CN114958600A - Device and method for integrating nucleic acid extraction and detection - Google Patents

Abstract

The device comprises a reagent card main body, wherein the nucleic acid extraction and elution process can be completed through an extraction tube, a magnetic bead tube, a cleaning tube and an elution tube on the reagent card main body; the reagent card main body and the rotary valve are provided with a first passage and a second passage, and the communication state of the first passage and the second passage can be changed through the rotary valve; when the first path is switched on, the nucleic acid extract in the elution tube can be transferred to the nucleic acid collection tube; when the second channel is communicated, the nucleic acid extract in the nucleic acid collecting tube can be transferred to the PCR reaction tube, so that the nucleic acid can be transferred without interference through the sealing channel in different processes and the PCR reaction can be carried out in a sealed environment.

Description

A device and method for integrating nucleic acid extraction and detection

technical field

The present application relates to the technical field of nucleic acid amplification and detection, and in particular, to an integrated device and method for nucleic acid extraction and detection.

Background technique

Nucleic acid analysis is an important experimental method for studying molecular biology. In terms of experimental steps, the general nucleic acid analysis process includes several parts: sample pretreatment, nucleic acid extraction and PCR amplification detection.

There are different processing methods such as centrifugation, filtration, and grinding according to different samples to be tested. Due to the variety of processing methods, it is usually done semi-manually with the help of common laboratory equipment such as centrifuges and thermostats.

The nucleic acid extraction process requires the lysis, cleaning and purification of the nucleic acid after the sample pretreatment. Commonly used methods include boiling method, spin column method and magnetic bead method. Since the magnetic bead method is easy to automate integration, the current automated nucleic acid extraction instrument adopts Most of them are nuclear extraction methods by magnetic bead method. The samples after nucleic acid extraction need to go through the elution and concentration step, but the concentration is still low after elution and concentration, and most of them cannot be directly used for hybridization for subsequent analysis. section.

PCR amplification and detection can be completed by a quantitative PCR instrument.

With the development of self-industrial automation, the automation equipment required for the above three links, such as sample preparation instrument, nucleic acid extraction instrument, and quantitative PCR instrument, are constantly innovating. The sample is loaded into the quantitative PCR instrument for amplification, and almost all the transfer operation links in the middle need to be completed manually by the experimenter, and the operation efficiency is low. In recent years, some manufacturers have replaced human hands to complete the transfer of samples through manipulators. However, the combination of several discrete devices and robotic arms will make the system extremely large and complex, and the cost is extremely high. Generally, batch experiments are required. The efficiency of the entire experimental process still cannot be really improved. At the same time, in the existing transfer methods, whether it is manual transfer or robotic transfer, there is a greater risk of nucleic acid leakage and contamination.

SUMMARY OF THE INVENTION

In view of this, the purpose of this application is to provide a nucleic acid extraction and detection integrated device and method thereof, which are used to solve the problems of high cost, low efficiency and the risk of nucleic acid leakage in the existing sample transfer method in nucleic acid analysis.

In order to achieve the above technical purpose, the first aspect of the present application provides an integrated device for nucleic acid extraction and detection, including: a reagent card and a rotary valve;

The reagent card includes: a reagent card body, an extraction tube, a magnetic bead tube, a cleaning tube, an elution tube, a nucleic acid collection tube and a PCR reaction tube;

The extraction tube, the magnetic bead tube, the cleaning tube, the elution tube, the nucleic acid collection tube and the PCR reaction tube are all arranged on the main body of the reagent card;

The nucleic acid collection tube, elution tube and PCR reaction tube are all sealed and isolated from the outside;

A first passage sealed and isolated from the outside is provided between the nucleic acid collection tube and the elution tube;

A second passage sealed and isolated from the outside is provided between the nucleic acid collection tube and the PCR reaction tube;

The rotary valve is rotatably arranged on the reagent card body around the rotation center, and the rotary valve is used to control the opening and closing of the first passage and the opening and closing of the second passage.

