the amplification chamber is large, which will destroy the material to be amplified in the amplification chamber 10 and affect the subsequent amplification reaction, so the temperature of each stage needs to be adjusted to reduce the deformation amount of the memory metal, and it is necessary to ensure that the disturbance coefficient approaches to the standard disturbance coefficient.

Specifically, by comparing the difference in the perturbation coefficients in adjacent cycles, if the difference is greater than 0.1 × X0, the influence coefficient of the degree of mixing in the corresponding cycle on the amplification reaction result is adjusted. It can be understood by those skilled in the art that if the difference between the coefficients of the perturbation functions in adjacent cycles is too large and larger than 0.1 × X0, the perturbation in at least one of the two cycles is too large or too small, and the amplification reaction intensity is beyond the normal range, so that the influence coefficient of the mixing degree in the two cycles on the whole amplification reaction result needs to be adjusted, if 40 reaction cycles are included in the actual reaction, if the sum of the proportions of all the cycles is 1, the proportion of each cycle is 1/40 on average, if the proportion of the reaction cycles with an abnormal perturbation coefficient can be 0.8 × 1/40, and so on, so that the amplification reaction result in the amplification bin is accurate, and the influence of the abnormal data result in a certain cycle on the final experimental result is reduced. Specifically, by comparing the difference between the perturbations in any two cycles, if the difference is greater than 0.09 × X0, the influence coefficient of the degree of mixing in the corresponding cycle on the amplification reaction result is adjusted. In practice, the difference between the perturbations in any two periods is compared, and if the difference is greater than 0.09 × X0, the perturbation in at least one of the two periods is too large or too small, and the amplification reaction intensity is beyond the normal range, so that the influence coefficient of the mixing degree in the two periods on the whole amplification reaction result needs to be adjusted, so that the amplification reaction result in the amplification bin is accurate, and the influence of the abnormal data result in a certain period on the final experimental result is reduced.

Specifically, please refer to fig. 5 and 6, which are schematic diagrams of a three-dimensional structure and an explosion structure of a chip device for nucleic acid detection according to an embodiment of the present invention, respectively, the chip device for nucleic acid detection according to the embodiment of the present invention includes a sample addition layer 3 disposed at the uppermost end, a gasket 2 disposed at the lower side of the sample addition layer 3, a pipeline layer 101 disposed at the lower side of the gasket 2, and a sealing film 104 disposed at the lowermost side, wherein a sample addition hole 302 is disposed at the upper side of the sample addition layer 3 for adding a sample into the chip, and the sample injected into the chip undergoes a reaction of nucleic acid extraction, purification, and amplification. The sample adding layer 3 and the pipeline layer 101 of the present embodiment are movably connected to the limiting frame 106 disposed at the side of the pipeline layer 101 through the clamping strip 304, correspondingly, the inner side of the limiting frame 106 is provided with the first clamping groove 107, and the first clamping groove 107 is connected to each other through the clamping strip 304 in a matching manner, so as to realize the switching and fixing of the relative positions of the sample adding layer 3 and the pipeline layer 101. The switching of relative position indicates the change of the relative distance of application of sample layer 3 and pipeline layer 101 exactly, application of sample layer 3 is by the process of first draw-in groove 107 switching second draw-in groove for the distance between application of sample layer 3 and pipeline layer 101 has become nearly, take out gasket 2 after, application of sample layer 3 and pipeline layer 101 communicate, specifically speaking, gasket 2's primary function is protection application of sample layer 3 and pipeline layer 101 do not communicate, take out the gasket during the use again, wherein, seal membrane 104 is pasted in the downside of pipeline layer 101, in order to realize sealed. The assembled sample adding layer 3, the gasket 2, the pipeline layer 101 and the sealing film 104 form a completely closed whole, and viruses in the sample cannot leak. As shown in fig. 6, in the embodiment, a second engaging groove is further disposed on a side surface of the limiting frame 106 at the lower side of the first engaging groove 107, the second engaging groove is located at the lower side of the first engaging groove 107, when the sample adding layer 3 is transported or stored, the first card slot 107 is connected with the sample adding layer 3, when the reagent reaction is carried out, the gasket 3 is drawn out, the sample adding layer 3 is pressed downwards, so that the sample adding layer 3 is connected with the second card slot, at the same time, the pricking pin disposed on the pipeline layer 101 punctures the reagent disposed in the sample addition layer 3, so that the reagent and the sample can be mixed and reacted, and the puncture needle is arranged on the pipeline layer 101, and in the practical application process, the pipeline layer 101 is provided with an upright post, the puncture needle is arranged at the circle center of the upright post, the upper end surface of the upright post is oval, and the end surface of the upright post is inclined, so that the puncture needle on the upright post can be conveniently matched with the tail end of the reagent tube, the reagent tube can be smoothly punctured, and the reagent can be filled. According to the chip device for nucleic acid detection provided by the embodiment of the invention, the first clamping groove 107 and the second clamping groove are arranged, so that the sample adding layer 3 and the pipeline layer 101 can be pressed to generate relative position change after the gasket 2 is extracted, and meanwhile, the pricker punctures the reagent in the sample adding layer 3 to realize sample adding, so that a sample and the reagent perform a series of reactions.

