CN110804649A - Optical excitation and detection method of nucleic acid amplification instrument - Google Patents
Optical excitation and detection method of nucleic acid amplification instrument Download PDFInfo
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Abstract
The invention provides an optical excitation and detection method of a nucleic acid amplification instrument, which comprises the steps of injecting nucleic acid into a first reaction cavity; the heating device works to ensure that the nucleic acid in the first reaction cavity is heated and split, and the first control valve is opened so that the split nucleic acid enters the second reaction cavity through the flow guide pipeline; the output matter control valve is opened so that the intermediate matter in the output matter storage bin flows into the second reaction cavity; the controller controls the heat dissipation device to start working so as to cool the second reaction cavity; the heating device in the second reaction cavity starts to work, the controller controls the second control valve to be opened, and meanwhile, the controller also controls the pump body to work so as to convey the nucleic acid in the second reaction cavity back to the first reaction cavity; the controller analyzes the data collected by the fluorescence detection unit and calculates the relationship between the system stability value deltas and the measurement interval K so as to select a corresponding working mode; the above reaction was repeated 20-30 times.
Description
Technical Field
The present invention relates generally to nucleic acid amplification apparatus, and more particularly to an optical excitation and detection method for a nucleic acid amplification apparatus.
Background
Nucleic acid amplification is a generic term for a broad class of technical methods, currently including conventional PCR, real-time fluorescent PCR, isothermal nucleic acid amplification techniques, etc., which are very useful in molecular biology and have wide applicability in every aspect of biology, therapeutics, diagnostics, forensics, and research. Typically, one or more primers are used to generate an amplicon from a starting template, wherein the amplicon corresponds to or is complementary to the template from which the amplicon was generated. Multiplex amplification also simplifies the process and reduces costs.
The existing nucleic acid amplification technology mainly comprises the following steps: heating to uncoil the double-stranded DNA, hybridizing the primer with the template DNA at annealing temperature, reacting with Taq DNA polymerase, dNTPs, Mg2+And extending the primer in the presence of a proper PH buffer solution, repeating the process of denaturation-annealing-primer extension to 25-40 cycles, and exponentially increasing the copy number of the nucleic acid in the sample to be detected. The fluorescence detection system mainly comprises an excitation light source and a detector, and the current mainstream is multicolor multi-channel detection, the more excitation channels, the more types of used fluorescein and the wider application range of the instrument. The real-time fluorescent quantitative PCR technology is to add specific fluorescent dye into the PCR reaction systemOr the probe, the change of the fluorescence signal truly reflects the increase of the template in the system, and the quantitative purpose is achieved by detecting the fluorescence signal.
The existing nucleic acid amplification instrument has low intelligent degree, and in the nucleic acid amplification process, the addition of components such as primers, buffer solution, dNTP and the like all needs manual addition of workers; meanwhile, in the nucleic acid amplification process, the amplified nucleic acid is often required to be repeated, the temperature control is required in each nucleic acid amplification process, and the operations are manually performed, so that the time and the labor are wasted, and the production and the research are not facilitated; to this end, the present invention provides a method for optically exciting and detecting a nucleic acid amplification instrument, which at least partially solves the above problems.
Disclosure of Invention
In this summary, concepts in a simplified form are introduced that are further described in the detailed description section. This summary of the invention is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
To at least partially solve the above technical problems, the present invention provides an optical excitation and detection method for a nucleic acid amplification apparatus, comprising:
step 1, injecting nucleic acid into a first reaction cavity;
step 2, the heating device works to enable the nucleic acid in the first reaction cavity to be heated and split, the first temperature sensor group starts to detect the temperature in the first reaction cavity, when the first temperature sensor group detects that the temperature in the first reaction cavity is 85-95 ℃ for the first time, the controller starts to time, and after the controller times for 5-8 minutes, the first control valve is opened, so that the split nucleic acid enters the second reaction cavity through the flow guide pipeline;
step 3, the second liquid level sensor group starts to detect the liquid level height in the second reaction cavity, when the real-time liquid level height H2 detected by the second liquid level sensor group meets a first liquid level standard value, the intermediate output device starts to work, the output control valve is opened, so that the intermediate in the output storage bin flows into the second reaction cavity, and the primer, the buffer solution and the dNTP are stored in the output storage bin;
step 4, the second liquid level sensor group continues to detect the liquid level height in the second reaction cavity, and when the real-time liquid level height H2 detected by the second liquid level sensor group meets a second liquid level standard value, the controller controls the heat dissipation device to start working so as to cool the second reaction cavity;
step 5, the second temperature sensor group starts to work, when the second temperature sensor group detects that the temperature in the second reaction cavity is reduced to 50 ℃ for the first time, the heating device in the second reaction cavity starts to work, and the heating device and the heat dissipation device work together to maintain the temperature in the second reaction cavity to be 45-55 ℃;
when the second temperature sensor group detects that the temperature in the second reaction cavity is reduced to 50 ℃ for the first time, the controller starts timing, controls a second control valve to be opened after timing for 1-3 minutes, and simultaneously controls the pump body to work so as to convey the nucleic acid in the second reaction cavity back to the first reaction cavity;
and 6, analyzing the data acquired by the fluorescence detection unit by the controller, and calculating the relationship between a system stable value delta s and a measurement interval K to select a corresponding working mode:
when the system stability value delta s is within the measurement interval K, the controller continuously keeps a normal working mode;
when the system stable value deltas is not in the measurement interval K, the controller enters a diagnostic program;
and 7, setting the steps 1 to 6 as a cyclic reaction, and repeating the cyclic reaction for 20-30 times.
