High-speed PCR amplification heating device

Abstract

The invention discloses a high-speed PCR (Polymerase Chain Reaction) amplification heating device which comprises a PCR amplification reagent conveying line, a high-temperature deformation heating system, a low-temperature annealing heating system, a medium-temperature extension heating system, a Taq DNA (Deoxyribose Nucleic Acid) polymerase adding mechanism and a computer, the high-temperature deformation heating system, the low-temperature annealing heating system and the medium-temperature extension heating system are sequentially arranged in the conveying direction of the PCR amplification reagent conveying line, the high-temperature deformation heating system performs heating at the temperature of 91-95 DEG C, the low-temperature annealing heating system performs heating at the temperature of 53-57 DEG C, the medium-temperature extension heating system performs heating at the temperature of 68-72 DEG C, and the high-temperature deformation heating system performs heating at the temperature of 55-65 DEG C; the Taq DNA polymerase adding mechanism is arranged on one side of the medium-temperature extension area of the PCR amplification reagent conveying line and is used for adding Taq DNA polymerase into the PCR amplification reagent test tube. The PCR reaction efficiency is high, the heating rate is high, the amplification rate is high, the heating uniformity is high, and the detection rate and the detection precision can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of PCR amplification, and in particular to a high-speed PCR amplification heating device. Background Art

[0002] Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments. It can be seen as a special DNA replication outside the body. The greatest feature of PCR is that it can greatly increase trace amounts of DNA. PCR instrument is a temperature control device based on polymerase. It can control the denaturation temperature, annealing temperature, and extension temperature very well. It combines high-temperature denaturation, low-temperature annealing, and appropriate temperature extension into a cycle, which is cyclically carried out to rapidly amplify the target DNA. It has the characteristics of strong specificity, high sensitivity, simple operation, and time saving. It can not only be used for basic research such as gene isolation, cloning, and nucleic acid sequence analysis, but also for disease diagnosis. According to the purpose of DNA amplification and the standard of detection, PCR instruments can be divided into four categories: ordinary PCR instruments, gradient PCR instruments, in situ PCR instruments, and real-time fluorescence quantitative PCR instruments.

[0003] PCR tube is a reaction vessel commonly used in the implementation of PCR amplification reaction. A typical PCR amplification reaction is completed by repeatedly heating and cooling the PCR tube placed in the PCR thermal cycler. In this process, the temperature is transmitted by the PCR thermal cycler heating the metal block and then transferred to the PCR tube placed in it, and then the heat is transferred to the PCR amplification reagent in the tube through the tube wall of the PCR tube, thereby initiating the amplification reaction. Whether a PCR reaction can amplify the gene template quickly and efficiently depends not only on the quality of the gene template, the properties of the polymerase, and the composition of the reaction solution, but also on the heating and cooling speed of the reaction solution. The faster the heating and cooling speed, the higher the efficiency of the PCR reaction, and the less likely it is to produce non-specific amplification products. In addition, the fast heating and cooling speed can greatly shorten the PCR reaction time.

[0004] Although PCR amplification can be achieved by using a PCR thermal cycler, the efficiency of the PCR reaction cannot be improved due to the heating and cooling time. In addition, the test tubes used in the PCR thermal cycler are mostly PCR tubes or microplates. PCR test tubes are generally made of high-quality transparent polypropylene material. Due to the nature of the polypropylene material itself, the thermal conductivity of polypropylene is only about 0.2W / mK. The poor thermal conductivity makes the PCR tubes heat slowly and the heating uniformity is poor, which affects the amplification rate, thereby affecting the detection speed and detection accuracy. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a high-speed PCR amplification heating device.

