CN114480597A - Droplet detection method and device based on digital PCR - Google Patents
Droplet detection method and device based on digital PCR Download PDFInfo
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- CN114480597A CN114480597A CN202210148423.2A CN202210148423A CN114480597A CN 114480597 A CN114480597 A CN 114480597A CN 202210148423 A CN202210148423 A CN 202210148423A CN 114480597 A CN114480597 A CN 114480597A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a droplet detection method and a droplet detection device based on digital PCR, wherein the method comprises the following steps: controlling the sample injection needle to perform needle inserting operation on the sample micro-droplet test tube, and detecting whether the sample injection needle reaches a preset position; sampling the sample microdroplets when the sampling needle reaches a preset position; the sampled sample droplet is transported to the detection chip through the pipeline by the negative pressure formed by the flow velocity difference and is detected. Sampling is carried out again when the syringe needle is in preset position and is guaranteed that syringe needle and test tube are in suitable distance, and the syringe needle is too high or low excessively from test tube bottom position when can avoiding direct sampling to lead to sample droplet sampling difficulty or remain too much the condition and appear. The negative pressure formed by the flow velocity difference can suck the sample droplet directly to the detection chip without passing through other elements in the pipeline, and the integrity of the sample droplet is ensured. The bubble sensor is used for monitoring the position of the sample micro-droplet and adjusting the detection time in real time, so that the accuracy and the stability of the detection are further improved.
Description
Technical Field
The invention relates to the technical field of droplet detection, in particular to a droplet detection method and a droplet detection device based on digital PCR.
Background
The microfluidic droplet technology is a brand new technology for controlling micro-volume liquid developed on the basis of microfluidics. The liquid drops generated by the technology are micro-Reaction units with the volume of nanoliters or even picoliters, and are applied to the fields of protein crystallization, cell analysis, rapid enzyme Reaction kinetics research, digital PCR (Polymerase Chain Reaction), gene sequencing and the like. The microfluidic droplet platform can rapidly and stably generate droplets with uniform sizes, and compared with the traditional microplate method, the screening flux of the microfluidic droplet technology can be improved by 1000 times, so that the microfluidic droplet technology has great potential to become a next generation ultrahigh flux screening platform.
Digital PCR is a recent quantitative technique, which is an absolute quantitative method for nucleic acid quantification based on a single-molecule PCR method for counting. The method mainly adopts a micro-fluidic chip method or a microdroplet method in the current analytical chemistry popular research field to disperse a large amount of diluted nucleic acid solution into micro-reactors or microdroplets of the chip, wherein the number of nucleic acid templates in each reactor is less than or equal to 1. Thus, after PCR cycling, a reactor with a nucleic acid molecule template will give a fluorescent signal, and a reactor without a template will have no fluorescent signal. Based on the relative proportions and the volume of the reactor, the nucleic acid concentration of the original solution can be deduced.
When the microdroplets are detected, a sample introduction system is required to extract microdroplet samples to a detection area, and then the microdroplet samples on the detection area are detected through optical detection equipment; the height of the traditional sample introduction system between the sample introduction needle and the sample test tube fluctuates, and if the traditional sample introduction system walks a fixed distance, the sample introduction needle and the test tube are stuck too close and cannot suck samples, or the distance between the sample introduction needle and the test tube is large, so that excessive residues are caused. The sample inlet pipeline is usually provided with a switching valve for controlling on-off and switching direction, so that the droplets can pass through more pipeline paths and pipeline connection sections before entering a detection area, great damage is easily caused to the droplets, and the droplets are broken, mixed or incompletely absorbed in the conveying process, so that the detection is difficult and the result is inaccurate. Due to the fact that the volumes of the samples to be detected are different to a certain extent, detection time is insufficient or overlong. Meanwhile, due to the complexity of the liquid path system, the problems of high failure rate, difficult maintenance and the like exist in the PCR detection instrument.
Disclosure of Invention
The purpose of the invention is: the method and the device for droplet detection based on digital PCR are provided, and the accuracy of droplet detection is improved.