In order to monitor the real-time state of the sample adding layer 3, a second strain gauge (not shown in the figure) is arranged in the first card slot 107 and the second card slot, and the second strain gauge is used for testing the stress change of each part when the first card slot 107 or the second card slot is connected with the sample adding layer 3. In the practical application process, M points are transversely taken in the first card slot 107, wherein the M points are arranged at intervals, the state of the sample adding layer 3 is judged by detecting the stress change of the M points, when the sample adding layer 3 is stored or transported, the sample adding layer 3 is connected with the first card slot 107, the stress function of the first chip in the first card slot 107 is F (F1, F2 … … fm), wherein F1 and F2 … … fm represent the stress condition of each point, and the stress conditions of all the points are approximately the same because the sample adding layer 3 is uniformly stressed; at this time, stress detection is also performed at a corresponding position in the second card slot, and since the second card slot is not connected with the sample adding layer 3, a stress function of the second chip in the second card slot is F ' (F1 ', F2 ', … … fm '), wherein F1 ', F2 ', … … fm ' are all 0; and when the sample adding layer 3 is connected with the second card slot, F1 and F2 … … fm in the first chip stress function F (F1 and F2 … … fm) in the first card slot are both 0, so that the current position of the sample adding layer 3 can be judged according to the first chip stress function and the second chip stress function, the problem that the experimenter leaves midway and cannot continuously complete subsequent experiments is solved, and other experimenters can continuously complete the subsequent experiments according to the experiment progress of the previous experimenter.

Furthermore, when the stress values at the positions in the second chip stress function are all 0, that is, the sample-adding layer 3 is connected to the first card slot 107, a first chip stress function F (F1, F2 … … fm) is obtained, the stress value F1 at the first position in the first chip stress function is compared with the stress value fm at the mth position in the first chip stress function, a first positive difference value is obtained, if the first positive difference value is lower than a first preset difference value, it indicates that the sample-adding layer 3 is uniformly stressed in the first card slot 107, and then the subsequent operation of extracting the gasket 2 for pressing and the like can be performed; if the first positive difference is higher than the first predetermined difference, the difference between the stress value at the first position and the stress value at the mth position is larger, and it may be that the clip strip 304 of the sample-adding layer 3 or the first clip groove 107 on the limiting frame 106 has a defect or has impurities, etc., and the sample-adding layer 3 needs to be checked and adjusted, so that the first positive difference is lower than the first predetermined difference. And when the first positive difference value is lower than the first preset difference value, the back gasket 2 is output, the sample adding layer 3 is pressed, so that the sample adding layer 3 enters the second card slot under the action of external force, when the sample adding layer 3 is connected with the second card slot, a second chip stress function F ' (F1 ', F2 ', … … fm ') is obtained, the stress value F1 ' of the first position in the second card slot is compared with the stress value fm ' of the M position to obtain a second positive difference value, and if the second positive difference value is lower than the second preset difference value F0 ', the sample adding layer 3 is pressed to the second card slot and then is uniformly stressed, and no matter whether the card strip 304 or the second card slot has no obvious abnormality. If the second positive difference is higher than the second preset difference f 0', the sample addition layer 3 needs to be adjusted, which specifically refers to the adjustment when the stress value in the first slot 107 is abnormal, and is not described again.

In the practical application process, the stress response difference value between the first position and the mth position can be selected, and the positive difference value comparison can be performed on the positions of any two other points, so that the positions of all points in the first clamping groove 101 and the second clamping groove can be analyzed and checked, and the accuracy of the detection result can be further ensured.

When the sample adding layer 3 is connected with the first card slot 107, the stress difference value of the first position and the Mth position in the stress function of the first chip is detected, so that the state of the sample adding layer 3 in the first card slot 107 is checked and judged, the uniform stress of the sample adding layer in the first card slot 107 is ensured, and the states of all parts of the chip device are in normal states; correspondingly, when the sample adding layer 3 is connected with the second clamping groove, the stress difference value of the first position and the Mth position in the stress function of the second chip is detected, so that the state of the sample adding layer 3 in the second clamping groove is checked and judged, the uniform stress of the sample adding layer in the second clamping groove is ensured, and the states of all parts of the chip device are in normal states. In the practical application process, the sample adding layer 3 is physically pressed in the process of being pressed from the first card slot 107 to the second card slot, and abrasion to each structural component of the chip device easily occurs in the process, so that the first card slot 107, the second card slot, the card strip 304 and other related components need to be checked to ensure the accuracy of the mixed reaction result of the sample and the reagent.