[0006] The technical solution of the present invention is as follows:

[0007] A high-speed PCR amplification heating device comprises a PCR amplification reagent conveying line, a high-temperature deformation heating system, a low-temperature annealing heating system, a medium-temperature extension heating system, a Taq DNA polymerase adding mechanism and a computer. A plurality of PCR amplification reagent test tubes are sequentially conveyed through the PCR amplification reagent conveying line. The high-temperature deformation heating system, the low-temperature annealing heating system and the medium-temperature extension heating system are sequentially arranged on one side of the PCR amplification reagent conveying line along the conveying direction of the PCR amplification reagent conveying line. The PCR amplification reagent conveying line is sequentially provided with a high-temperature deformation area, a low-temperature annealing area and a medium-temperature extension area corresponding to the high-temperature deformation heating system, the low-temperature annealing heating system and the medium-temperature extension heating system. The high-temperature deformation heating system is used to perform 91° annealing of the PCR amplification reagent in the PCR amplification reagent test tube passing through the high-temperature deformation area. The low temperature annealing heating system is used to heat the PCR amplification reagent in the PCR amplification reagent test tube passing through the low temperature annealing area at 53° to 57°. The medium temperature extension heating system is used to heat the PCR amplification reagent in the PCR amplification reagent test tube passing through the medium temperature extension area at 68° to 72°. The template DNA double strand in the PCR amplification reagent or the double strand DNA formed by PCR amplification will be separated into single strand DNA after passing through the low temperature annealing area. When passing through the low temperature annealing area, the primer in the PCR amplification reagent will be paired with the complementary sequence of the DNA single strand to form a DNA template-primer conjugate. The Taq The DNA polymerase adding mechanism is arranged at one side of the medium-temperature extension area of ​​the PCR amplification reagent conveying line, and is used for adding Taq DNA polymerase to the PCR amplification reagent test tube passing through the medium-temperature extension area. When the PCR amplification reagent test tube passes through the medium-temperature extension area, the DNA template-primer conjugate in the PCR amplification reagent thereof will synthesize a new semi-conservative replication chain complementary to the template DNA chain under the action of Taq DNA polymerase. The computer is respectively connected with the PCR amplification reagent conveying line, the high-temperature deformation heating system, the low-temperature annealing heating system, the medium-temperature extension heating system and the Taq DNA polymerase adding mechanism signal.

[0008] Furthermore, the high-temperature deformation heating system includes a first temperature controller and a high-temperature deformation heater, the signal input end of the first temperature controller is connected to a computer, the signal output end of the first temperature controller is connected to the high-temperature deformation heater, and the high-temperature deformation heater is located on one side of the PCR amplification reagent delivery line; the low-temperature annealing heating system includes a second temperature controller and a low-temperature annealing heater, the signal input end of the second temperature controller is connected to a computer, the signal output end of the second temperature controller is connected to the low-temperature annealing heater, and the low-temperature annealing heater is located on one side of the PCR amplification reagent delivery line; the medium-temperature extension heating system includes a third temperature controller and a medium-temperature extension heater, the signal input end of the third temperature controller is connected to a computer, the signal output end of the third temperature controller is connected to the medium-temperature extension heater, and the medium-temperature extension heater is located on one side of the PCR amplification reagent delivery line.

[0009] Furthermore, the PCR amplification reagent conveying line includes a loading track, a circular track and a unloading track, one end of the circular track is connected to the loading track, and the other end of the circular track is connected to the unloading track, and an arc-shaped receiving groove is provided on the inner side of the circular track. The high-temperature deformation heater, the low-temperature annealing heater and the medium-temperature extension heater are arranged in an arc shape on the inner side of the circular track and are adapted to the arc-shaped receiving groove, and the arc-shaped heating surfaces of the high-temperature deformation heater, the low-temperature annealing heater and the medium-temperature extension heater are respectively facing the high-temperature deformation area, the low-temperature annealing area and the medium-temperature extension area of ​​the PCR amplification reagent conveying line.