In order to achieve the above object, the present invention provides a droplet detecting method based on digital PCR, comprising: controlling the sample injection needle to perform needle inserting operation on the sample micro-droplet test tube, and detecting whether the sample injection needle reaches a preset position;
sampling a sample droplet in the sample droplet test tube when the sample needle reaches the preset position;
and transmitting the sampled sample droplet to a detection chip through a pipeline by virtue of negative pressure formed by the flow velocity difference, so that the detection chip detects the sample droplet.
Further, the control advances to carry out the operation of inserting the needle to the sample microdroplet test tube to whether detect the injection needle and reach preset position, specifically do:
controlling the sample injection needle to perform needle descending operation on the sample droplet test tube, and detecting whether the sample injection needle is positioned at the bottom of the sample droplet test tube according to data collected by the pressure sensor; the pressure sensor is arranged on a sampling arm of the sample injection needle;
when the sample injection needle does not reach the bottom of the sample microdroplet test tube, controlling the sample injection needle to continue to downwards probe until the bottom of the sample microdroplet test tube is reached;
when the sample injection needle is positioned at the bottom of the sample microdroplet test tube, performing sample suction through a sample injection hole on the side surface of the sample injection needle; when the sample injection needle is positioned at the bottom of the sample microdroplet test tube, the sample injection needle can be lifted to a preset position according to preset lifting parameters, and samples are sucked through the sample injection hole in the bottom surface of the sample injection needle.
Further, the sampled sample droplet is transported to the detection chip through a pipeline by negative pressure formed by flow velocity difference, specifically:
the first injector is positioned at the input end of the detection chip and is controlled to input detection oil to the detection chip through a pipeline;
the second injector is positioned at the output end of the detection chip and is controlled to extract the detection oil and the sample microdroplet from the output end of the detection chip;
the sample droplet is transported to the detection chip through a conduit by a negative pressure created by a difference in flow rate between the first syringe and the second syringe.
Further, before controlling the sample injection to perform a needle inserting operation on the sample droplet test tube, the method further includes:
before controlling the sample injection needle to perform a needle-down operation on the sample droplet test tube, injecting air from the sample injection needle port by using a power element so that the sample droplet can be separated from the detection oil in the pipeline when being conveyed to the detection chip.
Further, before the sampled sample droplet is transported to the detection chip through the pipeline, the method further includes:
controlling the sampled sample microdroplet to pass through a bubble sensor so that the bubble sensor detects whether the sampled sample microdroplet is conveyed to the detection chip or not, and monitoring the sample introduction condition of the sampled sample microdroplet in real time;
adjusting the detection time of the detection chip according to the monitoring condition of the sampled sample droplet by the bubble sensor.
Furthermore, an electromagnetic valve is respectively arranged between the first injector and the detection chip and between the second injector and the detection chip; the electromagnetic valve is used for controlling the on-off state of the liquid path and the direction conversion of the pipeline.
Further, the structure of the detection chip specifically includes:
the detection chip comprises a threaded joint and a pipeline; wherein, the edge of the pipeline is of a flanging structure;
the pipeline of the detection chip is connected with the detection chip through a threaded joint;
and the screwed joint compresses the flanging structure to the detection chip.
Further, the embodiment of the present invention also provides a droplet detecting apparatus based on digital PCR, including: the device comprises a control module, a sampling module and a detection module;
the control module is used for controlling the sample injection needle to carry out needle inserting operation on the sample micro-droplet test tube and detecting whether the sample injection needle reaches a preset position;
the sampling module is used for sampling the sample microdroplets in the sample microdroplet test tube when the sample injection needle reaches the preset position;
the detection module is used for transmitting the sampled sample microdroplets to a detection chip through a pipeline by virtue of negative pressure formed by flow speed difference, so that the detection chip detects the sample microdroplets.