Further, when the sample adding layer 3 is connected to the second card slot, a second chip stress function F '(F1', F2 ', … … fm') is obtained, and then absolute values of stress difference values at positions corresponding to one another in the first chip stress function F (F1, F2 … … fm) and the second chip stress function F '(F1', F2 ', … … fm') are compared, where the first chip stress function F (F1, F2 … … fm) is a first chip stress function generated when the sample adding layer 3 is connected to the first card slot 107, and whether the absolute value of each stress difference value is smaller than a preset standard error F0 is determined, that is, whether | F1 '-F1 |, | F2' -F2|, | F3 '-F3 |, | F4' -F4|, … …, and | fm '-fm' are respectively determined, and the size of the preset standard error F68628, 9 '-F599 | F9 |, i' -F639 | F … … |, and | F 'of the size of | F6342 |, i' F596 'F' is determined when the absolute value of | is determined, If the absolute value of the stress difference value at a certain position is found to be greater than the standard error F0, the reason of data abnormality needs to be searched until the relevant abnormality is eliminated, and the detection is carried out again.

In practical application, in order to determine whether the chip device is abnormal or not according to the stress values at the respective positions, rough estimation may be performed, for example, absolute values of stress differences at the M positions are obtained, and if the absolute values of stress differences at more than half of the M positions are smaller than the standard difference F0, that is, if the stress at most of the M positions is uniform, the subsequent operations may be performed; of course, the user can also select the judgment criteria to be M/2 positions or 4M/5 positions or the number of other position points according to actual needs, which are not listed here.

In practical application process, probably because the pressing power degree is inhomogeneous for some of application of sample layer 3 has pressed the second draw-in groove, and some has remained in first draw-in groove or because press and lead to application of sample layer 3 to incline, detect f1 ' and fm ' in the second draw-in groove this moment, the stress of first two positions at the end must be different very greatly, not in presetting the difference within range, just need readjust application of sample layer 3' this moment. In practical application, the stress difference of any two points in the M points can be detected, so that the position of the sample adding layer 3 is ensured to be accurate and stable. In addition, the stress value of the first position in the first card slot 107 and the stress value of the first position in the second card slot can be detected, when the stress on the sample adding layer 3 is not uniform, f1 and f 1' have slight difference, and it can be understood that, besides the stress difference of the first position, the stress difference of other M-1 positions can be detected, which is not described one by one here. In this embodiment, the second strain gauge provides multiple modes for detecting the stress uniformity of the sample adding layer 3 in the chip device, thereby ensuring the accuracy of subsequent nucleic acid detection.

In the practical application process, possible factors causing slight changes of stress values of the second strain gauges in the first clamping groove 107 and the second clamping groove include abrasion of the sample adding layer 3, inclination, external impurities, abrasion of the second strain gauge and the like, and the inspection needs to be carried out according to data of the stress function of the first chip and the stress function of the second chip, so that the sample adding layer 3 and the pipeline layer 101 are ensured to be stably butted to ensure that subsequent fluid enters a preset liquid injection port and a preset pipeline, and after the sample adding layer is butted with the pipeline layer, the uniformity of stress of the sample adding layer and the pipeline layer after being pressed is determined through extrusion force received by the first strain gauge, stress fatigue of the sample adding layer or the pipeline layer due to overlarge local stress is prevented, and the service life of the sample adding layer or the pipeline layer is shortened.

Particularly, through setting up the second foil gage, make in the pressfitting process, can assess the steady of pressfitting in-process, so that confirm the unstable factor in the pressfitting process, and after the block, through first foil gage, through the stress variation of first foil gage, make in the experimentation, add the sample and extract the purification and amplify the reaction process, carry out real-time supervision to the block condition of the two, prevent in the reaction process because stress variation leads to the problem of leakproofness, influence the experiment progress, through the stress variation who sets up first foil gage, can in time discover abnormal conditions, in time adjust. The sealing condition of the sample adding layer and the pipeline layer can affect the efficiency of the amplification reaction, so that the arrangement of the first strain gauge is necessary.

