WO2020062315A1

WO2020062315A1 - Pcr pipeline

Info

Publication number: WO2020062315A1
Application number: PCT/CN2018/109326
Authority: WO
Inventors: 车团结; 徐红; 李琳; 陈游
Original assignee: 苏州百源基因技术有限公司
Priority date: 2018-09-30
Filing date: 2018-10-08
Publication date: 2020-04-02
Also published as: CN109337807A

Abstract

The present invention belongs to the technical field of medical automation equipment, and discloses a PCR pipeline, comprising a nucleic acid extraction unit, a sample adding unit, a PCR reaction unit and a data analysis unit. The nucleic acid extraction unit, the sample adding unit, the PCR reaction unit and the data analysis unit are connected in series and are all closably disposed. Movement of reagents along the units is achieved by a moving device. The nucleic acid extraction unit, the sample adding unit, the PCR reaction unit and the data analysis unit are connected and disposed in series, such that the PCR pipeline of the present invention permits focusing on a particular operation in every unit, and enables increased operation efficiency and enhances throughput of a PCR reaction by means of automation. Since the nucleic acid extraction unit, the sample adding unit, the PCR reaction unit and the data analysis unit are closably disposed, aerosol contamination among the units can be avoided. Since movement of reagents along the units is achieved by a moving device, biohazards can be avoided.

Description

PCR pipeline

Technical field

The invention belongs to the technical field of medical automation equipment, and particularly relates to a PCR pipeline.

Background technique

Polymerase chain reaction (PCR) is a method for enzymatic synthesis of specific DNA fragments in vitro, and is one of the most commonly used molecular biology techniques. It consists of high temperature denaturation, low temperature annealing (refolding), and appropriate temperature extension to form a cycle, and the cycle is performed, so that the target DNA can be rapidly amplified. It has the characteristics of strong specificity, high sensitivity, simple operation, and time saving.

At present, PCR experiments are usually performed by PCR laboratories. The usual PCR laboratories are divided into four areas: reagent preparation area, sample processing area, nucleic acid amplification area, and product analysis area. In these four areas, equipment such as centrifuges, refrigerators, heating units, water baths, PCR instruments, and electrophoresis instruments will be placed.

However, when the reaction tube is moved between various areas, it is currently manually moved by the operator. The reaction tube and other containers generate large vibrations during the movement, thereby forming an aerosol, and the sample nucleic acid template, microorganism or Viruses can spread with aerosols or aerosols, causing operators to bear a higher risk of biological hazards. In addition, the efficiency of manual operations is also low.

Summary of the Invention

Therefore, the technical problem to be solved by the present invention lies in the fact that the reaction tube needs to be manually transferred during the existing PCR experiment process, so that the operator bears a higher risk of biological hazards and a low operation efficiency, thereby providing a PCR pipeline.

The PCR pipeline provided by the present invention includes a nucleic acid extraction unit with a nucleic acid extraction instrument for extracting DNA;

A sample adding unit for adding a DNA extract to a PCR reaction reagent to form a reaction solution;

PCR reaction unit with PCR instrument for PCR reaction of the reaction solution after sample loading;

Data analysis unit, which is used for data analysis of the PCR reactants;

The nucleic acid extraction unit, the loading unit, the PCR reaction unit and the data analysis unit are connected in sequence, and all of them can be closed. The movement of the reagents between the units is realized by a mobile device.

As a preferred solution, an isolation chamber is connected between the nucleic acid extraction unit, the sample loading unit, the PCR reaction unit and the data analysis unit, which are sequentially connected, and the mobile device is disposed in the isolation chamber.

As a preferred solution, after the moving device moves the reagent located in each unit to the isolation chamber, the isolation chamber is replaced by the internal air to prevent aerosol contamination between the reagents that enter in sequence.

As a preferred solution, it further includes,

The computer control module is configured to connect and control the nucleic acid extraction unit, the sample application unit, the PCR reaction unit and the data analysis unit, respectively.

As a preferred solution, a data port is provided in the nucleic acid extraction unit, the sampling unit, the PCR reaction unit, and the data analysis unit, and is used to electrically connect with the computer control module.