Further, control module control advances kind and carries out the operation of inserting the needle to sample droplet test tube to whether detect the needle of advancing and arrive preset position, specifically do:
the control module controls the sample injection needle to perform needle inserting operation on the sample microdroplet test tube, and detects whether the sample injection needle is positioned at the bottom of the sample microdroplet test tube according to data collected by the pressure sensor; the pressure sensor is arranged on a sampling arm of the sample injection needle;
when the sample injection needle does not reach the bottom of the sample microdroplet test tube, controlling the sample injection needle to continue to downwards probe until the bottom of the sample microdroplet test tube is reached;
when the sample injection needle is positioned at the bottom of the sample microdroplet test tube, performing sample suction through a sample injection hole on the side surface of the sample injection needle; when the sample injection needle is positioned at the bottom of the sample microdroplet test tube, the sample injection needle can be lifted to a preset position according to preset lifting parameters, and then the sample is sucked through the sample injection hole in the bottom surface of the sample injection needle. Further, the detection module is configured to transmit the sampled sample droplet to a detection chip through a pipeline by using a negative pressure formed by a flow velocity difference, and specifically includes:
the first injector is positioned at the input end of the detection chip and is controlled to input detection oil to the detection chip through a pipeline;
the second injector is positioned at the output end of the detection chip and is controlled to extract the detection oil and the sample microdroplet from the output end of the detection chip;
the sample droplet is transported to the detection chip through a conduit by a negative pressure created by a difference in flow rate between the first syringe and the second syringe.
Compared with the prior art, the droplet detection method and the droplet detection device based on the digital PCR have the beneficial effects that: the embodiment of the invention controls the sample injection needle to perform needle inserting operation on the sample micro-droplet test tube, detects the position of the sample injection needle in the sample micro-droplet test tube, samples the sample micro-droplet when the sample injection needle is at the preset position, and transmits the sampled sample micro-droplet to the detection chip for detection through the negative pressure formed by the flow speed difference. By detecting the position of the sampling needle, sampling is carried out when the sampling needle is at a preset position, so that the sampling needle and the test tube are in a proper distance and a proper amount of sample microdroplets are obtained. After sampling, the sample microdroplets are directly sucked to the detection chip through negative pressure formed by flow speed difference, the sample microdroplets are ensured to completely enter the detection chip, and the accuracy and the stability of detection can be improved.
In addition, the detection chip of the embodiment of the invention adopts a threaded connection method, and a pipeline flanging technology is added on the basis of the mature ferrule joint sealing technology, so that the sealing performance of the pipeline and the chip is ensured. The method can ensure that the sealing effect can still be achieved when the size is reduced due to the fact that pipelines are in different batches. Need wait for glue to solidify when detecting the chip mounting with the mode of traditional viscose, must change with all pipelines that the chip is connected again during the change, need wait for glue to solidify the debugging of carrying on the instrument again after good simultaneously and compare, the simple and the convenience of operation effectively improve, and the operation requirement is lower.
Drawings
FIG. 1 is a schematic flow diagram of an embodiment of a digital PCR-based droplet detection method according to an embodiment of the present invention;
FIG. 2 is a schematic flow chart of a bubble sensor according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of an embodiment of a digital PCR-based droplet detection apparatus according to an embodiment of the present invention;
fig. 4 is a schematic structural diagram of a detection chip according to an embodiment of the present invention.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to the accompanying drawings, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Referring to fig. 1, fig. 1 is a schematic flow chart of an embodiment of a digital PCR-based droplet detection method provided by an embodiment of the present invention, as shown in fig. 1, the method includes steps 101 to 103, which are as follows:
step 101: and controlling the sample injection needle to perform needle inserting operation on the sample micro-droplet test tube, and detecting whether the sample injection needle reaches a preset position.
In an embodiment of the present invention, a pressure sensor is disposed on the sampling arm of the needle for detecting the position of the needle in the sample droplet test tube, and when the needle reaches the bottom of the sample droplet test tube, the sample is aspirated through the sample inlet hole on the side of the needle. When the sample injection needle is positioned at the bottom of the sample microdroplet test tube, the sample injection needle can be lifted to a preset position according to preset lifting parameters, and then the sample is sucked through the sample injection hole in the bottom surface of the sample injection needle.