Further, the first passage includes an elution tube through-hole, a nucleic acid tube through-hole, a first through-hole and a second through-hole, all of which are sealed and isolated from the outside;

The through hole of the elution tube is arranged on the main body of the reagent card and communicated with the elution tube;

The nucleic acid tube through-hole is arranged on the reagent card body and communicated with the nucleic acid collection tube;

The first through hole and the second through hole are both arranged on the rotary valve and communicate with each other;

The rotary valve is used to rotate and control the first through hole to communicate with the nucleic acid tube through hole and the second through hole to communicate with the elution tube through hole, so that the first passage is connected;

The second passage includes a PCR tube through-hole, the nucleic acid tube through-hole, the first through-hole and the second through-hole, which are sealed and isolated from the outside;

The PCR tube through hole is arranged on the main body of the reagent card and communicated with the PCR reaction tube;

The rotary valve is also used to control the first through hole to communicate with the nucleic acid tube through hole and the second through hole to communicate with the PCR tube through hole, so that the second passage is connected.

Further, the second passage further includes: a nucleic acid transfer through hole and a nucleic acid discharge through hole;

The nucleic acid discharge through hole is arranged on the main body of the reagent card and above the PCR reaction tube;

The nucleic acid transfer through hole is arranged on the main body of the reagent card, a communication groove is arranged between the bottom end and the PCR tube through hole, and a communication groove is arranged between the top end and the nucleic acid discharge through hole;

Each of the communication grooves is hermetically isolated from the outside.

Further, a communication groove is provided between the first through hole and the second through hole;

The communication groove is sealed off from the outside.

Further, a third passage is also included;

The third passage is communicated with the PCR reaction tube, and the third passage is used for adding reaction material to the PCR reaction tube;

The rotary valve is also used to control the opening and closing of the third passage.

Further, the third passage includes: a third through hole and a reaction material feeding through hole;

The reaction material feeding through hole is arranged on the main body of the reagent card and communicates with the PCR reaction tube;

the third through hole is arranged on the rotary valve;

The rotary valve is also used to control the communication between the third through hole and the reactant feed through hole, so that the third passage is connected.

Further, the third passage further includes: a reaction material discharge through hole and a reaction material transfer through hole;

The reaction material discharge through hole is arranged on the main body of the reagent card, and is located above the PCR reaction tube;

The reaction material transfer through hole is arranged on the main body of the reagent card, a communication groove is arranged between the bottom end and the reaction material feed through hole, and a communication groove is arranged between the top end and the reaction material discharge through hole groove;

Each of the communication grooves is hermetically isolated from the outside.

Further, the method of sealing and isolation is to cover with a sealing film.

A second aspect of the present application provides a method for integrating nucleic acid extraction and detection, which is applied to the device for integrating nucleic acid extraction and detection according to any one of the first aspects, and includes the following steps:

S1. Extract the magnetic beads in the magnetic bead tube into the extraction tube, and mix the magnetic beads with the nucleic acid extract in the extraction tube;

S2, extracting the mixed magnetic beads into the cleaning tube for cleaning;

S3, extracting the cleaned magnetic beads to an elution tube for magnetic separation to obtain a purified nucleic acid extract;

S4, rotating the rotary valve to make the first passage connected, so that the purified nucleic acid extract is transferred from the elution tube to the nucleic acid collection tube;

S5. Turn the rotary valve to turn on the second passage, so that the purified nucleic acid extract is transferred from the nucleic acid collection tube to the PCR reaction tube.

Further, the integrated device for nucleic acid extraction and detection also includes a third channel;

The third pathway is communicated with the PCR reaction tube;

The rotary valve is also used to control the opening and closing of the third passage;

Also includes:

S6. Turn the rotary valve to connect the third passage, so that the reaction material enters the PCR reaction tube through the third passage.

It can be seen from the above technical solutions that the present application provides an integrated device for nucleic acid extraction and detection and a method thereof. The device includes a reagent card main body. The tube can complete the extraction and elution process of nucleic acid; the main body of the reagent card and the rotary valve are provided with a first passage and a second passage, and the connection state of the first passage and the second passage can be changed through the rotary valve; when the first passage is turned on , the nucleic acid extract in the elution tube can be transferred to the nucleic acid collection tube; when the second channel is turned on, the nucleic acid extract in the nucleic acid collection tube can be transferred to the PCR reaction tube, so that the nucleic acids in different processes can be exchanged through the sealed channel. Non-interfering transfer and performing PCR reaction in a sealed environment can reduce costs and reduce aerosol pollution to the environment compared to automatic arm transfer, effectively solving the problem that the existing sample transfer method in nucleic acid analysis is expensive and expensive. Low efficiency and the risk of nucleic acid leakage.