As shown in fig. 6, the lower side of the gasket 2 according to the embodiment of the present invention is further provided with a slide rail 202, and correspondingly, the upper side of the pipeline layer 101 is provided with a slide groove 108, and the slide rail 202 is connected with the slide groove 108 in a matching manner, so as to realize the sliding connection between the gasket 2 and the pipeline layer 101. The slide groove 108 of the present embodiment is provided inside the stopper 106 on the pipe layer 101. The end of the gasket 2 is provided with a plurality of notches and protrusions arranged at intervals, wherein the slide rail 202 is arranged on the bottom surface of the outermost protrusion.

As shown in fig. 6, the sample application hole 302 of the present embodiment is provided with a sample application hole cover 303 for sealing. Still set up buckle structure at application of sample layer 3 and pipeline layer 101, be provided with first buckle 301 in one side of application of sample layer 3, the downside of first buckle 301 stretches out the end and stretches out application of sample layer 3's bottom is installing application of sample layer 3 and pipeline layer 101 cooperation back together, through first buckle 301 joint on the side of pipeline layer 101 to prevent application of sample layer 3 and pipeline layer 101 separation.

As shown in fig. 6, the pipeline layer 101 of the present embodiment is provided with two first single valves 102 for controlling the stopping or flowing of the liquid in the pipeline layer 101 during the reaction process; the pipeline layer 101 is further provided with a double valve 103 for cutting off a road for fluid in the pipeline or allowing the fluid to pass through, the double valve 103 is communicated with the amplification bin through the pipeline, and the double valve 103 is used for controlling two ends of the amplification bin to be closed or opened simultaneously so that a closed cavity is formed inside the amplification bin. In fig. 6, handles 201 are further provided on both sides of the gasket 2 to facilitate extraction of the chip device for nucleic acid detection. In the embodiment of the invention, the amplification bin is arranged at the edge of the pipeline layer 101, and the amplification bin is of a semi-elliptical structure, so that not only can the reaction of the reaction reagent be realized, but also the convenient positioning and installation can be realized through the convex semi-elliptical structure when in use.

Continuing to refer to fig. 6, in the present embodiment, a row of pricker 105 is disposed on the pipeline layer 101, after the sample-adding layer 3 and the gasket 2 are clamped together, the handle 201 is operated to slide the gasket 2 along the sliding groove 108, when the gasket 2 is pulled out when the gasket slides to a position where the gasket cannot advance, the sample-adding layer 3 is pressed into the second clamping groove from the first clamping groove 107, so that the pricker 105 can prick a reagent tube in the sample-adding layer 3, and further the pricker 105 is communicated with a reagent in the sample-adding layer 3, and when a fluorescence sequence marked in the reagent is complementarily matched with a nucleic acid pricker at a corresponding position, a group of probe sequences with completely complementary sequences is obtained by determining a probe position with the strongest fluorescence intensity. A baffle is disposed outside the needle 105, and it acts as a stop and a positioning function when the sample injection layer 3 is matched with the pipeline layer 101.

Specifically, in the embodiment of the present invention, in the sample loading state, a plurality of sets of reagent tubes are disposed in the sample loading layer 3, the sample loading layer 3 is clamped with the first clamping groove 107 through the clamping strip 304 thereon, in the initial installation state, the sample loading layer 3 is matched with the pipeline layer 101 from top to bottom, and the reagent in the reagent tubes is isolated from the lancet through the gasket 2, so that the lancet and the reagent are prevented from being mixed due to vibration in the transportation process, the sample loading layer and the pipeline layer are protected from being disconnected, and puncturing is avoided. When needs are tested, outwards take gasket 2 out along spout 108, outwards take back along spout 108 for gasket 2 presses application of sample layer 3 downwards for card strip 304 and the second draw-in groove joint on application of sample layer 3, and at this moment, felting needle 105 on setting up on pipeline layer 101 mixes with application of sample layer 3's reagent, introduces reagent into pipeline layer 101 and tests.

Specifically, the gasket structure is arranged, so that the chip device for nucleic acid detection can be stored perfectly in the processes of reagent storage and transportation, and when the chip device is used, the reagent can be introduced into the pipeline layer only by drawing out the gasket and pressing the sample adding layer downwards.

FIG. 7 is a schematic view of a sample-adding layer according to an embodiment of the present invention; in this embodiment, a sample loading chamber is located below the sample loading hole 302, the sample loading chamber can be connected to a reagent tube for loading a reagent or a sample, a reagent outlet 312 is located at the lower portion of the sample loading chamber, a sealing structure 313 is located between the reagent outlet 312 and the sample loading chamber for sealing, and when a reagent needs to be loaded, the spike 105 can puncture 313 to allow the reagent to enter the fluid pipeline along the reagent outlet 312. A pressurizing structure is arranged on one side of the sample adding bin and comprises a tube wall 305, a piston 308 is arranged in the tube wall 305, and the piston 308 moves towards the sample adding bin to push the reagent in the sample adding bin to flow out to a reagent outlet 312; of course, when the reagent is required to be withdrawn, the piston 308 may also withdraw the reagent or other waste liquid, and a sealing ring 311 is provided at the end of the piston rod of the piston 308 for sealing.