As a preferable scheme of the embodiment of the invention, a moving shaft mechanism is further provided to be connected with the sample injection needle, and is used for lifting the sample injection needle to a proper position. When the pressure sensor detects that the sampling needle is positioned at the bottom of the sample micro-drop test tube, the sampling needle is lifted to a lifting parameter distance away from the bottom of the test tube according to the set lifting parameter. The lifting parameters are determined based on the particular sample droplet tube size, the amount of sample droplets aspirated, and the like. In the embodiment of the invention, the movable shaft mechanism controls the sample injection to perform the needle inserting operation, when the pressure sensor detects that the sample injection needle reaches the bottom of the test tube, the movable shaft mechanism stops moving downwards, the lifting parameter is 1mm, the movable shaft mechanism is lifted upwards by 1mm, and the distance between the sample injection needle and the bottom of the test tube is 1 mm. In the action of the sample suction of the lower needle of the sample injection needle, the method of firstly detecting the bottom and then lifting by the pressure sensor is adopted, so that the distance between the position of the needle opening and the bottom of the test tube can be adjusted according to corresponding lifting parameters under the condition that the test tube and a clamp for placing the test tube have dimension errors, and the situation that the height is not too high and the height is not too low is ensured.
As a preferred scheme of the embodiment of the invention, before the sample injection needle enters the sample micro-drop test tube for needle inserting operation, a small segment of air is injected from the sample injection needle port through the power element. In the digital PCR detection, the sample injection needle and the pipeline between the sample injection needle and the detection chip are filled with detection oil, and before the lower needle absorbs the sample microdroplets, a small segment of air is injected into the sample injection needle by using an injector so that the absorbed sample microdroplets and the detection oil between the pipelines are separated by a certain distance.
Step 102: sampling a sample droplet in the sample droplet test tube when the sample needle reaches the preset position.
When the moving shaft lifts the sample injection needle to a position which is away from the bottom of the sample droplet test tube by the lifting parameter, namely the sample injection needle is at a preset position, the sample droplet is sucked into a liquid path pipeline of the detection system through the sample injection needle.
Step 103: and transmitting the sampled sample droplet to a detection chip through a pipeline by virtue of negative pressure formed by the flow velocity difference, so that the detection chip detects the sample droplet.
In the embodiment of the invention, the input end and the output end of the detection chip are respectively provided with an injector, after sampling is finished, the injector at the input end of the detection chip inputs detection oil into the detection chip, the injector at the output end of the detection chip starts to suck the detection oil in the detection chip, and sample droplets in the pipeline are sucked into the detection chip through negative pressure formed by flow speed difference between the two injectors. When the sample droplet is sucked into the detection chip, the optical detection system detects the sample droplet, and the output end injector extracts the detection oil in the detection chip and the detected sample droplet.
As a preferable scheme of the embodiment of the invention, the input end injector, the output end injector and a pipeline in front of the detection chip are respectively provided with an electromagnetic valve for controlling the on-off state of a liquid path in the pipeline and reversing the pipeline. Meanwhile, in the embodiment of the invention, the sample micro-droplets do not need to pass through the electromagnetic valve, so that the risk of damage to the sample micro-droplets due to a plurality of sharp-angled structures in the electromagnetic valve is avoided. Considering that a complete fluid path system needs a plurality of solenoid valves to implement control, as an alternative to the embodiment of the present invention, a ten-way valve may be used instead of the solenoid valves.