Furthermore, the diversion pipeline is connected with the first reaction chamber and the second reaction chamber, the first control valve and the intermediate output device are both arranged on the diversion pipeline, and the first reaction chamber and the second reaction chamber are respectively arranged in the first reaction tank and the second reaction tank;
wherein the level of the first reaction chamber is higher than the level of the second reaction chamber; the diversion pipeline is arranged between the first reaction cavity and the second reaction cavity with a certain gradient, so that the solution in the first reaction cavity can enter the second reaction cavity through the diversion pipeline under the action of gravity;
the tops of the first reaction tank and the second reaction tank are provided with ventilation openings, and the ventilation openings are provided with the heat dissipation devices; the bottom of the first reaction tank is provided with an input end communicated with the first reaction cavity, and the bottom of the second reaction tank is provided with an output end communicated with the second reaction cavity;
heating devices are arranged in the first reaction tank and the second reaction tank, and the heating devices in the first reaction tank are arranged in a gap region enclosed by the outer surface of the first reaction cavity and the inner surface of the first reaction tank; the heating device in the second reaction tank is arranged in a gap interval enclosed by the outer surface of the second reaction cavity and the inner surface of the second reaction tank; the heating device comprises at least two heating lamps and a lamp control group, the lamp control group is electrically connected with the heating lamps, the lamp control group is used for controlling the heating power of the heating lamps, and the heating lamps are vertically arranged in a gap interval and surround the first reaction chamber and the second reaction chamber;
the backflow pipeline is connected with the first reaction cavity and the second reaction cavity, and materials in the second reaction cavity return to the first reaction cavity through the backflow pipeline; the device that is equipped with on the return line includes: the pump body, second control valve and restriction valve are followed by the material in the direction of second reaction chamber flow direction first reaction chamber, the sequence of arrangement of the device of installation on the backflow pipeline is in proper order: the second control valve, the pump body and the flow limiting valve;
the second control valve is arranged near an interface of the backflow pipeline and the second reaction cavity and used for controlling the connection and disconnection of the second reaction cavity and the backflow pipeline, the pump body is used for conveying materials in the backflow pipeline into the first reaction cavity, and the flow limiting valve is used for controlling the flow rate of the materials in the backflow pipeline;
first reaction chamber with all be equipped with agitating unit in the second reaction chamber, agitating unit includes motor, pivot and stirring piece, the stirring piece sets up in the pivot, motor drive the pivot rotates, and then makes the stirring piece is right first reaction chamber with the material in the second reaction chamber stirs.
Furthermore, the heat dissipation device comprises a heat dissipation blocking piece and a heat dissipation fan, wherein the heat dissipation blocking piece comprises a fixed piece, a rotating column and a rotating piece;
the heat dissipation blocking piece is arranged below the heat dissipation fan, the fixed sheet and the rotating sheet are respectively connected with the rotating column, when the heat dissipation fan does not work, the fixed sheet and the rotating sheet can completely shield the ventilation opening to prevent dust from entering the first reaction chamber and the second reaction chamber, when the heat dissipation fan works, the controller controls the rotating column to rotate, the rotating sheet rotates under the driving of the rotating column, the second reaction chamber is communicated with the heat dissipation fan, and the controller controls the heat dissipation fan to work so as to cool the second reaction chamber.
Furthermore, the heat dissipation blocking piece is provided with a vent hole, so that the atmospheric pressure on the first reaction tank and the atmospheric pressure on the second reaction tank are always the same as the outside.
Further, the detection unit includes a first fluorescence detector and a second fluorescence detector. The first fluorescence detector detects the sample passing through the drainage pipeline, and real-time data N1 are detected; the second fluorescence detector detects the sample passing through the return pipeline and detects real-time data N2;
wherein Δ s ═ N2-N1The range of the measurement interval K is set to [2N ]1/3，N1]。
Further, the controller is connected toContinuously collecting real-time data N1' and real-time data N2', and N1' and N1Making a difference or dividing N2' and N2Making a difference value; wherein N is1' and N2' reflecting the real-time data detected by the detection unit for the next cycle;
and if the controller detects that the system stability value delta s is not in the range of the measurement interval K in the three continuous circulating reactions, the controller sends out a reminding alarm so that a worker can detect the temperature of the material flowing through the diversion pipeline and the backflow pipeline.
Further, the control system comprises a control group and a controlled element, the control group comprises the controller and a human-computer interaction panel, the controlled element is respectively electrically connected with the controller, the controlled element comprises the first temperature sensor group, the second temperature sensor group, the first flow sensor group, the second flow sensor group, the third flow sensor group, a first liquid level sensor group, the second liquid level sensor group, the lamp control device, the heat radiation fan, the first control valve, the pump body, the second control valve, the flow limiting valve, the rotary column and the output control valve;
wherein the first temperature sensor group is arranged in the first reaction cavity to detect the real-time temperature T1 in the first reaction cavity; the second temperature sensor group is arranged in the second reaction cavity to detect the real-time temperature T2 in the second reaction cavity; the first flow sensor group is arranged on the diversion pipeline and at the front end of the intermediate output device so as to detect the real-time flow rate V1 of the material flowing out of the first reaction cavity; the second flow sensor group is arranged on the output control pipeline to detect the real-time flow rate V2 of the intermediate; the third flow sensor group is arranged on the return pipeline to detect the real-time flow velocity V3 of the material flowing out of the second reaction cavity; the first liquid level sensor group is arranged in the first reaction cavity to detect the real-time liquid level height H1 of the first reaction cavity; the second liquid level sensor group is arranged in the second reaction cavity to detect the real-time liquid level height H2 of the second reaction cavity.
Further, the level values stored in the controller include:
the first liquid level standard values H11, H12 and H13 are repeated until the 20 th to 30 th standard liquid level value is reached;
the second liquid level standard values H21, H22, H23 and the like till the 20 th to 30 th standard liquid level values;
limit level H0;
the calculation method of the first liquid level standard value comprises the following steps: h11 is H10- Δ 1, H1n is H2(n-1) - Δ 1- Δ 2, Δ 1 is the loss of material flowing through the diversion pipeline, Δ 2 is the loss of material flowing through the return pipeline, and n is a positive integer greater than or equal to 2;
the method for calculating the second liquid level standard value comprises the following steps: h2n ═ H1n + G, G is the height of the liquid level of the intermediate flowing into the second reaction chamber, and n is a positive integer equal to or greater than 1.
Further, when the real-time liquid level H1 detected by the first liquid level sensor or the real-time liquid level H2 detected by the second liquid level sensor exceeds the limit liquid level H0, the reaction is stopped, and the materials in the first reaction chamber and the second reaction chamber exit the nucleic acid amplification instrument through the output end.
Further, the controller also calculates the flow rates of the diversion pipeline, the output control pipeline and the return pipeline, and the flow rate calculation formula is as follows:
wherein S is the cross-sectional area of the pipeline, V is the real-time flow rate of the material, and T is the time of flowing through the pipeline;
substituting the cross-sectional area S1 of the diversion pipeline, the real-time flow velocity V1 measured by the first flow velocity sensor group and the time T1 of the material passing through the first flow velocity sensor group into a flow calculation formula to calculate a first flow Q1;
substituting the cross-sectional area S2 of the output control pipeline, the real-time flow velocity V2 measured by the second flow sensor group and the time T2 of the material passing through the second flow sensor group into a flow calculation formula to calculate a second flow Q2;
substituting the cross-sectional area S3 of the return pipeline, the real-time flow velocity V3 measured by the third flow velocity sensor group and the time T3 of the material passing through the third flow velocity sensor group into a flow calculation formula to calculate a third flow Q3;
the working personnel can judge the working state of the nucleic acid amplification instrument by detecting the first flow Q1, the second flow Q2 and the third flow Q3, and the detection formula comprises the following steps: k (Q1+ Q2) ═ Q3, k is the reaction coefficient.