Continuing to refer to fig. 7, the piston rod of this embodiment is further provided with a nut 307, which is in threaded connection with the nut 307 to realize relative rotation, and correspondingly, an output structure, such as an air cylinder and an oil cylinder, is provided at one end of the piston rod, or connected to the piston rod by rotating the output structure, such as a motor and a lead screw, at this time, the piston rod rotates, and only the reagent needs to be pushed to flow out of the reagent outlet. Correspondingly, a guide sleeve 306 is sleeved outside the nut 307, and a corresponding shaft shoulder is arranged inside the pipe wall 305 to position and fix the guide sleeve 306; snap rings 314 are further provided at the outer sides of both ends of the guide sleeve 306 to catch the corresponding guide sleeve 306. A sheath 309 is also provided outside the guide sleeve 306 to protect the piston rod, the nut 307 and the guide sleeve 306. When reagent is injected into the pipeline layer 101, the piston moves toward the sample loading bin to increase the pressure therein, so as to push the reagent to flow toward the reagent outlet 312, thereby realizing reagent injection. In the embodiment of the invention, a plurality of groups of reagent tubes are arranged, in the embodiment, five groups of reagent tubes are arranged, and different or same reagents are sequentially applied to the pipeline layer according to experiment requirements, so that the use efficiency can be greatly improved.

As shown in fig. 7, a second buckle 310 is disposed below the sample adding layer 3, and the second buckle 310 is disposed on a side opposite to the first buckle 301 to prevent the sample adding layer 3 from sliding.

It can be seen that this embodiment is integrated at chip pipeline layer to complicated experimentation, can control the liquid trend to can improve work efficiency effectively.

Referring to fig. 8, as shown in fig. 8, it should be noted in advance that, in fig. 8, the first single valve 102 and the double valve 103 are both disposed on the pipeline layer 101, and fig. 8 also includes the first single valve and the double valve, which are different reference numerals, the first single valves 210 and 211, and the first portion 271 and the second portion 281 of the double valve constitute the double valve 103.

As can be understood by those skilled in the art, the tube structure disposed on the tube layer is unable to perform related nucleic acid detection tests when the sample adding layer is not connected to the tube layer, so that it is necessary to perform nucleic acid extraction, purification and amplification reactions when the tube layer is in contact with the sample adding layer.

Specifically, as shown in fig. 8, the pipeline layer includes a first sample inlet 21, a first reagent inlet 22, a second reagent inlet 23, a third reagent inlet 24, a fourth reagent inlet 25, a purification chamber 26 and an amplification chamber 27, the first sample inlet 21 and the first reagent inlet 22 are connected through a first pipeline, a first single valve 211 is disposed on the first pipeline, the purification chamber 26 includes an inlet 261 and an outlet 262, the first sample inlet 21 and the inlet 261 are connected through a second pipeline 16, the first reagent inlet 22 and the outlet 262 are connected through a third pipeline 126, and the second reagent inlet 23, the third reagent inlet 24 and the fourth reagent inlet 25 are all connected with the inlet 262 through a fourth pipeline 610; the first end of the amplification chamber 27 is provided with a first double-valve portion 271, the second end of the amplification chamber is provided with a second double-valve portion 281, the first double-valve portion 271 is connected with the other single valve 212 through a fifth pipeline, the other single valve 212 is connected with the outlet 262 through a sixth pipeline 126, and the first double-valve portion 271 and the second double-valve portion 281 are opened and closed simultaneously.

Referring to fig. 8, a first sample inlet 21, a first reagent inlet 22, a second reagent inlet 23, a third reagent inlet 24, a fourth reagent inlet 25, a purification bin 26 and a PCR amplification bin 27 are further described, the first sample inlet 21 and the first reagent inlet 22 are connected through a first pipeline, a second single valve 211 is disposed on the first pipeline, the purification bin 26 includes an inlet 261 and an outlet 262, the first sample inlet 21 and the inlet 261 are connected through a second pipeline 16, the first reagent inlet 22 and the outlet 262 are sequentially connected through a seventh pipeline 92, a first buffer bin 29, a first single valve 212 and a third pipeline 126, and the second reagent inlet 23, the third reagent inlet 24 and the fourth reagent inlet 25 are all connected with the inlet 261 through a fourth pipeline 610; the first end of the PCR amplification chamber 27 is connected to the first part 271 of the double valve, the second end of the PCR amplification chamber is connected to the second part 281 of the double valve, the second part 281 of the double valve is connected to the outlet 262 of the purification chamber 26 through the eighth pipeline 86, the first part 271 of the double valve is connected to the first reagent port 22 through the second buffer chamber 210, the buffer chamber pipeline 97 and the first buffer chamber 29, and the first part 271 of the double valve and the second part 281 of the double valve are operated in a manner of being simultaneously closed or simultaneously opened.