As a preferred solution, it further includes,

A data scanning device, which is arranged in the nucleic acid extraction unit, sample loading unit, PCR reaction unit or data analysis unit, and is used for scanning the identification code located on the reagent bottle and sending data information to the data in the computer control module Identify the unit.

As a preferred solution, the sampling unit has a plurality of preset PCR reaction reagents, and a bottle of the PCR reaction reagent has a preset identification code. After the reaction solution is formed, all the reagents are processed by the computer control module. The identification code on the bottle of the PCR reaction reagent corresponds to the identification code on the bottle of the DNA extract.

As a preferred solution, the PCR instrument adopts a side-opening door arrangement.

As a preferred solution, the PCR instrument in the PCR reaction unit has a plurality of reaction chambers, and the reaction conditions in each of the reaction chambers can be individually controlled by the computer control module.

As a preferred solution, it further includes,

The storage unit is provided with a refrigerating device, and is used for storing the remaining reaction solution in the sample adding unit that has not been subjected to the PCR reaction, and is used as a backup for re-examination.

As a preferred solution, it further includes,

A sample mixing unit is provided between the nucleic acid extraction unit and the sample adding unit, and is used to mix a plurality of groups of DNA extracts to form a group of DNA extraction solution.

As a preferred solution, the data analysis of the data analysis unit includes performing DNA sequencing on the PCR reaction product by a sequencer.

The technical solution provided by the present invention has the following advantages:

1. The PCR pipeline provided by the present invention is sequentially connected with a set of nucleic acid extraction unit, sample loading unit, PCR reaction unit and data analysis unit. Each unit only focuses on the work of a certain fragment, and can improve the work through automated production. Efficiency and increase the throughput of PCR reaction; Nucleic acid extraction unit, sample loading unit, PCR reaction unit and data analysis unit are all closable settings, so aerosol contamination between units can be avoided; reagents in each unit The movement between them is realized by a mobile device, so that biological hazards to the operator can be avoided.

2. In the PCR pipeline provided by the present invention, an isolation chamber is respectively connected between the nucleic acid extraction unit, the sampling unit, the PCR reaction unit and the data analysis unit, and the mobile device is set in the isolation chamber. When the device moves the reagent between units, no aerosol will diffuse to the outside, thereby effectively preventing the aerosol pollution from spreading, and avoiding biological hazards for operators outside the world; and using a mobile device to move the reagent can improve Operating efficiency further increases the throughput of PCR reactions.

3. According to the PCR pipeline provided by the present invention, the air inside the isolation chamber can be replaced as needed, thereby avoiding cross-contamination of aerosol generated in each unit and ensuring the accuracy of the experiment.

4. The PCR pipeline provided by the present invention has a computer control module, through which the nucleic acid extraction unit, sample loading unit, PCR reaction unit and data analysis unit can be individually controlled, and the above-mentioned each can be set by setting a specific program. The unit runs automatically, thus realizing the automated operation of the assembly line, improving operation efficiency, and further increasing the throughput of performing PCR reactions.

5. The PCR pipeline provided by the present invention is provided with a data port in a nucleic acid extraction unit, a sample loading unit, a PCR reaction unit and a data analysis unit, for electrically connecting with the computer control module, so that the above units can be connected to a computer. Control module data transmission.

6. The PCR pipeline provided by the present invention has a data scanning device, which is arranged in a nucleic acid extraction unit, a sample loading unit, a PCR reaction unit or a data analysis unit, and is used to scan an identification code located on a reagent bottle and send data information to The data recognition unit in the computer control module can connect the reagents transported in each unit through the data recognition unit. By reading the identification code information, the sample information and detection index information corresponding to the test result can be obtained, which is beneficial to the realization of PCR. The detection results are automatically matched with the sample and index information, thereby realizing the automation of the PCR detection experiment. At the same time, in the case of a large amount of sample detection, the unique identification code on the PCR reaction tube can be used to prevent the detection information from being disrupted. When the sample information does not correspond to the detection result, the error rate of the detection result is reduced.