Compared with the prior art, the invention has the beneficial effects that:
compared with the existing amplification instrument equipment which is provided with the first reaction cavity and the second reaction cavity, the nucleic acid is circulated from the first reaction cavity to the second reaction cavity and returns to the first reaction cavity from the second reaction cavity, the nucleic acid can continuously repeat the circulating operation under the action of the controller, and the controller controls the intermediate output device to periodically convey components such as primers, buffer solution, dNTP and the like to the second reaction cavity, so that the amplification reaction can be continuously carried out.
Further, only the enzymes for decomposing nucleic acids circulating together with the nucleic acids in the first reaction chamber and the second reaction chamber decompose the nucleic acids only when a suitable temperature is provided in the first reaction chamber; the intermediate output device is arranged on a drainage pipeline which is connected with the first reaction tank and the second reaction tank, and when a nucleic acid amplification reaction cycle is carried out, the intermediate output device can provide materials required by the amplification reaction cycle so as to ensure that all nucleic acid amplification is completed in the second reaction chamber, and further ensure that the first reaction chamber and the second reaction chamber keep specific temperature, wherein the first reaction chamber keeps the optimal activity temperature of the nucleic acid decomposition enzyme, and the second reaction chamber keeps the optimal activity temperature of the nucleic acid polymerase.
Furthermore, the heat radiating fan and the heating device arranged in each reaction cavity can work together to control the temperature of the reaction cavity, so that the temperature control sensitivity is improved.
Further, the controller also calculates the flow rates of the diversion pipeline, the output control pipeline and the return pipeline, and the flow rate calculation formula is as follows:
wherein S is the cross-sectional area of the pipeline, V is the real-time flow rate of the material, and T is the time of flowing through the pipeline;
substituting the cross-sectional area S1 of the diversion pipeline, the real-time flow velocity V1 measured by the first flow velocity sensor group and the time T1 of the material passing through the first flow velocity sensor group into a flow calculation formula to calculate a first flow Q1;
substituting the cross-sectional area S2 of the output control pipeline, the real-time flow velocity V2 measured by the second flow sensor group and the time T2 of the material passing through the second flow sensor group into a flow calculation formula to calculate a second flow Q2;
substituting the cross-sectional area S3 of the return pipeline, the real-time flow velocity V3 measured by the third flow velocity sensor group and the time T3 of the material passing through the third flow velocity sensor group into a flow calculation formula to calculate a third flow Q3;
the working personnel can judge the working state of the nucleic acid amplification instrument by detecting the first flow Q1, the second flow Q2 and the third flow Q3, and the detection formula comprises the following steps: k (Q1+ Q2) ═ Q3, k is the reaction coefficient.
Furthermore, the invention can also be provided with a control panel, and the control panel and the controller can be provided with signal receiving and sending devices, so that the control panel and the controller can be accessed to a local area network or the Internet, and the control panel can remotely control the work of the controller through the communication of the Internet.
Furthermore, the heating device comprises a plurality of heating lamps, and the working modes of the heating lamps comprise a whole working mode and a partial working mode, so that the practicability of the amplification instrument is improved.
Furthermore, the invention is also provided with a fluorescence detection unit, wherein the fluorescence detection unit comprises a first fluorescence detector and a second fluorescence detector, and the fluorescence detectors detect the materials in the drainage pipeline and the return pipeline so as to judge the effect of nucleic acid amplification through the controller; meanwhile, the controller is also internally provided with a diagnostic program to analyze and judge the data detected by the first fluorescence detector and the second fluorescence detector, and the controller can automatically or manually control the analysis result to timely adjust the heating temperature in each reaction cavity of the nucleic acid amplification instrument so as to ensure the activity of the enzyme in the reaction cavity.
Drawings
In order that the advantages of the invention will be readily understood, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments that are illustrated in the appended drawings. Understanding that these drawings depict only typical embodiments of the invention and are not therefore to be considered to be limiting of its scope, the invention will be described and explained with additional specificity and detail through the use of the accompanying drawings.
FIG. 1 is a schematic front view of a nucleic acid amplification apparatus according to the present invention;
FIG. 2 is a schematic top view of the nucleic acid amplification apparatus according to the present invention;
FIG. 3 is a schematic view of the heating apparatus shown in FIG. 1;
FIG. 4 is a schematic structural view of the stirring device shown in FIG. 1;
fig. 5 is a schematic structural view of the heat dissipation blocking sheet shown in fig. 1;
FIG. 6 is a schematic front view of the structure of FIG. 5;
fig. 7 is a schematic structural diagram of a control system according to the present invention.
Description of reference numerals:
1: first reaction tank 2: second reaction tank
3: and (4) diversion pipeline: first control valve
5: intermediate output device 6: return conduit
7: a pump body 8: second control valve
9: the flow limiting valve 10: controller
11: first reaction chamber 12: input terminal
13: heat sink device
141: rotating shaft 142: stirring sheet
143: the motor 131: heat dissipation blocking piece
132: cooling fan 1311: fixing sheet
1312: rotation column 1313: rotating sheet
1511: first heating lamp 1512: second heating lamp
1513: third add light 1514: fourth heating lamp
1511': fifth heating lamp 1512': sixth heating lamp
1513': seventh heating lamp 1514': eighth heating lamp
152: lamp control device 21: second reaction chamber
22: output terminal 31: first sampling port
32: second sampling port 51: output storage bin
52: output control valve 53: output control pipeline
101: first temperature sensor group 102: second temperature sensor group
103: first flow rate sensor group 104: second flow rate sensor group
105: third flow rate sensor group 106: first liquid level sensor group
107: second liquid level sensor group
Detailed Description
In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art, that embodiments of the invention may be practiced without one or more of these specific details. In other instances, well-known features have not been described in detail so as not to obscure the embodiments of the invention.
In the following description, a detailed structure will be presented for a thorough understanding of embodiments of the invention. It is apparent that the implementation of the embodiments of the present invention is not limited to the specific details familiar to those skilled in the art. The following detailed description of preferred embodiments of the invention, however, the invention is capable of other embodiments in addition to those detailed.