Specifically, the purification and amplification device provided by the embodiment of the present invention further includes a first buffer bin 29, the first buffer bin 29 may also be referred to as a waste liquid bin, a high water absorption sponge is disposed in the first buffer bin, and mainly functions that after the lysate in the first reagent port 22 is injected, a small amount of liquid will remain in the lysate bin, the first reagent port 22 is used as a driven bin matched with other reagents and enters the purification bin, in this process, the piston structure connected to the first reagent port 22 will perform a suction motion, in order to avoid mixing a small amount of overflowing waste liquid into the whole liquid path system, the first buffer bin is disposed to absorb a small amount of waste liquid, one end of the first buffer bin 29 is connected to the first reagent port 22 through a seventh pipeline 92, the other end of the first buffer bin 29 is connected to a fifth pipeline to form a three-way pipeline, a second buffer bin 210 is further disposed between the three-way pipeline and the PCR amplification bin 27, be provided with the sponge in the second surge bin 210 for strengthen protection PCR and amplify storehouse 27, the second portion 281 of bivalve through eighth pipeline 86 with outlet 262 connects, be provided with second single valve 211 on the first pipeline, the one end of second single valve 211 pass through ninth pipeline 111 with first introduction port 21 connects, the other end of second single valve 211 pass through tenth pipeline 112 with first reagent mouth 22 connects.

When the amplification reaction is performed, firstly, injecting a sample, which may be blood or a swab, into the first sample inlet 21, injecting a first reagent, which is a lysate, into the first reagent inlet 22, opening the second single valve 211 to mix the sample and the first reagent via the first pipeline, so as to obtain a first product, which includes a liquid, the liquid enters the purification chamber via the second pipeline 16, the purification chamber 26 contains magnetic beads, the sample is decomposed by the lysate, nucleic acid substances and proteins are separated, the purification chamber is a reaction chamber for nucleic acid extraction and purification, the extraction is a reaction chamber for extracting nucleic acid substances from a mixture of nucleic acid substances and proteins, the purification is a step of cleaning the extracted nucleic acid substances, then closing the second single valve, opening the first single valve 212, and injecting a second reagent into the second reagent inlet 23, the second reagent is a cleaning solution, the second reagent enters the purification chamber through the fourth pipeline 610 and reacts with the liquid to obtain a second product, the third reagent is injected into the third reagent port 24, the third reagent is a cleaning solution, the third reagent enters the purification chamber through the fourth pipeline 610 and reacts with the second product to obtain a third product, the fourth reagent is injected into the fourth reagent port 25 and is an eluent, so that the nucleic acid substance is separated from the magnetic beads, the fourth reagent enters the purification chamber through the fourth pipeline 610 and reacts with the third product to obtain a fourth product, and the fourth product is introduced into the PCR amplification chamber 27 through the sixth pipeline 126, the first check valve 212 and the fifth pipeline to perform an amplification reaction.

Adding a sample into the first sample port 21, closing the first single valve 212, the first part 271 of the double valve and the second part 281 of the double valve, opening the second single valve 211, and then pushing a first reagent into the first reagent port 22, wherein the sample may be blood, nasopharyngeal swab, sample along the ninth pipeline 111, and the first reagent along the tenth pipeline 112, mixing in the first pipeline, in order to make the mixing of the sample and the first reagent more sufficient, in the practical application process, a push-suction device, specifically a piston structure, may be added at the first reagent port and the first sample port, to accelerate the micro-flow of the sample or reagent in the ninth pipeline 111 and the tenth pipeline 112, and to fully mix and react, so as to obtain a first biological product in the first pipeline, and the first biological product fills the first pipeline.