7. The PCR pipeline provided by the present invention has preset multiple groups of PCR reaction reagents in the sample addition unit. The reaction solution is prepared by incorporating DNA extracts into each group of PCR reaction reagents and mixing the samples. The preparation time of the PCR reaction reagent improves the efficiency of preparing the reaction solution by mixing the samples, so the throughput of the PCR reaction can be further increased; the preset PCR reaction reagent bottle has a preset identification code, After the reaction solution is formed, the PCR reaction is performed according to the identification code sequence through computer control, so that the computer can conveniently record the reagents for the PCR reaction, avoiding manual operation and improving work efficiency.

8. The PCR pipeline provided by the present invention, the PCR instrument in the PCR reaction unit adopts a side-opening door arrangement. Compared with the top opening arrangement of the prior art, it can accommodate more reaction reagents, so it can carry out more reaction solutions at a time. PCR reaction, increase the throughput of PCR reaction.

9. In the PCR pipeline provided by the present invention, the PCR instrument in the PCR reaction unit has multiple reaction chambers, and the reaction conditions in each reaction chamber are individually controlled by a computer. Therefore, when a PCR instrument is used for the reaction, it can Performing multiple sets of reactions in different reaction chambers improves the efficiency of PCR experiments and further increases the throughput of PCR reactions.

10. The PCR pipeline provided by the present invention has a storage unit. Only a small portion of the reaction solution prepared by mixing samples in the loading unit is required for the PCR reaction. The remaining reaction solution that has not undergone the PCR reaction can pass through the storage unit. Refrigerators are stored as a backup for retests.

11. The PCR pipeline provided by the present invention has a sample mixing unit. A plurality of groups of DNA extracts are mixed in the sample mixing unit to form a group of DNA extracts, and then the above-mentioned group of DNA extracts are added to the subsequent sample adding unit. The reaction solution is formed in the PCR reaction reagent, and the PCR reaction is performed on the above reaction solution in the subsequent PCR reaction unit, which can complete the PCR reaction of the reaction solution containing multiple groups of DNA extracts. If the reaction result shows that the DNA is free of pathogenic bacteria Infection, the detection of the above multiple sets of DNA is completed at one time, so the efficiency of the PCR reaction can be improved and the throughput of the PCR reaction can be increased.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the specific embodiments or the description of the prior art are briefly introduced below. Obviously, the appendixes in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without paying creative labor.

FIG. 1 is a front view of a PCR pipeline provided by the present invention.

Reference sign description:

1. Nucleic acid extraction unit; 2. Sample loading unit; 3. PCR reaction unit; 4. Data analysis unit; 5. Mobile device; 6. Isolation chamber; 7. Storage unit.

detailed description

The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are part of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

In the description of the present invention, it should be noted that the directions indicated by the terms "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside" or the like The positional relationship is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplified description. It does not indicate or imply that the device or element referred to must have a specific orientation, structure and operation in a specific orientation. Therefore, it cannot be understood as a limitation to the present invention.

In the description of the present invention, it should be noted that the terms "installation", "connected", and "connected" should be understood in a broad sense unless otherwise specified and limited. For example, they may be fixed connections or removable. Connected or integrated; it can be mechanical or electrical; it can be directly connected, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood on a case-by-case basis.

In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

Example 1

This embodiment provides a PCR pipeline, as shown in FIG. 1, including a nucleic acid extraction unit 1, a loading unit 2, a PCR reaction unit 3, a data analysis unit 4, and a storage unit 7.

The nucleic acid extraction unit 1, the loading unit 2, the PCR reaction unit 3, and the data analysis unit 4 are connected in sequence, and all of them can be closed. The movement of the reagents between the units is realized by the mobile device 5.