Referring to fig. 1 and 2, the nucleic acid amplification apparatus according to the present invention includes a first reaction tank 1, a second reaction tank 2, a flow guide pipe 3, a first control valve 4, and an intermediate output device 5; the diversion pipeline 3 is connected with the first reaction tank 1 and the second reaction tank 2, and the first control valve 4 and the intermediate output device 5 are both arranged on the diversion pipeline 3; wherein, be equipped with first reaction chamber 11 and second reaction chamber 21 in first retort 1 and the second retort 2 respectively, communicate through water conservancy diversion pipeline 3 between first reaction chamber 11 and the second reaction chamber 12, and the level of first reaction chamber 11 is higher than the level of second reaction chamber 12, and water conservancy diversion pipeline 3 has the setting of certain slope between first reaction chamber 11 and second reaction chamber 21 promptly to make the solution in first reaction chamber 11 accessible water conservancy diversion pipeline 3 enter second reaction chamber 21 under the effect of gravity only. The top of the first reaction tank 1 is provided with a vent, and a heat dissipation device 13 is arranged on the vent; heating devices 15 are arranged in the first reaction tank 1 and the second reaction tank 2, and the heating devices 15 in the first reaction tank 1 are arranged in a gap interval enclosed by the outer surface of the first reaction cavity 11 and the inner surface of the first reaction tank 1; the heating device 15 in the second reaction tank 2 is arranged in a gap interval enclosed by the outer surface of the second reaction cavity 21 and the inner surface of the second reaction tank 2; the reflux pipeline 6 is connected with the first reaction tank 1 and the second reaction tank 2, and the material in the second reaction chamber 21 can return to the first reaction chamber 11 through the reflux pipeline 6; the pump body 7, the second control valve 8 and the flow limiting valve 9 are arranged on the return pipeline 6, and the arrangement sequence of the devices arranged on the return pipeline 6 is that in the direction from the second reaction tank to the first reaction tank by the materials: a second control valve 8, a pump body 7 and a flow limiting valve 9; the second control valve 8 is arranged near the interface of the return pipeline 6 and the second reaction chamber 21 and used for controlling the connection and disconnection of the second reaction chamber 21 and the return pipeline 6, the pump body 7 is used for conveying materials in the return pipeline 6 to the first reaction chamber 11, and the flow limiting valve 9 is used for controlling the flow rate of the materials in the return pipeline 6.
Specifically, the intermediate output device 5 includes an output storage bin 51, an output control valve 52, and an output control pipe 53; the output storage chamber 51 communicates with the pilot line 3 through an output control line 53, and an output control valve 52 is provided on the output control line 53.
In some embodiments of the present invention, the bottom of the first reaction tank 1 is provided with a stirring device extending to the middle of the first reaction chamber 11; the heat sink and the stirring device provided in the second reaction tank 2 are similar to those of the first reaction tank 1, so that the present invention will be described in detail only with respect to the heat sink 13 and the stirring device in the first reaction tank 1. The bottom of the first reaction tank 1 is provided with an input end 12 communicated with the first reaction chamber 11, and the bottom of the second reaction tank 2 is provided with an output end 22 communicated with the second reaction chamber 21. The stirring device comprises a motor 143, a rotating shaft 141 and a stirring sheet 142, the stirring sheet 142 is arranged on the rotating shaft 141, and the motor 143 drives the rotating shaft 141 to rotate, so that the stirring sheet 142 can stir the materials in the first reaction chamber 11.
Referring to fig. 4, in some embodiments of the present invention, the first reaction tank may further include a motor 143 ' for driving the first reaction chamber 11 to rotate, the motor 143 ' is connected to the reaction chamber 11 via a rotating shaft 141 ', and the motor 143 ' drives the rotating shaft 141 ' to rotate so as to achieve a stirring effect on the first reaction chamber 11.
As shown in fig. 3, the heating device 15 includes a plurality of heating lamps vertically disposed in the first reaction tank 1 and annularly distributed around the first reaction chamber 11; in the embodiment of the present invention, the heating device 15 includes a first heating lamp 1511, a second heating lamp 1512, a third heating lamp 1513, a fourth heating lamp 1514, a fifth heating lamp 1511 ', a sixth heating lamp 1512', a seventh heating lamp 1513 ', an eighth heating lamp 1514', and the lamp control device 152; in some embodiments of the invention, the heating device 15 operates in two modes: (1) the lamp control device 152 controls the first heating lamp 1511, the third heating lamp 1513, the fifth heating lamp 1511 ', and the seventh heating lamp 1513' to operate, and the remaining heating lamps do not operate. (2) The lamp control device 152 controls the operation of all the heating lamps. It is obvious that the different operation modes of the heating device 15 of the present invention are designed to have different heating efficiencies, and those skilled in the art can understand that the heating power of each heating lamp of the present invention can be adjusted, and the heating efficiency of the above operation mode (2) is significantly greater than that of the operation mode (1) on the premise that the heating powers of all the heating lamps are the same. The heating means 15 installed in the second reaction tank 2 is the same as that of the first reaction tank 1, and the present invention will not be described in detail herein.
As shown in fig. 5 and fig. 6, the heat dissipation device 13 includes a heat dissipation baffle 131 and a heat dissipation fan 132, the heat dissipation baffle 131 includes a fixed plate 1311, a rotating column 1312, and a rotating plate 1313; the heat dissipation blocking sheet 131 is arranged below the heat dissipation fan 132, the fixed sheet 1311 and the rotating sheet 1313 are respectively connected to the rotating column 1312, wherein when the heat dissipation fan 132 does not work, the fixed sheet 1311 and the rotating sheet 1313 can completely block the ventilation opening to prevent dust from entering the first reaction chamber 11, and when the heat dissipation fan 132 works, the rotating sheet 1313 is driven by the rotating column 1312 to rotate, so that the first reaction chamber 11 is communicated with the heat dissipation fan 132.
Specifically, the fixed plate 1311 is connected to the rotation column 1312 at one end thereof, and fixed to the inner wall of the first reaction chamber 11 at the other end thereof, and the rotation plate 1313 is connected to a part of the rotation column 1312 'so that the rotation plate 1313 is rotated by the rotation column 1312' when a rotation command is received.
In some embodiments of the present invention, the heat dissipation blocking plate 131 is further provided with a vent hole to ensure that the atmospheric pressure on the first reaction tank 1 and the second reaction tank 2 is always the same as the outside.