The second single valve 211 is closed, the first portion 271 of the double valve and the second portion 281 of the double valve are closed, and the first single valve is opened. At this time, the first product is respectively left in the ninth pipeline 111 and the tenth pipeline 112, the first product comprises liquid and gas, the liquid enters the purifying bin 26 from the first sample inlet 21 through the second pipeline 16, the liquid fills the purifying bin 26 and overflows in the sixth pipeline 126, meanwhile, under the buffering action of the second buffer bin 29, the gas of the first product enters the sixth pipeline 126 through the seventh pipeline 92 and the first single valve 212, and the liquid and the gas are merged in the sixth pipeline 126. When the second reagent is added, the second reagent is pushed into the second reagent port 23, the second reagent actually enters the purification cartridge 26 from the inlet 261 along the fourth pipeline 610, magnetic beads are arranged in the purification cartridge 26 so that the second reagent enters the purification cartridge 26 filled with the liquid, the magnetic beads in the purification cartridge 26 are scattered under the action of ultrasonic waves, so that the nucleic acid in the liquid in the purification cartridge is sufficiently contacted and adsorbed with the magnetic beads, the first reagent port 22 is sucked in while the second reagent is pushed in, the first product is sucked into the first reagent port so that the second reagent smoothly enters the purification cartridge 26, the second reagent reacts with the first product to obtain a second product, and the purification cartridge 26 is filled with the second product. Some waste liquid may be generated while the second product is being produced, and the waste liquid may be discharged to the first reagent port 22 and/or the first sample port 21, and optionally, the waste liquid may be discharged to the first reagent port 22 through 126, or the waste liquid may be discharged to the first sample port 21 through 16.

When the third reagent and the second product are mixed, the second reagent and the third reagent clean the liquid in the purification bin 26 to realize the extraction and purification of nucleic acid, when the third reagent port 24 pushes the third reagent through the fourth pipeline 610, the second reagent port 23 sucks part of the second product, then when the second reagent port 23 pushes the second product, the third reagent port 24 sucks the third reagent, and finally, the waste liquid of the reaction is sucked into the second reagent port 23 and/or the first reagent port 22 and/or the first sample port 21; when the fourth reagent and the third product are mixed, and the fourth reagent port 25 is pushed in the fourth reagent, the third reagent port sucks a part of the third product, and then when the third reagent port 24 is pushed in the third product, the fourth reagent port 25 sucks the fourth reagent, the fourth reagent is an eluent, and when the purification cartridge is filled with the fourth product after the reaction of the third reagent and the third reactant, the first single valve 212 and the second single valve 211 are closed, the first part 271 of the double valve and the second part 281 of the double valve are opened, and the fourth product fills the PCR amplification cartridge 26 from the outlet 262 along the eighth pipeline 86, and further the first part 271 of the double valve and the second part 281 of the double valve are closed. Compared with the previous embodiment of the present invention, the fourth product is introduced into the PCR amplification chamber 27 through the eighth pipeline 86, and compared with the PCR amplification chamber 27 through the sixth pipeline 126 and the fifth pipeline, the eighth pipeline 86 is not contaminated by other liquid or gas during the reaction process, and is clean, so that the purity of the substance entering the PCR amplification chamber 27 can be ensured.

In particular, it will be understood by those skilled in the art that the features of the technical solutions may be combined without departing from the natural laws and technical conflicts, which are not limited herein.

So far, the technical solutions of the present invention have been described in connection with the preferred embodiments shown in the drawings, but it is easily understood by those skilled in the art that the scope of the present invention is obviously not limited to these specific embodiments. Equivalent changes or substitutions of related technical features can be made by those skilled in the art without departing from the principle of the invention, and the technical scheme after the changes or substitutions can fall into the protection scope of the invention.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention; various modifications and alterations to this invention will become apparent to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (8)

1. A stirring control method based on a PCR amplification bin is characterized by comprising the following steps:

when a reactant to be amplified enters an amplification bin, recording a first length and a second length of a memory metal at a first temperature and a second temperature;

establishing a first disturbance function T1(T1i, L1i, Q1i) and a second disturbance function T2(T2i, L2i, Q2i), wherein T1i and T2i respectively represent a first temperature and a second temperature in the ith period, and L1i and L2i respectively represent the length of the corresponding memory metal in the ith period; q1i and Q2i respectively represent the mixing degree of the reactants to be amplified and the freeze-dried spheres in the ith period;

comparing the first perturbation function with the second perturbation function to obtain a first comparison function T12(T1i-T2i, L1i-L2i, Q1i-Q2 i); setting a first comparison standard function T120, if all parameters of the first comparison function are within the range of the first comparison standard function T120, continuing the subsequent amplification reaction, otherwise, adjusting the length parameters at the corresponding temperature;

in a plurality of reaction cycles, adjusting the current reaction temperature according to the first length and the second length of the memory metal so as to enable the reactant to be amplified to be carried out at a preset temperature, and obtaining the mixing degree of the reactant to be amplified and the freeze-dried ball which meets the requirement;