Description of drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only These are some embodiments of the present application, and for those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort.

1 is a top view of the overall structure of a nucleic acid extraction and detection integrated device provided in an embodiment of the present application;

FIG. 2 is a top view of the main body of the reagent card in a nucleic acid extraction and detection integrated device provided in an embodiment of the present application;

3 is a bottom view of the main body of the reagent card in a nucleic acid extraction and detection integrated device provided in an embodiment of the present application;

4 is a cross-sectional view of the overall structure of an integrated device for nucleic acid extraction and detection provided in an embodiment of the present application;

FIG. 5 is a flowchart of a nucleic acid extraction and detection method provided in an embodiment of the present application;

In the figure: 1. Main body of reagent card; 2. Extraction tube; 3. Magnetic bead tube; 4. First cleaning tube; 5. Second cleaning tube; 6. Third cleaning tube; 7. Elution tube; 8. Nucleic acid Collection tube; 9. Rotary valve; 10. Reaction material feed through hole; 11. Reaction material second transfer hole; 12. Reaction material discharge through hole; 13. Nucleic acid discharge through hole; 14. Nucleic acid transfer 15. Second through hole; 16. Operational connection position; 17. First through hole; 18. Elution tube through hole; 19. Nucleic acid tube through hole; 20, Third through hole; 21, PCR tube through hole hole; 22. PCR reaction tube; 23. Magnetic sleeve rod.

Detailed ways

The technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, but not all of the embodiments. Based on the embodiments in the embodiments of the present application, all other embodiments obtained by persons of ordinary skill in the art without creative work fall within the scope of the claimed protection of the present application.

In the description of the embodiments of the present application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" The orientation or positional relationship indicated by ” etc. is based on the orientation or positional relationship shown in the accompanying drawings, which is only for the convenience of describing the embodiments of the present application and simplifying the description, rather than indicating or implying that the indicated device or element must have a specific orientation, It is constructed and operated in a specific orientation, and therefore should not be construed as a limitation on the embodiments of the present application. Furthermore, the terms "first", "second", and "third" are used for descriptive purposes only and should not be construed to indicate or imply relative importance.

In the description of the embodiments of the present application, it should be noted that, unless otherwise expressly specified and limited, the terms "installed", "connected" and "connected" should be understood in a broad sense, for example, it may be a fixed connection or a The interchangeable connection, or the integral connection, can be a mechanical connection, an electrical connection, a direct connection, an indirect connection through an intermediate medium, or an internal communication between two components. Those of ordinary skill in the art can understand the specific meanings of the above terms in the embodiments of the present application in specific situations.

The embodiment of the present application discloses a nucleic acid extraction and detection integrated device and a method thereof.

Referring to FIGS. 1 to 4 , an integrated device for nucleic acid extraction and detection provided in the embodiment of the present application includes: a reagent card and a rotary valve 9 . The reagent card includes: a reagent card body 1 , an extraction tube 2 , a magnetic bead tube 3 , a cleaning tube, an elution tube 7 , a nucleic acid collection tube 8 and a PCR reaction tube 22 . The extraction tube 2 , the magnetic bead tube 3 , the cleaning tube, the elution tube 7 , the nucleic acid collection tube 8 and the PCR reaction tube 22 are all arranged on the reagent card main body 1 .

Among them, the extraction tube 2 can contain nucleic acid extraction reagents, and can also contain cell lysing solution for performing the nucleic acid extraction process.

The magnetic bead tube 3 contains magnetic beads, and the magnetic beads are used to absorb the nucleic acid extract in the extraction tube 2; Pipette into the extraction tube 2, and transfer the magnetic beads mixed with the nucleic acid extract to the washing tube for washing through the magnetic sleeve rod 23.