[0010] Furthermore, the PCR amplification reagent conveying line conveys PCR amplification reagent test tubes through a plurality of rotating jigs fixedly arranged at intervals, the rotating jigs being composed of a rotating positioning block and a fixed base, the rotating positioning block being rotatably connected to the fixed base, the PCR amplification reagent test tubes being placed on the rotating positioning block for positioning, the width of the annular track being the same as the outer diameter of the PCR amplification reagent test tubes, and the PCR amplification reagent test tubes passing through the high-temperature deformation zone, the low-temperature annealing zone, and the medium-temperature extension zone having their tube walls in conflict with the arc-shaped heating surfaces of the high-temperature deformation heater, the low-temperature annealing heater, and the medium-temperature extension heater and rotating under the conflicting force.

[0011] Furthermore, the PCR amplification reagent test tube is a metal test tube.

[0012] Furthermore, the metal material used for the metal test tube is silver, copper, aluminum, magnesium, zinc, aluminum alloy, aluminum-magnesium alloy, copper alloy or copper-zinc alloy.

[0013] Furthermore, an annular heat-insulating side plate is provided on the outer side of the annular track, and the annular heat-insulating side plate contacts the PCR amplification reagent tube when the tube passes through the high-temperature deformation area, the low-temperature annealing area, and the medium-temperature extension area.

[0014] Furthermore, an upper cover is provided between the top end of the annular heat-insulating side plate and the top end of the arc-shaped heating surfaces of the high-temperature deformation heater, the low-temperature annealing heater and the medium-temperature extension heater.

[0015] Compared with the prior art, the beneficial effect of the present invention is that the present invention conveys the PCR amplification reagent metal test tube through a PCR amplification reagent conveying line, and the PCR amplification reagent conveying line is composed of a loading track, a circular track and a unloading track. The inner side of the circular track is sequentially provided with a high-temperature deformation heater, a low-temperature annealing heater and a medium-temperature extension heater along the conveying direction. The PCR amplification reagent in the PCR amplification reagent metal test tube is first subjected to DVA denaturation through high-temperature deformation, and then subjected to low-temperature annealing for primer and template DNA combination, and finally subjected to medium-temperature extension for DNA synthesis, without the need for heating and cooling in between, and a direct transition is sufficient. Since there is no heating and cooling time, the PCR reaction efficiency can be higher. When the PCR amplification reagent metal test tube enters the high-temperature deformation area, the low-temperature annealing area and the medium-temperature extension area, it will conflict with the arc-shaped heating surfaces of the high-temperature deformation heater, the low-temperature annealing heater and the medium-temperature extension heater and rotate under the conflicting force. Rotating heating and the use of metal test tubes are helpful to improve the heating rate, amplification rate and heating uniformity of the heating reagent, thereby improving the detection rate and detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 A schematic diagram of the external structure of a high-speed PCR amplification heating device provided by the present invention;

[0018] Figure 2 A schematic diagram of the internal structure of a high-speed PCR amplification heating device provided by the present invention;

[0019] Figure 3 A connection diagram of the computer of the present invention;

[0020] Figure 4 This is a schematic diagram of the placement of the PCR amplification reagent tubes of the present invention. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] In order to illustrate the technical solution of the present invention, a specific embodiment is provided below for illustration.