Specifically, the nucleic acid extraction unit 1 has a nucleic acid extraction device inside for extracting DNA; the sample adding unit 2 is used to add a DNA extract to a PCR reaction reagent to form a reaction solution; the PCR reaction unit 3 has a PCR instrument inside for performing PCR reactions on the reaction solution after the sample is added; the data analysis unit 4 has a sequencer inside for DNA sequencing of the PCR reactants; the reactions performed in the above units and the The functions are all existing technologies, and will not be described in detail here. In this application, the above units are formed into an assembly line. The nucleic acid extraction unit 1, the sampling unit 2, the PCR reaction unit 3, and the data analysis unit 4 are sequentially passed through the isolation chamber 6. Connected, a mobile device 5 is provided in the isolation chamber 6 that communicates between the units, and the reagent located in each unit is moved into or out of the isolation chamber 6 through the mobile device 5, thereby realizing the reagent in the Transfer between each reaction unit.

Specifically, the moving device 5 may be a robot, a conveyor, or other existing mechanical transfer devices.

There is a refrigerating device in the storage unit 7 for storing the remaining reaction solution that has not been subjected to the PCR reaction in the sampling unit 2 as a backup when performing a re-examination.

In order to prevent aerosol pollution between the reaction units, an air replacement device is provided in the isolation chamber 6 for replacing the internal air of the isolation chamber 6. Specifically, the air replacement device may use an existing exhaust fan Or exhaust device.

In order to improve the automation of the assembly line operation, the nucleic acid extraction unit 1, the sampling unit 2, the PCR reaction unit 3, the data analysis unit 4, the mobile device 5, and the isolation chamber 6 are all connected and controlled by a computer control module, respectively.

A data identification unit is provided in the computer control module, and the data identification unit is configured to receive the identification code on the reagent bottle from the data scanning device located in the nucleic acid extraction unit 1, the sample application unit 2, the PCR reaction unit 3, and the data analysis unit 4. Scanning information, by scanning the identification code on the reagent bottle, one-to-one correspondence can be made to the sample, reaction solution or reaction product transmitted in each reaction unit.

The PCR instrument is provided with a side-opening door, and the PCR instrument of the PCR reaction unit 3 has a plurality of reaction chambers, and the reaction conditions in each of the reaction chambers are individually controlled by a computer.

The sample adding unit 2 has preset multiple groups of PCR reaction reagents, and the reaction solution is prepared by incorporating a DNA extract into each group of PCR reaction reagents and mixing samples.

A sample mixing unit is provided between the nucleic acid extraction unit and the sample adding unit, and a plurality of groups of DNA extracts are mixed in the sample mixing unit to form a group of DNA extraction solution, and then the above-mentioned one is added in a subsequent sample adding unit. The group DNA extraction solution is added to the PCR reaction reagent to form a reaction solution, and the PCR reaction is performed on the above reaction solution in a subsequent PCR reaction unit, which can complete the PCR reaction on the reaction solution containing multiple groups of DNA extracts. If the reaction result shows The DNA is free of pathogenic bacteria, and the detection of the above multiple groups of DNA is completed at one time, so the PCR reaction efficiency can be improved and the throughput of the PCR reaction can be increased.

work process:

When performing a PCR experiment through the PCR pipeline of the present application, the following steps are sequentially included:

In the nucleic acid extraction unit, the reagent to be reacted is subjected to DNA extraction.

Use a mobile device to move the DNA extract to the loading unit.

In the loading unit, the DNA extract is added to the PCR reaction reagent to form a reaction solution; part of the reaction solution is used for performing the PCR reaction, and part of the reaction solution is used for storage.

A part of the reaction solution used for storage and reserve in the sample adding unit is moved to the storage unit through a moving device, and refrigerated and stored in the storage unit.

A mobile device is used to move a part of the reaction solution used for performing the PCR reaction to the PCR reaction unit.

A PCR reaction is performed on the reaction solution in a PCR reaction unit.

The reaction sample is moved to the data analysis unit by a mobile device.

DNA sequencing was performed on the samples in the data analysis unit.

In the above reaction step, in order to prevent aerosol pollution between the units, each time the reagent is moved between the units, it first passes through the isolation chamber, and air replacement is performed at least once in the isolation chamber.