As another preferable scheme of the embodiment of the invention, a bubble sensor is arranged in front of the input end of the detection chip and used for positioning the sample sucking position. Referring to fig. 2, fig. 2 is a schematic view of a working flow of a bubble sensor according to an embodiment of the present invention. Because the sample injection needle and the liquid path pipeline are filled with detection oil, according to the flowing characteristic of liquid in the pipeline (the flowing speed of the pipeline center is maximum), part of sample droplets can move in advance relative to a sample main body, and meanwhile, part of samples can enter other pipelines or injectors, so that the part of sample droplets cannot be detected, and the accuracy of PCR detection is influenced. The bubble sensor can ensure that the sample droplet is sucked to a specified pipeline position and is not influenced by the volume change of the sample droplet, the flow characteristic of the pipeline and the like. When the bubble sensor detects the first section of air injected into the sample injection needle in advance, whether the sample microdroplet sucks the pipeline to be detected or not is judged, the sample injection condition of the sample microdroplet is monitored in real time, and when the sample microdroplet is injected completely, the bubble sensor detects the air in the sample injection needle at the moment. When the bubble sensor detects the second section of air, the bubble sensor indicates that the sample droplet completely enters the detection chip for detection. The detection time can be adjusted in due time by monitoring the sample micro-droplet feeding condition through the bubble sensor. If the volume of the preset detection sample droplet is less than or equal to 50 microliters, the corresponding detection time is 100 seconds, and when the sample droplet is greater than 50 microliters, the detection time has elapsed for 100 seconds, but the sample droplet does not completely enter the detection chip at this time, so that the bubble sensor does not detect the second section of air yet, at this time, the mobile terminal receives the detection progress of the bubble sensor, sends a corresponding instruction to prolong the detection time, and other elements in the pipeline continue to execute the sample droplet detection operation until the bubble sensor detects the second section of air, so as to ensure that all the sample droplets enter the detection chip for detection. In addition, when the bubble sensor does not detect the second section of air after the detection time exceeds the specified value, the failure of the instrument in the droplet detection device can be checked as a judgment standard of the failure of the instrument, and the development of the droplet detection work is promoted.
According to the droplet detection method based on the digital PCR, the moving shaft is arranged to control the sample injection to perform needle inserting operation on the sample droplet test tube, the pressure sensor is arranged to detect the position of the sample injection needle in the sample droplet test tube, the sample injection needle is lifted to a position which is proper to the bottom of the test tube according to the lifting parameters to perform sample absorption, and the problem that when the size error exists in the test tube and a clamp for placing the test tube, the distance between the sample injection needle and the bottom of the test tube is too low or too high, so that the sampling difficulty or too much sampling is caused can be avoided. After the sample microdroplets are sucked, the sample microdroplets are directly adsorbed to the detection chip for detection through negative pressure formed by flow speed difference of the injectors at the two ends of the detection chip, the sample microdroplets can be ensured to completely enter the detection chip, and the accuracy and the stability of detection can be improved.
Example 2
Referring to fig. 3, fig. 3 is a schematic structural diagram of an embodiment of a droplet detecting device based on digital PCR according to an embodiment of the present invention, including: the device comprises a sample droplet 1, a moving shaft mechanism 2, a pressure sensor 3, a sampling arm 4, a sample injection needle 5, a bubble sensor 6, a detection chip 7, a waste liquid bottle 8, a first electromagnetic valve 9, a second electromagnetic valve 10, a first injector 11, a second injector 12 and an oil bottle 13.
The sample microdroplets 1 are stored in the test tube, and before the sample injection needle enters the sample microdroplets to be stored, any power element is used, in the embodiment of the invention, a small section of air is injected into the needle port of the sample injection needle by using an injector, so that when the sample injection needle performs sample suction, the sample microdroplets can not be mixed with detection oil in the sample injection needle and can be separated by a certain distance. After injecting the air, through removing 2 control sampling needle 5 entering sample microdroplet test tubes of axle mechanism, pressure sensor 3 sets up on the sampling arm 4 of being connected with sampling needle 5, a position for detecting sampling needle 5 in sample microdroplet test tube, after sampling needle 5 is located sample microdroplet test tube bottom, pressure sensor 3 sends the suggestion, it stops downstream to remove 2 axle mechanisms this moment, lifting parameter according to the backstage provides will advance 5 lifting of needle to apart from sample microdroplet test tube bottom lifting parameter distance department, be suitable for the preset position of sampling promptly.
When the needle 5 is in the predetermined position, suction of the sample droplet 1 through the needle 5 into the channel is started. After the sample droplet 1 is completely sucked, the switches of the first electromagnetic valve 9 and the second electromagnetic valve 10 are opened, the first injector 11 injects the detection oil to the input end of the detection chip, and the detection oil is stored in the oil bottle 13. At the same time, the second syringe 12 starts to draw the detection oil at the output end of the detection chip, and the sample droplet in the pipeline is sucked into the detection chip for detection through the negative pressure formed by the flow speed difference between the first syringe 11 and the second syringe 12. When the test is started, the second syringe 12 extracts the sample droplet and the test oil from the test chip and transfers the waste liquid generated during the test to the waste liquid bottle 8. As an alternative to the embodiments of the present invention, a ten-way valve may be used instead of a solenoid valve, reducing the complexity and failure rate of the instrument structure.