The cleaning tube contains a washing solution for cleaning the magnetic beads mixed with the nucleic acid mixture; in this embodiment, the cleaning tube includes a plurality of tubes, namely the first cleaning tube 4, the second cleaning tube 5 and the third cleaning tube Tube 6, by setting up multiple cleaning tubes, the magnetic beads can be cleaned multiple times to ensure the cleaning degree of impurities. The magnetic beads washed through the washing tube can be transferred to the elution tube through the magnetic sleeve rod 23 again.

Nucleic acid eluent is contained in the elution tube 7 for magnetic separation of the magnetic beads transferred to the elution tube 7; under the action of the eluent, the nucleic acid is detached from the magnetic beads and dissolved in the eluent , the purified nucleic acid extract was obtained.

The PCR reaction tube 22 contains the reaction system required for the PCR reaction, such as lyophilized powder containing Max, primers, probes and other substances.

In addition, the extraction tube 2, the magnetic bead tube 3, the cleaning tube and the elution tube 7 are all provided with openings on the reagent card body 1, and the openings are sealed and isolated from the outside; Before using the reagent card, each opening can be closed by the sealing film; when using the reagent card, the sealing film must be torn first to avoid the deterioration of the reagents in each tube.

The nucleic acid collection tube 8 and the elution tube 7 are provided with a first passage that is sealed and isolated from the outside; wherein, between the nucleic acid collection tube 8 and the elution tube 7 means that the first passage in the communication relationship can connect the nucleic acid collection tube 8 to the elution tube 7. The communication between the elution tubes 7 is not a positional relationship in a narrow sense. That is to say, the first passage may not be located between the nucleic acid collection tube 8 and the elution tube 7, so as to connect the nucleic acid collection tube 8 and the elution tube 7 when the first passage is connected. . Between the nucleic acid collection tube 8 and the PCR reaction tube 22, a second passageway sealed and isolated from the outside is provided; the rotary valve 9 is rotatably arranged on the reagent card main body 1 around the center of rotation; wherein, the center of rotation is the center of the rotation of the rotary valve 9 . Specifically, the rotary valve 9 can be a multi-hole valve, and by rotating the rotary valve 9, the connection of different through holes can be changed to change the connection of the first passage and the second passage.

In this embodiment, the first passage specifically includes an elution tube through-hole 18, a nucleic acid tube through-hole 19, a first through-hole 17, and a second through-hole 15, all of which are sealed and isolated from the outside; the elution tube through-hole 18 is provided in the The reagent card body 1 is connected to the elution tube 7; the nucleic acid tube through hole 19 is arranged on the reagent card body 1 and communicated with the nucleic acid collection tube 8; the first through hole 17 is communicated with the second through hole 15, and both Installed on the rotary valve 9 . The second passage includes a PCR tube through-hole 21, a nucleic acid tube through-hole 19, a first through-hole 17 and a second through-hole 15 which are sealed and isolated from the outside; The pipes 22 communicate with each other.

Specifically, the elution tube through-hole 18, the nucleic acid tube through-hole 19, the PCR tube through-hole 21, the first through-hole 17 and the second through-hole 15 are all sealed off from the outside world to ensure that reagents are not transferred during the experiment. tightness. The opening of the elution tube 7 can be closed by a structure such as a sealing membrane after obtaining the purified nucleic acid extract; the elution tube 7 can be provided with an opening at the bottom, and the hose is connected with the through hole 18 of the elution tube through the opening. Communication; the nucleic acid collection tube 8 can be provided with an opening at the bottom, and communicate with the nucleic acid tube through hole 19 through the opening connecting hose.

By rotating the rotary valve 9, the first through hole 17 can be communicated with the nucleic acid tube through hole 19 while the second through hole 15 is communicated with the elution tube through hole 18, that is, the first passage is connected, and the elution tube The nucleic acid extract in 7 can be transferred to the nucleic acid collection tube 8; continue to rotate the rotary valve 9, the first through hole 17 can be communicated with the nucleic acid tube through hole 19 while the second through hole 15 is communicated with the PCR tube through hole 21, and also Even if the second channel is turned on, the nucleic acid extract in the nucleic acid collection tube 8 can be transferred to the PCR reaction tube for reaction.