In the above reaction step, in order to improve the efficiency of the experiment and increase the throughput of the PCR reaction, a mixing unit is provided between the nucleic acid extraction unit and the loading unit, and multiple sets of DNA extracts are mixed in the mixing unit to form a group. DNA extraction solution; in this way, multiple groups of DNA extracts are introduced into the same PCR reaction reagent at one time in the loading unit to form a reaction solution; and then the PCR reaction is performed once in the PCR reaction unit, If the reaction result shows that the DNA is not infected by pathogenic bacteria, the above multiple groups of DNA tests can be completed at one time. If the reaction result shows that the DNA has pathogenic bacteria infection, re-sample the reaction solution with separate DNA extracts, one by one. Perform a secondary PCR test.

Obviously, the foregoing embodiment is merely an example for clear description, and is not a limitation on the implementation. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description. There is no need and cannot be exhaustive for all implementations. However, the obvious changes or variations introduced thereby are still within the protection scope created by the present invention.

Claims

PCR pipeline including:

Nucleic acid extraction unit (1), which has a nucleic acid extraction instrument for extracting DNA;

A sample adding unit (2) for adding a DNA extract to a PCR reaction reagent to form a reaction solution;

The PCR reaction unit (3) has a PCR instrument for performing a PCR reaction on the reaction solution after the addition;

A data analysis unit (4), configured to perform data analysis on the PCR reactants;

It is characterized in that the nucleic acid extraction unit (1), the sample application unit (2), the PCR reaction unit (3) and the data analysis unit (4) are connected in sequence, and all of them can be closed, and the reagents between the units Movement is achieved by a mobile device (5).
The PCR pipeline according to claim 1, characterized in that the nucleic acid extraction unit (1), the sample application unit (2), the PCR reaction unit (3), and the data analysis unit (4) are connected in order. An isolation chamber (6) is provided with the moving device (5) inside the isolation chamber (6).
The PCR pipeline according to claim 2, characterized in that, after the moving device (5) moves the reagent located in each unit to the isolation chamber (6), the isolation chamber (6) passes through the internal air. Replacement prevents aerosol contamination between successive reagents.
The PCR pipeline according to claim 1, further comprising:

The computer control module is configured to connect and control the nucleic acid extraction unit (1), the sample application unit (2), the PCR reaction unit (3), and the data analysis unit (4), respectively.
The PCR pipeline according to claim 4, characterized in that a data port is provided in the nucleic acid extraction unit (1), the sample application unit (2), the PCR reaction unit (3) and the data analysis unit (4). And is electrically connected to the computer control module.
The PCR pipeline according to claim 5, further comprising:

A data scanning device is provided in the nucleic acid extraction unit (1), the sample application unit (2), the PCR reaction unit (3) or the data analysis unit (4), and is used for scanning the identification code located on the reagent bottle, and The data information is sent to a data identification unit in the computer control module.
The PCR pipeline according to claim 6, wherein:

The sample adding unit (2) has a plurality of preset PCR reaction reagents, and a preset identification code is provided on a bottle of the PCR reaction reagents. After the reaction solution is formed, the computer control module converts the The identification code on the bottle of the PCR reaction reagent corresponds to the identification code on the bottle of the DNA extract.
The PCR pipeline according to claim 4, wherein the PCR instrument is provided with a side-opening door.
The PCR pipeline according to claim 8, characterized in that the PCR instrument in the PCR reaction unit (3) has a plurality of reaction chambers, and the reaction conditions in each of the reaction chambers can be controlled by the computer control module. For individual control.
The PCR pipeline according to claim 1, further comprising:

The storage unit (7) is provided with a refrigerating device, and is used for storing the remaining reaction solution in the sample adding unit (2) that has not been subjected to the PCR reaction, and is used as a backup during the re-examination.
The PCR pipeline according to claim 1, further comprising:

The sample mixing unit is arranged between the nucleic acid extraction unit (1) and the sample adding unit (2), and is used for mixing a plurality of groups of DNA extracts to form a group of DNA extraction solution.
The PCR pipeline according to claim 1, wherein the data analysis of the data analysis unit (4) comprises performing DNA sequencing on the PCR reaction product by a sequencer.