As shown in FIG. 7, the present invention is provided with a control system to solve the problems of complicated means, low nucleic acid amplification efficiency and low intelligence degree in the existing nucleic acid amplification process. The control system comprises a control group and a controlled element. Wherein, the control group comprises a controller 10 and a control panel (not shown in the figure), the controller 10 and the control panel can be disposed on the nucleic acid amplification apparatus of the present invention, or disposed at other places beneficial to the control of the staff, and the controller 10 is electrically connected to the control panel. In some embodiments of the present invention, a signal transceiver may be further disposed on the control panel and the controller 10, so that the control panel and the controller 10 can access a local area network or the internet. The controller 10 may be provided on the nucleic acid amplification apparatus, and the control panel may remotely control the operation of the controller 10 through internet communication. The controlled elements are respectively electrically connected with the controller 10, and comprise a first temperature sensor group 101, a second temperature sensor group 102, a first flow rate sensor group 103, a second flow rate sensor group 104, a third flow rate sensor group 105, a first liquid level sensor group 106, a second liquid level sensor group 107, a lamp control device 152, a heat radiation fan 132, a first control valve 4, a pump body 7, a second control valve 8, a flow limiting valve 9, a rotating column 1312, a motor 143 and an output control valve 52.
Specifically, the first temperature sensor group 101 is disposed in the first reaction chamber 11 to detect the real-time temperature T1 in the first reaction chamber 11; the second temperature sensor group 102 is arranged in the second reaction chamber to detect the real-time temperature T2 in the second reaction chamber 21; the first flow sensor group 103 is arranged on the diversion pipeline 3 and at the front end of the intermediate output device 5 to detect the real-time flow rate V1 of the material flowing out of the first reaction chamber 11; a second flow sensor group 104 is arranged on the output control pipeline 53 to detect the real-time flow rate V2 of the intermediate; the third flow sensor group 105 is arranged on the return pipeline 6 to detect the real-time flow velocity V3 of the material flowing out of the second reaction chamber 21; the first liquid level sensor group 106 is arranged in the first reaction chamber 11 to detect the real-time liquid level height H1 of the first reaction chamber 11; the second liquid level sensor group 107 is disposed in the second reaction chamber 21 to detect the real-time liquid level height H2 of the second reaction chamber 21. The sensor group can be one sensor or a plurality of same sensors, the sensors are distributed according to a certain array, and the value obtained by the sensor group is the average value of the values measured by the sensors.
With continued reference to FIGS. 1, 2 and 7, the present invention provides a method for optical excitation and detection of a nucleic acid amplification apparatus, the method comprising:
step 1, injecting nucleic acid into a first reaction cavity 11;
step 2, the heating device works to enable the nucleic acid in the first reaction cavity 11 to be heated and split, the first temperature sensor group starts to detect the temperature in the first reaction cavity 11, when the first temperature sensor group 101 detects that the temperature in the first reaction cavity 11 is between 85 and 95 ℃ for the first time, the controller 10 starts to time, and the first control valve 4 is opened after the controller 10 times for 5 to 8 minutes, so that the split nucleic acid enters the second reaction cavity 21 through the flow guide pipeline 3;
step 3, the second liquid level sensor group 107 starts to detect the liquid level height in the second reaction cavity, when the real-time liquid level height H2 detected by the second liquid level sensor group 107 meets the first liquid level standard value, the intermediate output device 5 starts to work, the output control valve 52 is opened, so that the intermediate in the output storage bin 51 flows into the second reaction cavity 21, and the primers, the buffer solution and the dNTP are stored in the output storage bin 51;
step 4, the second liquid level sensor group 107 continues to detect the liquid level height in the second reaction chamber 21, and when the real-time liquid level height H2 detected by the second liquid level sensor group 107 meets a second liquid level standard value, the controller 10 controls the heat dissipation device to start working to cool the second reaction chamber 21;
step 5, the second temperature sensor group 101 starts to work, when the second temperature sensor group 101 detects that the temperature in the second reaction cavity 21 is reduced to 50 ℃ for the first time, the heating device in the second reaction cavity 21 starts to work, and the heating device and the heat dissipation device 13 work together to maintain the temperature in the second reaction cavity 21 to be 45-55 ℃;
when the second temperature sensor group 102 detects that the temperature in the second reaction chamber 21 is reduced to 50 ℃ for the first time, the controller 10 starts timing, the controller 10 controls the second control valve 8 to be opened after timing for 1-3 minutes, and simultaneously the controller 10 also controls the pump body 7 to work so as to convey the nucleic acid in the second reaction chamber 21 back to the first reaction chamber 11;
and 6, setting the steps 1 to 5 as a cyclic reaction, and repeating the cyclic reaction for 20-30 times.
Specifically, the liquid level values stored in the controller 10 include:
the first liquid level standard values H11, H12 and H13, and the like until the 20 th to 30 th standard liquid level values;
second liquid level standard values H21, H22, H23 and the like until the 20 th to 30 th standard liquid level values;
limit level H0;
the calculation method of the first liquid level standard value comprises the following steps: h11 is H10- Δ 1, H1n is H2(n-1) - Δ 1- Δ 2, Δ 1 is the loss of the material flowing through the diversion pipeline 3, Δ 2 is the loss of the material flowing through the return pipeline 6, and n is a positive integer greater than or equal to 2;
the method for calculating the second liquid level standard value comprises the following steps: h2n ═ H1n + G, G is the height of the liquid level of the intermediate flowing into the second reaction chamber 21, and n is a positive integer equal to or greater than 1.
When the real-time liquid level H1 detected by the first liquid level sensor 106 or the real-time liquid level H2 detected by the second liquid level sensor 107 exceeds the limit liquid level H0, the reaction is stopped, and the materials in the first reaction chamber 11 and the second reaction chamber 21 leave the nucleic acid amplification instrument through the output end 22.
Further, the controller 10 calculates the flow rates of the diversion pipeline 3, the output control pipeline 53 and the return pipeline 6, and the flow rate calculation formula is as follows:
wherein S is the cross-sectional area of the pipeline, V is the real-time flow rate of the material, and T is the time of flowing through the pipeline;
substituting the cross-sectional area S1 of the diversion pipeline 3, the real-time flow velocity V1 measured by the first flow velocity sensor group 103 and the time T1 of the material passing through the first flow velocity sensor group 103 into a flow calculation formula to calculate a first flow Q1;
substituting the cross-sectional area S2 of the output control pipeline 53, the real-time flow velocity V2 measured by the second flow velocity sensor group 104 and the time T2 of the material passing through the second flow velocity sensor group 104 into a flow calculation formula to calculate a second flow Q2;
substituting the cross-sectional area S3 of the return pipeline 6, the real-time flow velocity V3 measured by the third flow velocity sensor group 105 and the time T3 of the material passing through the third flow velocity sensor group 105 into a flow calculation formula to calculate a third flow Q3;
the working personnel can judge the working state of the nucleic acid amplification instrument by detecting the first flow Q1, the second flow Q2 and the third flow Q3, and the detection formula comprises the following steps: k (Q1+ Q2) ═ Q3, k is the reaction coefficient.