Wherein, the first through hole 17 and the nucleic acid tube through hole 19 may both be disposed at the center of rotation, that is, the two are kept in a connected state. The positions of the PCR tube through hole 21 and the nucleic acid tube through hole 19 correspond to the positions of the first through hole 17 and the second through hole 15; the positions of the elution tube through hole 18 and the nucleic acid tube through hole 19 Corresponding to the positions of both the first through hole 17 and the second through hole 15; the corresponding position means that the first through hole 17 and the second through hole 15 can be respectively connected to the nucleic acid tube through hole 19 by rotating the rotary valve 9. It is overlapped and communicated with the PCR tube through-hole 21, or the first through-hole 17 and the second through-hole 15 are overlapped and communicated with the nucleic acid tube through-hole 19 and the elution tube through-hole 18 respectively, so as to control the communication between the first channel and the second channel. break.

In this embodiment, after controlling the communication between the first passage or the second passage by rotating the rotary valve 9, the power for the circulation of the reagent between the passages can be provided by a power pump, that is, through each through hole or The reagent push is realized by setting a microfluidic pump inside the reagent tube.

In practical applications, the lyophilized powder made from the reaction material can be pre-placed in the PCR reaction tube 22 , so that the nucleic acid extract only needs to be transferred to the PCR reaction tube 22 to perform the reaction.

In this embodiment, in order to facilitate the regulation of the ratio of the reaction material to the nucleic acid extract, a third passage is also included; the third passage is communicated with the PCR reaction tube 22 for adding reaction material to the PCR reaction tube 22; the rotary valve 22 is also used for It is used to control the opening and closing of the third passage.

The third passage includes: a third through hole 20 and a reaction material feeding through hole 10; the reaction material feeding through hole 10 is disposed on the reagent card body 1 and communicates with the PCR reaction tube 22; the third through hole 20 is disposed in the rotating Through the rotation of the rotary valve 9, the third through hole 20 can be controlled to communicate with the reaction material feeding through hole 10, so that the third passage is connected, so that the reaction material can be transferred to the PCR reaction tube 22 through the third passage.

Specifically, the distance between the third through hole 20 and the rotation center is equal to the distance between the reaction material feeding through hole 10 and the rotation center, that is, by rotating the rotary valve 9, the reaction material feeding through hole 10 and the third through hole can be 20 coincides. Wherein, the positional relationship of the first through hole 17 , the second through hole 15 and the reaction material feeding through hole 10 may be the same as the positional relationship of the nucleic acid tube through hole 19 , the third through hole 20 and the PCR tube through hole 21 . Correspondingly, the process of transferring the nucleic acid extract and the reaction material to the PCR reaction tube can be performed simultaneously.

As a further improvement, in this embodiment, an operation connection position 16 may be provided on the rotary valve 9 for directly operating the rotary valve 9 or connecting a rotary tool to rotate the rotary valve 9 . The rotary valve 9 can be arranged above the reagent card main body 1 to facilitate the rotary operation of the rotary valve 9 . The PCR reaction tube 22 is arranged below the main body of the reagent card, so as to facilitate the storage of reagents.

Since the PCR reaction tube 22 can be connected to an image acquisition device for signal detection, it can be determined whether the sample after the PCR reaction is positive.

In practical applications, the third through hole 20 and the PCR tube through hole 21 can also be connected to the PCR reaction tube 22 by means of hoses or the like; in order to reduce the complexity of the external pipeline connection of the PCR reaction tube 22, the main body of the reagent card 1 is reduced to the size of the PCR reaction tube. The structural volume of the tube 22 position, in this embodiment, the test tube station is also provided with a nucleic acid discharge through hole 13, a reaction material discharge through hole 12, a nucleic acid transfer through hole 14 and a second reaction material transfer through hole 11.

The PCR tube through hole 21 communicates with the PCR reaction tube 22 through the nucleic acid discharge through hole 13 and the nucleic acid transfer through hole 14; the third through hole 20 passes through the reaction material discharge through hole 12 and the second reaction material transfer through hole 11 communicates with the PCR reaction tube 22 .