[0023] Example

[0024] See also Figures 1 to 3 This embodiment provides a high-speed PCR amplification heating device, including a PCR amplification reagent delivery line 1, a high-temperature deformation heating system 2, a low-temperature annealing heating system 3, a medium-temperature extension heating system 4, a Taq DNA polymerase adding mechanism 5 and a computer 6. A plurality of PCR amplification reagent test tubes 10 are sequentially transported through a PCR amplification reagent conveying line 1; a high temperature deformation heating system 2, a low temperature annealing heating system 3, and a medium temperature extension heating system 4 are sequentially arranged on one side of the PCR amplification reagent conveying line 1 along the conveying direction of the PCR amplification reagent conveying line 1, and the PCR amplification reagent conveying line 1 is sequentially provided with a high temperature deformation area, a low temperature annealing area, and a medium temperature extension area corresponding to the high temperature deformation heating system 2, the low temperature annealing heating system 3, and the medium temperature extension heating system 4; the high temperature deformation heating system 2 is used to heat the PCR amplification reagent in the PCR amplification reagent test tube 10 passing through the high temperature deformation area at 91° to 95°, the low temperature annealing heating system 3 is used to heat the PCR amplification reagent in the PCR amplification reagent test tube 10 passing through the low temperature annealing area at 53° to 57°, and the medium temperature extension heating system 4 is used to heat the PCR amplification reagent in the PCR amplification reagent test tube 10 passing through the medium temperature extension area at 68° to 72°; a Taq DNA polymerase adding mechanism 5 is arranged on one side of the medium temperature extension area of ​​the PCR amplification reagent conveying line 1, and is used to add Taq DNA polymerase to the PCR amplification reagent test tube 10 passing through the medium temperature extension area. DNA polymerase; the computer 6 is respectively connected with the PCR amplification reagent delivery line 1, the high temperature deformation heating system 2, the low temperature annealing heating system 3, the medium temperature extension heating system 4 and the Taq DNA polymerase adding mechanism 5, and the computer 6 is used to control the PCR amplification reagent delivery line 1, the high temperature deformation heating system 2, the low temperature annealing heating system 3, the medium temperature extension heating system 7, and the Taq DNA polymerase adding mechanism 5 to realize the automation of the whole process.

[0025] The principle of PCR amplification is as follows: the double-stranded DNA template in the PCR amplification reagent or the double-stranded DNA formed by PCR amplification will be separated into single-stranded DNA after passing through the low-temperature annealing area. When passing through the low-temperature annealing area, the primers in the PCR amplification reagent will be paired with the complementary sequence of the single-stranded DNA to form a DNA template-primer conjugate. Finally, when passing through the medium-temperature extension area, the DNA template-primer conjugate in the PCR amplification reagent, under the action of Taq DNA polymerase, uses dNTP as the reaction raw material and the target sequence as the template. According to the principle of base complementary pairing and semi-conservative replication, a new semi-conservative replication chain complementary to the template DNA chain is synthesized; during implementation, the three processes of high-temperature denaturation → low-temperature annealing → medium-temperature extension can be repeated, so that more semi-conservative replication chains can be obtained, and the newly synthesized chain can be used as a template for the next cycle. Compared with the use of a PCR thermal cycler, there is no heating and cooling time between high-temperature denaturation → low-temperature annealing → medium-temperature extension, and the transition can be directly transported, which improves the efficiency of the PCR reaction.

[0026] Among them, the PCR amplification reagent conveying line 1 includes a loading track 11, a circular track 12 and a unloading track 13, one end of the circular track 12 is connected to the loading track 11, and the other end of the circular track 12 is connected to the unloading track 13. An arc-shaped receiving groove is provided on the inner side of the circular track 12, and an annular insulation side plate 14 is provided on the outer side of the circular track 12. The high temperature deformation heating system 2 includes a first temperature controller 21 and a high temperature deformation heater 22. The signal input end of the first temperature controller 21 is connected to the computer 6, and the signal output end of the first temperature controller 21 is connected to the high temperature deformation heater 22. The temperature is set by the computer 6, and then the first temperature controller 21 controls the high temperature deformation heater 22 to heat to 91°~95°; the low temperature annealing heating system 3 includes a second temperature controller 31 and a low temperature annealing heater 32. The signal input end of the second temperature controller 31 is connected to the computer 6, and the signal output end of the second temperature controller 31 is connected to the low temperature annealing heater 32. The temperature is set by the computer 6, and then the second temperature controller 31 controls the high temperature deformation heater 32 to heat to 53°~57°; the medium temperature extension heating system 4 includes a third temperature controller 41 and a medium temperature extension heater 42. The signal input end of the third temperature controller 41 is connected to the computer 6, and the signal output end of the third temperature controller 41 is connected to the medium temperature extension heater 42. The temperature is set by the computer 6, and then the third temperature controller 41 controls the high temperature deformation heater 42 to heat to 68°~72°. The high temperature deformation heater 22, the low temperature annealing heater 32, and the medium temperature extension heater 42 are arranged in an arc shape on the inner side of the annular track 12 and are adapted to the arc-shaped mounting groove. The arc-shaped heating surfaces of the high temperature deformation heater 22, the low temperature annealing heater 32, and the medium temperature extension heater 42 are respectively oriented toward the high temperature deformation area, the low temperature annealing area, and the medium temperature extension area of ​​the PCR amplification reagent delivery line 1. An upper cover 15 is provided between the top of the annular heat-insulating side plate 14 and the top of the arc-shaped heating surfaces of the high temperature deformation heater 22, the low temperature annealing heater 32, and the medium temperature extension heater 42.