Specifically, the first reaction chamber 11 may further contain therein an undivided nucleic acid and an enzyme for dissociating the nucleic acid into single strands. In some embodiments of the present invention, deoxynucleotide triphosphates are present in the first reaction chamber 11 for dissociating nucleic acids into single strands, and sufficient deoxynucleotide triphosphates are present in the first reaction chamber to ensure that these nucleotides are sufficient to allow the nucleic acids to be cleaved 20 to 30 times.
Specifically, when the intermediate output device 5 starts operating; the controller 10 controls the output control valve 52 to be opened to flow the intermediate in the output storage chamber 51 into the second reaction chamber 21; the output storage bin 51 stores primers, buffer solution and dNTP; in some embodiments of the invention, the primer is predominantly TaqDNA polymerase enzyme and the buffer comprises KCI, Mg2+Gelatin, non-ionic detergent, etc., dNTPs including: dATP, dTTP, dGTP and dCTP.
Specifically, the intermediate material in the output material storage bin 51 entering the second reaction chamber 21 each time is determined by the first liquid level standard value, and the controller 10 calculates the specific amount of the intermediate material flowing into the second reaction chamber 21 according to the first liquid level standard values H11, H12, H13, and so on until the values of the 20 th to 30 th standard liquid level values, and further calculates the corresponding intermediate material liquid level height G.
In some embodiments of the present invention, in order to improve the working effect of the primer, a temperature control manner of the second reaction chamber 21 is further provided, which includes: under the combined action of the cooling fan 132 and the heating device, the temperature is reduced and raised, and the temperature is reduced by 1 ℃ each time until the temperature is reduced to 45 ℃, and then the cooling fan 132 stops working; after the temperature gradually rises again, the temperature reduction and rise control operation is repeated for 2-3 times.
Specifically, when the real-time temperature T2 measured by the second temperature sensor group 102 for the first time is 55 ℃, the controller 10 controls the heat dissipation fan 132 to operate, when the real-time temperature in the second reaction chamber 21 is reduced to 54 ℃ by the heat dissipation fan 132, the heat dissipation fan 132 stops operating for 5 to 10 seconds, and then continues operating, and the above-mentioned operation is repeated until the real-time temperature T2 in the second reaction chamber 21 is reduced to 45 ℃, the heat dissipation fan 132 completely stops operating, and the controller 10 controls the heating device to perform a temperature raising operation. When the real-time temperature T2 measured by the second temperature sensor group 102 is 55 ℃, stopping heating, and at this time, restarting the cooling fan 132 to continue the cooling operation; the cooling fan 132 and the heating device perform the above operations for 2-3 cycles, thereby improving the working effect of the primers.
In some embodiments of the present invention, the mixture further comprises a monoclonal antibody of TaqDNA polymerase, and when the temperature in the second reaction chamber 21 is increased to a temperature that is high enough to denature and inactivate the antibody, the antibody neutralizes the activity of TaqDNA polymerase, and when the temperature is increased sufficiently, the antibody is inactivated and the amplification reaction is started.
In some embodiments of the present invention, the input end 12 and the output end 22 are also provided with control valves, and the input end 12 can be connected to a nucleic acid material tank, and the output end 22 is connected to a nucleic acid generation tank, and at this time, the controller 10 and the control panel of the system are both connected to the internet, so that the whole nucleic acid amplification process can be controlled anytime and anywhere by only putting the nucleic acid material into the nucleic acid material tank in advance for storage.
It can be understood by those skilled in the art that the control panel herein may be a control panel for man-machine interaction in industry, and may also be other electronic devices such as a mobile phone, a computer, a tablet computer, and the like.
With continued reference to fig. 1 and 2, the present invention is also provided with a fluorescence detection unit that includes a first fluorescence detector and a second fluorescence detector. Specifically, a first sampling port 31 is provided on the drainage tube 3, and a probe (not shown in the figure) is provided at the first sampling port 31 of the first fluorescence detector to detect a sample passing through the drainage tube 3; the return pipe 6 is provided with a second sampling port 32, and the second fluorescence detector is provided with a probe (not shown in the figure) at the second sampling port 32 to detect the sample passing through the return pipe 6.
The controller 10 is also in communication with the first fluorescence detector and the second fluorescence detector; the first fluorescence detector detects the real-time data N1The real-time data N detected by the second fluorescence detector is transmitted to the controller 102Transmitted to the controller 10, the controller 10 processes the real-time data N1And real-time data N2The processing method of the controller 10 includes calculating a real-time system stability value Δ s, where Δ s ═ N2-N1. The controller 10 further stores a measurement interval K, and the controller 10 compares the real-time system stability value Δ s with the measurement interval K.
Specifically, in each reaction cycle, real-time data N detected by the first fluorescence detector1Real-time data N detected by a second fluorescence detector for the number of nucleic acids in the secondary reaction before the amplification2Ideally, the real-time data N measured each time is the number of amplified nucleic acid divisions in the cycle2Should be real-time data N1Twice the value of (1), i.e. N2-N1＝N1. In the invention, considering the influence of the factors such as pipeline loss and uneven temperature distribution of materials, the range of the measurement interval K is set as [2N1/3，N1](ii) a When the system stability value Δ s calculated by the controller 10 is within the measurement interval K, the controller 10 indicates that the check calculation amplifier is working normally, and when the system stability value Δ s is not within the measurement interval K, the controller 10 enters a diagnostic procedure.
Specifically, the diagnostic procedure includes:
case one, when the system steady value Δ s calculated by the controller 10 is less than 2N1At time/3, the controller 10 continues to collect the next measured real-time data N1' and real-time data N2', and N1' and N1Making a difference or dividing N2' and N2And (6) making a difference value. It will be understood by those skilled in the art that the controller 10 operates to detect two adjacent reactionsAnd performing difference on the real-time data. If the difference is within the measurement interval K, it is confirmed that all amplification reactions are normal, and the error of the stable value Δ s calculated by the controller 10 may be misjudged due to the detection of reaction residue by the fluorescence detector.