The specific communication mode can be as follows: both the nucleic acid discharge through hole 13 and the reaction material discharge through hole 12 are disposed above the PCR reaction tube 22 through the reagent card body 1, and the nucleic acid discharge through hole 13 and the reaction material discharge through hole 13 are connected to each other. The top of the hole 12 is closed by a structure such as a sealing film; the tops of the nucleic acid discharge through hole 13 and the nucleic acid transfer through hole 14 are provided with a communication groove that communicates the two; the nucleic acid transfer through hole 14 communicates with the PCR tube. Between the bottoms of the two holes 21 is provided with a communication groove that makes the two communicated; the tops of the reactant material discharge through hole 12 and the second transfer through hole 11 of the reaction material are provided with a communication groove to communicate the two ; The bottom of the second through hole 11 and the third through hole 20 of the reaction material is provided with a communication groove that communicates the two.

By arranging a plurality of communication grooves, the end face of the reagent card body 1 can be kept flat and there is no need to set other pipes for connection outside, so that both the PCR tube through-hole 21 and the third through-hole 20 can react with PCR The pipes 22 communicate with each other.

Similarly, the first through hole 17 and the second through hole 15 may also communicate with each other by providing a communication groove.

It should be noted that each communication groove is closed and cut off from the outside of the reagent card main body 1 . In this embodiment, each communication groove is cut off from the outside by covering the sealing film.

The above is a nucleic acid extraction and detection integrated device provided in the first aspect of the embodiment of the present application, and the following is a nucleic acid extraction and detection integrated method provided in the second aspect of the embodiment of the present application, which is applied to any of the first aspect. The one described nucleic acid extraction and detection integrated device, and includes the following steps (refer to Figure 5):

S1. Extract the magnetic beads in the magnetic bead tube 3 into the extraction tube 2, and mix the magnetic beads with the nucleic acid extract in the extraction tube 2.

Specifically, after tearing off the sealing film on the extraction tube 2, the sample is added to the extraction tube 2, and nucleic acid extraction is performed by the nucleic acid extraction agent and the cell lysis solution in the extraction tube 2. The process of transferring the magnetic beads can be completed by the magnetic sleeve rod 23 .

S2, extracting the mixed magnetic beads into a washing tube for washing.

Specifically, the cleaning tube includes a plurality of tubes, and the magnetic beads mixed with the nucleic acid extraction solution can be washed in each cleaning tube in sequence to improve the degree of cleaning.

S3. Extract the cleaned magnetic beads to the elution tube 7 for magnetic separation to obtain a purified nucleic acid extract.

Specifically, among the existing nucleic acid extraction methods, the traditional manual magnetic bead method uses a magnetic stand, which requires a large amount of reagents, and the extraction process is time-consuming and labor-intensive. A magnetic unit is set in the cuvette, and the lysate, cleaning solution and eluent required for the extraction process are added to the cuvette sequentially through an automated pipetting device. In this way, during the process of removing the waste liquid, the bottom of the cuvette will always be A certain amount of waste liquid remains, resulting in low purity of samples after nucleic acid extraction. In this embodiment, the extraction tube 2 , the magnetic bead tube 3 , the cleaning tube and the elution tube 7 are separated, and multiple cleaning tubes are set up. The cleaned magnetic beads enter the elution tube 7 and pass through the elution tube 7 . The nucleic acid eluent is magnetically separated to obtain a purified nucleic acid extract, which improves the purity of the sample after nucleic acid extraction.

S4. Rotate the rotary valve 9 to connect the first passage, that is, the first through hole 17 is connected to the nucleic acid tube through hole 19 and the second through hole 15 is connected to the elution tube through hole 18, so that the purified nucleic acid extract is Transfer to nucleic acid collection tubes.

Specifically, through the microfluidic pump in the main body 1 of the reagent card, the nucleic acid in the elution tube 7 can flow through the elution tube through-hole 18 , the second through-hole 15 , and the first through-hole 17 in sequence through the elution tube 7 After passing through the hole 19 with the nucleic acid tube, it enters the nucleic acid collection tube 8 .

S5. Rotate the rotary valve 9 so that the second passage is connected, that is, the first through hole 17 is connected to the nucleic acid tube through hole 19 and the second through hole 15 is connected to the PCR tube through hole 21, so that the purified nucleic acid extract is collected from the nucleic acid Tube 8 was transferred to PCR reaction tube 22.