respectively comparing first perturbation functions T1(T1i +1, L1i +1, Q1i +1) -T1(T1i, L1i and Q1i) in adjacent periods to obtain a first perturbation function difference value; comparing the second perturbation functions T2(T2i +1, L2i +1, Q2i +1) -T2(T2i, L2i and Q2i) in adjacent periods to obtain a second perturbation function difference value; establishing a first disturbance standard difference value and a second disturbance standard difference value, if the first disturbance function difference value is in accordance with the first disturbance standard difference value, if the second disturbance function difference value is in accordance with the second disturbance standard difference value, continuing the periodic amplification reaction, otherwise, adjusting the length parameter of the memory metal at the corresponding temperature;

comparing the degree of mixing of the reactants to be amplified and the freeze-dried balls in the amplification bin in the current time node with the degree of mixing of the reactants to be amplified and the freeze-dried balls in the first period; if the mixing degree of the reactant to be amplified and the freeze-dried ball is not increased, the mixing is sufficient.

2. The PCR amplification chamber-based agitation control method of claim 1, wherein the mixing degree of the reactants to be amplified with the freeze-dried balls in the last cycle of the current time node is compared with the mixing degree of the reactants to be amplified with the freeze-dried balls in the second last cycle; if the mixing degree of the reactant to be amplified and the freeze-drying balls is not increased, the stirring and mixing are finished fully, and if the mixing degree is increased, the stirring is continued.

3. The PCR amplification chamber-based agitation control method of claim 1, wherein a mixedness matrix (Q1i, Q2i) is established, wherein Q1i, Q2i respectively represent the first mixedness and the second mixedness in the i-th period;

and comparing the mixing degree matrixes in the adjacent periods to obtain a mixing degree difference value, and if the mixing degree difference value is 0, fully mixing.

4. The PCR amplification chamber-based agitation control method of claim 1, wherein the original length of the memory metal is a half-circular ring length;

the amplification bin is arranged in the pipeline layer, the pipeline layer is provided with a sample adding layer, the lower side of the sample adding layer is provided with a gasket, and the lower side of the pipeline layer is also provided with a sealing film for realizing sealing;

wherein the content of the first and second substances,

the upper side of the sample adding layer is provided with a sample adding hole for adding a sample into the chip, and the sample injected into the chip is subjected to nucleic acid extraction, purification and amplification reaction in sequence;

the sample adding layer and the pipeline layer are movably connected with a limiting frame arranged on the side part of the pipeline layer through clamping strips, correspondingly, a first clamping groove is arranged on the inner side of the limiting frame and is mutually matched and connected through the clamping strips, so that the relative position switching and fixing of the sample adding layer and the pipeline layer are realized;

the second clamping groove is arranged on the inner side of the limiting frame and is positioned below the first clamping groove;

when in transportation or storage, the sample adding layer is connected with the first clamping groove;

when the sample injection device is used, the gasket is pulled out, the sample injection layer is pressed downwards to be connected with the second clamping groove, and meanwhile, the puncture needle is arranged on the upright column on the pipeline layer and is used for puncturing the reagent arranged in the sample injection layer so as to enable the reagent and the sample to be mixed and reacted;

after the sample adding layer and the pipeline layer are pressed, a first strain gauge arranged at the bottom of the sample adding layer detects the extrusion force between the sample adding layer and the pipeline layer so as to determine the stress uniformity of the sample adding layer and the pipeline layer in the pressing process.

5. The PCR amplification bin-based agitation control method of claim 3, wherein the first temperature is a start temperature and the second temperature is a deformation temperature.

6. The PCR amplification bin-based agitation control method according to claim 3, wherein the disturbance coefficient X in each period is | L1i-L2i |/L1i + | L2i-L3i |/L2i + | L3i-L1i |/L3i, a standard disturbance coefficient for each period is preset, and if X is greater than the standard disturbance coefficient X0, the first temperature, the second temperature and the third temperature are adjusted so that the disturbance coefficient approaches the standard disturbance coefficient.

7. The PCR amplification chamber-based agitation control method of claim 6, wherein the difference between the disturbance coefficients in adjacent cycles is compared, and if the difference is greater than 0.1 XX 0, the influence coefficient of the fluorescence intensity in the corresponding cycle on the amplification reaction result is adjusted.

8. The PCR amplification bin-based agitation control method according to claim 7, wherein the difference of the perturbations in any two cycles is compared, and if the difference is greater than 0.09X 0, the influence coefficient of the fluorescence intensity in the corresponding cycle on the amplification reaction result is adjusted.

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