Second, when the system stability value Δ s calculated by the controller 10 is greater than N1The time controller 10 continues to collect the next measured real-time data N1' and real-time data N2', and N1' and N1Making a difference or dividing N2' and N2And (6) making a difference value. It will be understood by those skilled in the art that the controller 10 operates to difference the real-time data detected by two adjacent loop reactions. If the difference is within the measurement interval K, it is confirmed that all amplification reactions are normal, and the error of the stable value Δ s calculated by the controller 10 may be misjudged due to the detection of reaction residue by the fluorescence detector.
When the first case and the second case occur three or more times in succession, the controller 10 recognizes that a problem occurs in the fluorescence detector and the diagnostic result is replacement of the fluorescence detector.
Case three, when N1' and N1The difference is not within the measurement interval K and N2' and N2When the difference is not within the measurement interval K, the controller 10 sends out an alarm prompt so that the temperature of the materials flowing through the diversion pipeline 3 and the backflow pipeline 6 can be detected by the staff; the staff can carry out temperature detection through the first sampling port 31 and the second sampling port 32, namely whether the temperature in the first reaction tank 1 meets the activity of the lyase or not and whether the temperature in the second reaction tank 2 meets the activity of TaqDNA polymerase or not are detected; if the temperatures in the first reaction tank 1 and the second reaction tank 2 do not satisfy the requirement of the activity of the corresponding enzymes in the reaction tanks, the temperatures in the first reaction chamber 11 and the second reaction chamber 21 are increased by reducing the rotation frequency of the cooling fan 132 and replacing the working mode of the heating lamps, thereby ensuring the activity of the enzymes. In some embodiments of the present invention, the maximum upper limit of the temperature in the first reaction chamber 11 may be increased to 100 ℃ and the maximum upper limit of the temperature in the second reaction chamber may be increased to 60 ℃.
The invention has been described by way of the above embodiments, but it is to be understood that the above embodiments are for purposes of illustration and description only and are not intended to limit the invention to the described embodiments. It will be appreciated by those skilled in the art that many variations and modifications may be made to the teachings of the invention, which fall within the scope of the invention as claimed.
Claims (10)
1. A method for optically exciting and detecting a nucleic acid amplification instrument, comprising:
step 1, injecting nucleic acid into a first reaction cavity;
step 2, the heating device works to enable the nucleic acid in the first reaction cavity to be heated and split, the first temperature sensor group starts to detect the temperature in the first reaction cavity, when the first temperature sensor group detects that the temperature in the first reaction cavity is 85-95 ℃ for the first time, the controller starts to time, and after the controller times for 5-8 minutes, the first control valve is opened, so that the split nucleic acid enters the second reaction cavity through the flow guide pipeline;
step 3, the second liquid level sensor group starts to detect the liquid level height in the second reaction cavity, when the real-time liquid level height H2 detected by the second liquid level sensor group meets a first liquid level standard value, the intermediate output device starts to work, the output control valve is opened, so that the intermediate in the output storage bin flows into the second reaction cavity, and the primer, the buffer solution and the dNTP are stored in the output storage bin;
step 4, the second liquid level sensor group continues to detect the liquid level height in the second reaction cavity, and when the real-time liquid level height H2 detected by the second liquid level sensor group meets a second liquid level standard value, the controller controls the heat dissipation device to start working so as to cool the second reaction cavity;
step 5, the second temperature sensor group starts to work, when the second temperature sensor group detects that the temperature in the second reaction cavity is reduced to 50 ℃ for the first time, the heating device in the second reaction cavity starts to work, and the heating device and the heat dissipation device work together to maintain the temperature in the second reaction cavity to be 45-55 ℃;
when the second temperature sensor group detects that the temperature in the second reaction cavity is reduced to 50 ℃ for the first time, the controller starts timing, controls a second control valve to be opened after timing for 1-3 minutes, and simultaneously controls the pump body to work so as to convey the nucleic acid in the second reaction cavity back to the first reaction cavity;
and 6, analyzing the data acquired by the fluorescence detection unit by the controller, and calculating the relationship between a system stable value delta s and a measurement interval K to select a corresponding working mode:
when the system stability value delta s is within the measurement interval K, the controller continuously keeps a normal working mode;
when the system stable value deltas is not in the measurement interval K, the controller enters a diagnostic program;
and 7, setting the steps 1 to 6 as a cyclic reaction, and repeating the cyclic reaction for 20-30 times.
2. The optical excitation and detection method for a nucleic acid amplification instrument according to claim 1, wherein the flow guide pipeline connects the first reaction chamber and the second reaction chamber, the first control valve and the intermediate output device are both disposed on the flow guide pipeline, and the first reaction chamber and the second reaction chamber are disposed in the first reaction tank and the second reaction tank, respectively;
wherein the level of the first reaction chamber is higher than the level of the second reaction chamber; the diversion pipeline is arranged between the first reaction cavity and the second reaction cavity with a certain gradient, so that the solution in the first reaction cavity can enter the second reaction cavity through the diversion pipeline under the action of gravity;
the tops of the first reaction tank and the second reaction tank are provided with ventilation openings, and the ventilation openings are provided with the heat dissipation devices; the bottom of the first reaction tank is provided with an input end communicated with the first reaction cavity, and the bottom of the second reaction tank is provided with an output end communicated with the second reaction cavity;
heating devices are arranged in the first reaction tank and the second reaction tank, and the heating devices in the first reaction tank are arranged in a gap region enclosed by the outer surface of the first reaction cavity and the inner surface of the first reaction tank; the heating device in the second reaction tank is arranged in a gap interval enclosed by the outer surface of the second reaction cavity and the inner surface of the second reaction tank; the heating device comprises at least two heating lamps and a lamp control group, the lamp control group is electrically connected with the heating lamps, the lamp control group is used for controlling the heating power of the heating lamps, and the heating lamps are vertically arranged in a gap interval and surround the first reaction chamber and the second reaction chamber;
a backflow pipeline is connected with the first reaction cavity and the second reaction cavity, and materials in the second reaction cavity return to the first reaction cavity through the backflow pipeline; the device that is equipped with on the return line includes: the pump body, second control valve and restriction valve are followed by the material in the direction of second reaction chamber flow direction first reaction chamber, the sequence of arrangement of the device of installation on the backflow pipeline is in proper order: the second control valve, the pump body and the flow limiting valve;
the second control valve is arranged near an interface of the backflow pipeline and the second reaction cavity and used for controlling the connection and disconnection of the second reaction cavity and the backflow pipeline, the pump body is used for conveying materials in the backflow pipeline into the first reaction cavity, and the flow limiting valve is used for controlling the flow rate of the materials in the backflow pipeline;
first reaction chamber with all be equipped with agitating unit in the second reaction chamber, agitating unit includes motor, pivot and stirring piece, the stirring piece sets up in the pivot, motor drive the pivot rotates, so that the stirring piece is right first reaction chamber with the material in the second reaction chamber stirs.