Specifically, through the microfluidic pump in the main body of the reagent card, the nucleic acid extract in the nucleic acid collection tube 8 can flow through the nucleic acid tube through-hole 19, the first through-hole 17, the second through-hole 17, and the PCR tube through-hole in sequence. After 21, it is transferred to the PCR reaction tube 22, and the process of quantitatively and controllably transferring the sample after nucleic acid extraction to the PCR reaction tube is completed.

After step S5, the rotary valve 9 can be rotated to make the PCR reaction tube 22 in a sealed state, and then the PCR reaction can be performed.

It should be noted that the PCR reaction tube 22 can store the reaction materials required by the PCR reaction system in advance. In this embodiment, in order to realize quantitative control of the reaction materials, the integrated device for nucleic acid extraction and detection further includes a third passage; the third passage is communicated with the PCR reaction tube 22; the rotary valve 9 is also used to control the passage of the third passage. close.

Specifically, the reagent card main body 1 is provided with a third through hole 20; the rotary valve 9 is also provided with a reaction material feeding through hole 10; the positions of the third through hole 20 and the nucleic acid tube through hole 19 are the same as the first The through hole 17 corresponds to the position of the reaction material feeding through hole 10; the third through hole 20 communicates with the PCR reaction tube 22;

After step S5, it also includes:

S6. Rotate the rotary valve 9 so that the third passage is connected, that is, the reaction material feed through hole 10 is connected to the third through hole 20, so that the reaction material can enter the PCR reaction tube 22 through the reaction material feed through hole 10.

Specifically, the on-off control of the reaction material feeding through hole 10 and the PCR reaction tube 22 can be realized through the rotary valve 9, and the connection between the reaction material feeding through hole 10 and the nucleic acid tube can be realized by setting the position of the through hole. The well 19 is connected to the PCR reaction tube 22 at the same time, so that the transfer of the nucleic acid extract and the addition of the reaction material are carried out simultaneously.

After step S6 is completed, the rotary valve 9 can be rotated so that the through holes on the rotary valve 9 and the through holes on the reagent card main body 1 are staggered from each other, so that the PCR reaction tube 22 is in a sealed state, and then the PCR reaction tube 22 can be in a sealed state. The heating device connected to the tube 22 gradually heats the PCR reaction tube 22 by program to complete the PCR reaction, and collects signals through the image acquisition device to determine whether the sample is positive.

With the integrated device and method for nucleic acid extraction and detection provided in this embodiment, the experimental steps of nucleic acid extraction and nucleic acid amplification can be completed on one reagent card through simple operations, which can effectively Compared with the method of test strip detection, it can effectively reduce the amount of reagents used, control the detection time, and improve the accuracy of detection, and in the PCR reaction and subsequent detection, the nucleic acid does not need to be transported. The sealed design avoids the problem of aerogel contamination.

The above are only the preferred embodiments of the application, and are not intended to limit the present invention. Although the application has been described in detail with reference to examples, those skilled in the art can still modify the technical solutions described in the foregoing examples. , or equivalently replace some of its technical features, but any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included within the protection scope of this application.

Claims (10)

1. An integrated nucleic acid extraction and detection device, comprising: a reagent card and rotary valve;

the reagent card comprises: the kit comprises a reagent card main body, an extraction tube, a magnetic bead tube, a cleaning tube, an elution tube, a nucleic acid collection tube and a PCR reaction tube;

the extraction tube, the magnetic bead tube, the cleaning tube, the elution tube, the nucleic acid collection tube and the PCR reaction tube are all arranged on the reagent card main body;

the nucleic acid collecting tube, the elution tube and the PCR reaction tube are sealed and isolated from the outside;

a first passage which is sealed and isolated from the outside is arranged between the nucleic acid collecting pipe and the elution pipe;

a second passage which is sealed and isolated from the outside is arranged between the nucleic acid collecting pipe and the PCR reaction pipe;

the rotary valve is rotatably arranged on the reagent card main body around a rotation center, and is used for controlling the opening and closing of the first passage and controlling the opening and closing of the second passage.