[0027] The PCR amplification reagent delivery line 1 delivers the PCR amplification reagent test tube 10 through a plurality of rotating fixtures 7 that are fixedly arranged at intervals. Figure 4As shown, the rotating fixture 7 is composed of a rotating positioning block 71 and a fixed base 72, the rotating positioning block 71 is rotatably connected to the fixed base 72, the PCR amplification reagent test tube 10 is placed on the rotating positioning block 71 for positioning, the width of the annular track 12 is the same as the outer diameter of the PCR amplification reagent test tube 10, and the PCR amplification reagent test tube 10 is in conflict with the arc heating surface of the high-temperature deformation heater 22, the low-temperature annealing heater 32, and the medium-temperature extension heater 42 and the annular insulation side plate 14 when passing through the high-temperature deformation area, the low-temperature annealing area, and the medium-temperature extension area, and rotates under the conflicting force. Preferably, the PCR amplification reagent test tube 10 adopts a metal test tube, and the metal material used in the metal test tube is silver, copper, aluminum, magnesium, zinc, aluminum alloy, aluminum-magnesium alloy, copper alloy or copper-zinc alloy, and the thermal conductivity of these materials is good. Rotating heating and using a metal test tube help to improve the heating rate, amplification rate and heating uniformity of the heating reagent, and can improve the detection rate and detection accuracy.

[0028] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A high-speed PCR amplification heating device, characterized in that: The invention comprises a PCR amplification reagent conveying line, a high-temperature deformation heating system, a low-temperature annealing heating system, a medium-temperature extension heating system, a Taq DNA polymerase adding mechanism and a computer. A plurality of PCR amplification reagent test tubes are conveyed in sequence through the PCR amplification reagent conveying line. The high-temperature deformation heating system, the low-temperature annealing heating system and the medium-temperature extension heating system are arranged in sequence on one side of the PCR amplification reagent conveying line along the conveying direction of the PCR amplification reagent conveying line. The PCR amplification reagent conveying line is provided with a high-temperature deformation area, a low-temperature annealing area and a medium-temperature extension area in sequence corresponding to the high-temperature deformation heating system, the low-temperature annealing heating system and the medium-temperature extension heating system. The high-temperature deformation heating system is used for annealing the PCR amplification reagent in the PCR amplification reagent test tube passing through the high-temperature deformation area by 91°. The low temperature annealing heating system is used to heat the PCR amplification reagent in the PCR amplification reagent test tube passing through the low temperature annealing area at 53° to 57°. The medium temperature extension heating system is used to heat the PCR amplification reagent in the PCR amplification reagent test tube passing through the medium temperature extension area at 68° to 72°. The template DNA double strand in the PCR amplification reagent or the double strand DNA formed by PCR amplification will be separated into single strand DNA after passing through the low temperature annealing area. When passing through the low temperature annealing area, the primer in the PCR amplification reagent will be paired with the complementary sequence of the DNA single strand to form a DNA template-primer conjugate. The Taq The DNA polymerase adding mechanism is arranged at one side of the medium-temperature extension area of ​​the PCR amplification reagent conveying line, and is used for adding Taq DNA polymerase to the PCR amplification reagent test tube passing through the medium-temperature extension area. When the PCR amplification reagent test tube passes through the medium-temperature extension area, the DNA template-primer conjugate in the PCR amplification reagent thereof will synthesize a new semi-conservative replication chain complementary to the template DNA chain under the action of Taq DNA polymerase. The computer is respectively connected with the PCR amplification reagent conveying line, the high-temperature deformation heating system, the low-temperature annealing heating system, the medium-temperature extension heating system and the Taq DNA polymerase adding mechanism signal.