3. The optical excitation and detection method of a nucleic acid amplification instrument according to claim 2, wherein the heat dissipation means comprises a heat dissipation blocking piece and a heat dissipation fan, the heat dissipation blocking piece comprises a fixing piece, a rotating column and a rotating piece;
wherein, the heat dissipation separation blade sets up the radiator fan below, the stationary blade with the rotor plate respectively with the rotation post links to each other when radiator fan is out of work, the stationary blade with the rotor plate can shelter from completely the vent to prevent that the dust from getting into first reaction chamber with second reaction chamber, work as radiator fan during operation, controller control the rotation post rotates, the rotor plate is in rotate under the drive of rotation post, so that second reaction chamber with the radiator fan intercommunication, controller control radiator fan work is in order to right the second reaction chamber cools down.
4. The optical excitation and detection method for a nucleic acid amplification instrument according to claim 3, wherein the heat dissipation plate is provided with a vent hole so that the atmospheric pressure on the first reaction tank and the second reaction tank is always the same as the outside.
5. The optical excitation and detection method for a nucleic acid amplification apparatus according to claim 2, wherein the detection unit comprises a first fluorescence detector and a second fluorescence detector; the first fluorescence detector detects the sample passing through the drainage pipeline, and real-time data N1 are detected; the second fluorescence detector detects the sample passing through the return pipeline and detects real-time data N2;
wherein Δ s ═ N2-N1The range of the measurement interval K is set to [2N ]1/3，N1]。
6. The optical excitation and detection method of a nucleic acid amplification apparatus according to claim 2, wherein the diagnostic procedure comprises:
the controller continuesCollecting real-time data N1' and real-time data N2', and N1' and N1Making a difference or dividing N2' and N2Making a difference value; wherein N is1' and N2' reflecting the real-time data detected by the detection unit for the next cycle;
and if the controller detects that the system stability value delta s is not in the range of the measurement interval K in the three continuous circulating reactions, the controller gives an alarm to enable a worker to detect the temperature of the material flowing through the diversion pipeline and the backflow pipeline.
7. The optical excitation and detection method for nucleic acid amplification instrument of claim 3 or 5, wherein the control system comprises a control group and a controlled element, the control group comprises the controller and a human-computer interaction panel, the controlled element is electrically connected to the controller, respectively, the controlled element comprises the first temperature sensor group, the second temperature sensor group, the first flow sensor group, the second flow sensor group, the third flow sensor group, a first liquid level sensor group, the second liquid level sensor group, the lamp control device, the heat dissipation fan, the first control valve, the pump body, the second control valve, the flow limiting valve, the rotary column and the output control valve;
wherein the first temperature sensor group is arranged in the first reaction cavity to detect the real-time temperature T1 in the first reaction cavity; the second temperature sensor group is arranged in the second reaction cavity to detect the real-time temperature T2 in the second reaction cavity; the first flow sensor group is arranged on the diversion pipeline and at the front end of the intermediate output device so as to detect the real-time flow rate V1 of the material flowing out of the first reaction cavity; the second flow sensor group is arranged on the output control pipeline to detect the real-time flow rate V2 of the intermediate; the third flow sensor group is arranged on the return pipeline to detect the real-time flow velocity V3 of the material flowing out of the second reaction cavity; the first liquid level sensor group is arranged in the first reaction cavity to detect the real-time liquid level height H1 of the first reaction cavity; the second liquid level sensor group is arranged in the second reaction cavity to detect the real-time liquid level height H2 of the second reaction cavity.
8. The optical excitation and detection method for a nucleic acid amplification apparatus according to claim 7, wherein the level value stored in the controller includes:
the first liquid level standard values H11, H12 and H13 are repeated until the 20 th to 30 th standard liquid level value is reached;
the second liquid level standard values H21, H22, H23 and the like till the 20 th to 30 th standard liquid level values;
limit level H0;
the calculation method of the first liquid level standard value comprises the following steps: h11 is H10- Δ 1, H1n is H2(n-1) - Δ 1- Δ 2, Δ 1 is the loss of material flowing through the diversion pipeline, Δ 2 is the loss of material flowing through the return pipeline, and n is a positive integer greater than or equal to 2;
the method for calculating the second liquid level standard value comprises the following steps: h2n ═ H1n + G, G is the height of the liquid level of the intermediate flowing into the second reaction chamber, and n is a positive integer equal to or greater than 1.
9. The optical excitation and detection method for a nucleic acid amplification instrument according to claim 8, wherein the reaction is stopped when the real-time liquid level H1 detected by the first liquid level sensor or the real-time liquid level H2 detected by the second liquid level sensor exceeds a limit liquid level H0, and the contents of the first reaction chamber and the second reaction chamber exit the nucleic acid amplification instrument through the output port.
10. The optical excitation and detection method for a nucleic acid amplification instrument according to claim 7, wherein the controller further calculates the flow rates of the diversion conduit, the output control conduit and the return conduit, and the flow rate calculation formula is as follows:
wherein S is the cross-sectional area of the pipeline, V is the real-time flow rate of the material, and T is the time of flowing through the pipeline;
substituting the cross-sectional area S1 of the diversion pipeline, the real-time flow velocity V1 measured by the first flow velocity sensor group and the time T1 of the material passing through the first flow velocity sensor group into a flow calculation formula to calculate a first flow Q1;
substituting the cross-sectional area S2 of the output control pipeline, the real-time flow velocity V2 measured by the second flow sensor group and the time T2 of the material passing through the second flow sensor group into a flow calculation formula to calculate a second flow Q2;
substituting the cross-sectional area S3 of the return pipeline, the real-time flow velocity V3 measured by the third flow velocity sensor group and the time T3 of the material passing through the third flow velocity sensor group into a flow calculation formula to calculate a third flow Q3;
the working personnel can judge the working state of the nucleic acid amplification instrument by detecting the first flow Q1, the second flow Q2 and the third flow Q3, and the detection formula comprises the following steps: k (Q1+ Q2) ═ Q3, k is the reaction coefficient.
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