In the embodiment of the invention, a bubble sensor 6 is arranged in front of the input end of the detection chip and used for positioning the sample sucking position, so that the sample droplet 1 can be sucked to the specified pipeline position and is not influenced by the volume change of the sample droplet, the flow characteristics of the pipeline and the like.
As a preferable scheme of the embodiment of the invention, the detection chip 7 is connected with the pipeline through threads. Referring to fig. 4, fig. 4 is a schematic structural diagram of a detection chip according to an embodiment of the present invention, where the detection chip includes a chip clamp 1, a threaded joint 2, and a pipeline 3. The chip clamp 1 is used for placing a detection chip and plays a role in protection. The edge of the pipeline 3 is also provided with a flanging structure, and the flanging structure of the pipeline 3 is tightly pressed onto the detection chip by the threaded connector 2, so that the sealing connection between the detection chip and the pipeline is realized. The method for detecting the connection between the chip and the pipeline is to adopt the technology of flanging the pipeline on the basis of the existing mature sealing technology of the ferrule joint, and the technology of flanging the pipeline is combined with the technology of flanging the pipeline, so that the connection tightness between the pipeline and the chip is further improved.
The more detailed working principle and the process flow of the droplet detecting device of this embodiment can be, but are not limited to, those described in connection with example 1.
According to the droplet detection device based on the digital PCR, the detection chip is in threaded connection, and a pipeline flanging technology is added on the basis of a mature ferrule joint sealing technology, so that the sealing performance of a pipeline and the chip is guaranteed. The method can ensure that the sealing effect can still be achieved when the size is reduced due to the fact that pipelines are in different batches. Need wait for glue to solidify when detecting the chip mounting with the mode of traditional viscose, must change with all pipelines that the chip is connected again during the change, need wait for glue to solidify again and carry out the debugging of instrument and compare after good simultaneously, can effectively improve the simplicity and convenience of operation, and the operation requirement is lower.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and substitutions can be made without departing from the technical principle of the present invention, and these modifications and substitutions should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for droplet detection based on digital PCR, comprising:
controlling the sample injection needle to perform needle inserting operation on the sample micro-droplet test tube, and detecting whether the sample injection needle reaches a preset position;
sampling a sample droplet in the sample droplet test tube when the sample needle reaches the preset position;
and transmitting the sampled sample droplet to a detection chip through a pipeline by virtue of negative pressure formed by the flow velocity difference, so that the detection chip detects the sample droplet.
2. The method according to claim 1, wherein the controlling of the sample injection is performed to the sample droplet test tube by a needle insertion operation and whether the sample injection needle reaches a predetermined position is detected by:
controlling the sample injection needle to perform needle descending operation on the sample micro-droplet test tube, and detecting whether the sample injection needle is positioned at the bottom of the sample micro-droplet test tube according to data collected by the pressure sensor; the pressure sensor is arranged on a sampling arm of the sample injection needle;
when the sample injection needle does not reach the bottom of the sample microdroplet test tube, controlling the sample injection needle to continue to downwards probe until the bottom of the sample microdroplet test tube is reached;
when the sample injection needle is positioned at the bottom of the sample microdroplet test tube, performing sample suction through a sample injection hole on the side surface of the sample injection needle; when the sample injection needle is positioned at the bottom of the sample microdroplet test tube, the sample injection needle can be lifted to a preset position according to preset lifting parameters, and samples are sucked through the sample injection hole in the bottom surface of the sample injection needle.