2. A high-speed PCR amplification heating device according to claim 1, characterized in that: The high-temperature deformation heating system includes a first temperature controller and a high-temperature deformation heater, the signal input end of the first temperature controller is connected to a computer, the signal output end of the first temperature controller is connected to the high-temperature deformation heater, and the high-temperature deformation heater is located on one side of the PCR amplification reagent delivery line; the low-temperature annealing heating system includes a second temperature controller and a low-temperature annealing heater, the signal input end of the second temperature controller is connected to a computer, the signal output end of the second temperature controller is connected to the low-temperature annealing heater, and the low-temperature annealing heater is located on one side of the PCR amplification reagent delivery line; the medium-temperature extension heating system includes a third temperature controller and a medium-temperature extension heater, the signal input end of the third temperature controller is connected to a computer, the signal output end of the third temperature controller is connected to the medium-temperature extension heater, and the medium-temperature extension heater is located on one side of the PCR amplification reagent delivery line.

3. A high-speed PCR amplification heating device according to claim 2, characterized in that: The PCR amplification reagent conveying line includes a loading track, a circular track and a unloading track, one end of the circular track is connected to the loading track, and the other end of the circular track is connected to the unloading track. An arc-shaped receiving groove is provided on the inner side of the circular track. The high-temperature deformation heater, the low-temperature annealing heater and the medium-temperature extension heater are arranged in an arc shape on the inner side of the circular track and are adapted to the arc-shaped receiving groove. The arc-shaped heating surfaces of the high-temperature deformation heater, the low-temperature annealing heater and the medium-temperature extension heater are respectively facing the high-temperature deformation area, the low-temperature annealing area and the medium-temperature extension area of ​​the PCR amplification reagent conveying line.

4. A high-speed PCR amplification heating device according to claim 3, characterized in that: The PCR amplification reagent conveying line conveys the PCR amplification reagent test tube through a plurality of rotating jigs fixedly arranged at intervals. The rotating jigs are composed of a rotating positioning block and a fixed base. The rotating positioning block is rotatably connected to the fixed base. The PCR amplification reagent test tube is placed on the rotating positioning block for positioning. The width of the annular track is the same as the outer diameter of the PCR amplification reagent test tube. When the PCR amplification reagent test tube passes through the high-temperature deformation area, the low-temperature annealing area, and the medium-temperature extension area, the tube wall of the PCR amplification reagent test tube contacts the arc-shaped heating surfaces of the high-temperature deformation heater, the low-temperature annealing heater, and the medium-temperature extension heater and rotates under the contact force.

5. A high-speed PCR amplification heating device according to claim 4, characterized in that: The PCR amplification reagent test tube adopts a metal test tube.

6. A high-speed PCR amplification heating device according to claim 5, characterized in that: The metal material used for the metal test tube is silver, copper, aluminum, magnesium, zinc, aluminum alloy, aluminum-magnesium alloy, copper alloy or copper-zinc alloy.

7. A high-speed PCR amplification heating device according to claim 4, characterized in that: An annular heat-insulating side plate is arranged on the outer side of the annular track, and the annular heat-insulating side plate abuts against the PCR amplification reagent tube when the tube passes through the high-temperature deformation area, the low-temperature annealing area, and the medium-temperature extension area.

8. A high-speed PCR amplification heating device according to claim 7, characterized in that: An upper cover is provided between the top end of the annular heat-insulating side plate and the top ends of the arc-shaped heating surfaces of the high-temperature deformation heater, the low-temperature annealing heater and the medium-temperature extension heater.

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