2. The integrated nucleic acid extraction and detection device according to claim 1, wherein the first channel comprises an elution tube through hole, a nucleic acid tube through hole, a first through hole and a second through hole;

the elution tube through hole is arranged on the reagent card main body and is communicated with the elution tube;

the nucleic acid tube through hole is arranged on the reagent card main body and is communicated with the nucleic acid collecting tube;

the first through hole and the second through hole are both arranged on the rotary valve and are communicated with each other;

the rotary valve is used for rotationally controlling the first through hole to be communicated with the nucleic acid tube through hole and the second through hole to be communicated with the elution tube through hole so that the first passage is communicated;

the second path includes: a PCR tube through hole, the nucleic acid tube through hole, the first through hole and the second through hole;

the PCR tube through hole is arranged on the reagent card main body and is communicated with the PCR reaction tube;

the rotary valve is also used to control the first through-hole to communicate with the nucleic acid tube through-hole and the second through-hole to communicate with the PCR tube through-hole so that the second passage is closed.

3. The integrated nucleic acid extraction and detection device according to claim 2, wherein the second channel further comprises: a nucleic acid transfer through hole and a nucleic acid discharge through hole;

the nucleic acid discharging through hole is arranged on the reagent card main body and is positioned above the PCR reaction tube;

the nucleic acid switching through hole is arranged on the reagent card main body, a communicating groove is arranged between the bottom end and the PCR tube through hole, and a communicating groove is arranged between the top end and the nucleic acid discharging through hole;

each of the communicating grooves is hermetically isolated from the outside.

4. The integrated nucleic acid extraction and detection device according to claim 2, wherein a communication groove is provided between the first through hole and the second through hole;

the communicating groove is sealed and isolated from the outside.

5. The integrated nucleic acid extraction and detection device according to claim 1, further comprising a third channel;

the third channel is communicated with the PCR reaction tube and is used for adding reaction materials to the PCR reaction tube;

the rotary valve is also used for controlling the opening and closing of the third passage.

6. The integrated nucleic acid extraction and detection device according to claim 5, wherein the third path comprises: the third through hole and the reaction material feeding through hole;

the reaction material feeding through hole is arranged on the reagent card main body and is communicated with the PCR reaction tube;

the third through hole is arranged on the rotary valve;

the rotary valve is also used for controlling the third through hole to be communicated with the reaction material feeding through hole, so that the third passage is communicated.

7. The integrated nucleic acid extraction and detection device according to claim 6, wherein the third path further comprises: the reaction material discharging through hole and the reaction material transferring through hole;

the reaction material discharging through hole is arranged on the reagent card main body and is positioned above the PCR reaction tube;

the reaction material transfer through hole is arranged on the reagent card main body, a communicating groove is arranged between the bottom end of the reagent card main body and the reaction material feeding through hole, and a communicating groove is arranged between the top end of the reagent card main body and the reaction material discharging through hole;

each communicating groove is sealed and isolated from the outside.

8. The integrated nucleic acid isolation apparatus according to any one of claims 1 to 7, wherein the sealing and isolation means is a sealing film.

9. A method for integrating nucleic acid extraction and detection, which is applied to the nucleic acid extraction and detection integrated device according to any one of claims 1 to 7, and comprises the steps of:

s1, extracting the magnetic beads in the magnetic bead tube into an extraction tube, and mixing the magnetic beads with the nucleic acid extract in the extraction tube;

s2, extracting the mixed magnetic beads into a cleaning tube for cleaning;

s3, extracting the washed magnetic beads to an elution tube for magnetic separation to obtain a purified nucleic acid extract;

s4, rotating the rotary valve to enable the first path to be connected, and further enabling the purified nucleic acid extract to be transferred from the elution tube to the nucleic acid collection tube;

s5, rotating the rotary valve to make the second path open, and then transferring the purified nucleic acid extract from the nucleic acid collection tube to the PCR reaction tube.

10. The method for integrating nucleic acid isolation and detection according to claim 9, wherein the nucleic acid isolation and detection integrated apparatus further comprises a third channel;

the third channel is communicated with the PCR reaction tube;

the rotary valve is also used for controlling the opening and closing of the third passage;

further comprising the steps of:

s6, rotating the rotary valve to open the third path, so that the reaction materials enter the PCR reaction tube through the third path.

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