3. A method for digital PCR-based droplet detection according to claim 1, wherein the sampled sample droplets are transported to the detection chip through a conduit by a negative pressure created by a difference in flow rates, in particular:
the first injector is positioned at the input end of the detection chip and is controlled to input detection oil to the detection chip through a pipeline;
the second injector is positioned at the output end of the detection chip and is controlled to extract the detection oil and the sample microdroplet from the output end of the detection chip;
the sample droplet is transported to the detection chip through the conduit by a negative pressure created by a difference in flow rate between the first syringe and the second syringe.
4. The method of claim 1, wherein before controlling the sample injection to perform a needle insertion operation on the sample droplet tube, the method further comprises:
before controlling the sample injection to perform a needle inserting operation on the sample droplet test tube, injecting air from the sample injection needle port by using a power element so that the sample droplet can be separated from the detection oil in the pipeline when being conveyed to the detection chip.
5. The method of claim 1, wherein the step of transporting the sampled sample droplet to the detection chip via a pipeline further comprises:
controlling the sampled sample microdroplet to pass through a bubble sensor so that the bubble sensor detects whether the sampled sample microdroplet is conveyed to the detection chip or not, and monitoring the sample introduction condition of the sampled sample microdroplet in real time;
adjusting the detection time of the detection chip according to the monitoring condition of the sampled sample droplet by the bubble sensor.
6. The method of claim 3, wherein a solenoid valve is disposed between each of the first and second syringes and the detection chip; the electromagnetic valve is used for controlling the on-off state of the liquid path and the direction conversion of the pipeline.
7. The digital PCR-based droplet detection method according to any one of claims 1 to 6, wherein the structure of the detection chip is specifically as follows:
the detection chip comprises a threaded joint and a pipeline; wherein, the edge of the pipeline is of a flanging structure;
the pipeline of the detection chip is connected with the detection chip through a threaded joint;
and the screwed joint compresses the flanging structure to the detection chip.
8. A digital PCR-based droplet detection apparatus, comprising: the device comprises a control module, a sampling module and a detection module;
the control module is used for controlling the sample injection needle to perform needle inserting operation on the sample micro-droplet test tube and detecting whether the sample injection needle reaches a preset position;
the sampling module is used for sampling the sample microdroplets in the sample microdroplet test tube when the sample injection needle reaches the preset position;
the detection module is used for transmitting the sampled sample microdroplets to a detection chip through a pipeline by virtue of negative pressure formed by flow speed difference, so that the detection chip detects the sample microdroplets.
9. The digital PCR-based droplet detection apparatus according to claim 8, wherein the control module is configured to control the needle inserting operation of the sample injection needle for the sample droplet test tube, and detect whether the sample injection needle reaches a predetermined position, specifically:
the control module controls the sample injection needle to perform needle inserting operation on the sample microdroplet test tube, and detects whether the sample injection needle is positioned at the bottom of the sample microdroplet test tube according to data collected by the pressure sensor; the pressure sensor is arranged on a sampling arm of the sample injection needle;
when the sample injection needle does not reach the bottom of the sample microdroplet test tube, controlling the sample injection needle to continue to downwards probe until the bottom of the sample microdroplet test tube is reached;
when the sample injection needle is positioned at the bottom of the sample microdroplet test tube, performing sample suction through a sample injection hole on the side surface of the sample injection needle; when the sample injection needle is positioned at the bottom of the sample microdroplet test tube, the sample injection needle can be lifted to a preset position according to preset lifting parameters, and a sample is sucked through the sample injection hole in the bottom surface of the sample injection needle.
10. The digital PCR-based droplet detection apparatus according to claim 8, wherein the detection module is configured to transmit the sampled sample droplet to the detection chip through a pipeline by using a negative pressure formed by a flow velocity difference, and specifically:
the first injector is positioned at the input end of the detection chip and is controlled to input detection oil to the detection chip through a pipeline;
the second injector is positioned at the output end of the detection chip and is controlled to extract the detection oil and the sample microdroplet from the output end of the detection chip;
the sample droplet is transported to the detection chip through a conduit by a negative pressure created by a difference in flow rate between the first syringe and the second syringe.
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| CN115414972A (en) * | 2022-08-08 | 2022-12-02 | 广东省科学院生物与医学工程研究所 | Portable coaxial focusing micro-droplet generation device and micro-droplet preparation method |
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