CN115369029A

CN115369029A - Quick PCR appearance

Info

Publication number: CN115369029A
Application number: CN202211161109.4A
Authority: CN
Inventors: 刘晓雷; 袁新旺; 闫媛媛; 徐超
Original assignee: Suzhou Yingze Biomedical Technology Co ltd
Current assignee: Suzhou Yingze Biomedical Technology Co ltd
Priority date: 2022-09-23
Filing date: 2022-09-23
Publication date: 2022-11-22

Abstract

The invention discloses a rapid PCR (polymerase chain reaction) instrument, which comprises a first heating module and a second heating module which are movably arranged in a PCR instrument device body along the vertical direction, wherein the two heating modules provide different reaction temperatures for a PCR reaction tube, holes for the insertion of the PCR reaction tube are formed in the first heating module and the second heating module, the diameter of each hole is larger than that of the PCR reaction tube, the holes in the first heating module and the holes in the second heating module are in one-to-one correspondence in position and quantity, the bottom of the PCR reaction tube is colorless and transparent, and the PCR instrument further comprises a heat cover. The PCR instrument can rapidly change the temperature of a reaction system, shorten the reaction time, improve the temperature uniformity of different reaction systems, ensure accurate and reliable results, and has a simple structure and greatly prolonged service life.

Description

Quick PCR appearance

Technical Field

The invention relates to the technical field of molecular biology detection, in particular to a rapid PCR instrument.

Background

In the prior art, a PCR apparatus (thermal cycler) is generally used by placing a tube or a plate in a fixed metal heating block, and controlling the temperature of the tube or the plate by heating or cooling the metal heating block. The problems thus posed are: (1) the heating and cooling required a relatively long time, so the time required to actually complete the reaction was much longer than the theoretically calculated (denaturation time + extension time) × cycle number (for example, the PCR reaction program was set to 95 degrees pre-denaturation for 5 minutes; 95 degrees denaturation for 1 second; 60 degrees extension for 10 seconds; 40 cycles; the reaction time calculated according to this program was only 12 minutes and 20 seconds, but in practice, the typical thermal cycler temperature variation rate was 3-5 degrees per second, and it took 25-30 minutes to actually run through this program). (2) The temperature of different positions of the metal heating block is different in the heating and cooling processes, and when a multi-tube reaction is carried out, the temperature in different tubes is different, so that the result is deviated. (3) The instrument needs frequent and rapid temperature rise and fall during working, the performance is attenuated after the instrument is used for a period of time, and faults are easy to occur. Based on the above technology, to accelerate the PCR reaction requires more powerful heating and cooling capabilities, so that the temperature of the metal heating block can change rapidly (for example, from 3/10/s), but this requires increasing the power of the apparatus, and more advanced heating and cooling systems are adopted, resulting in increased cost. Therefore, it is difficult to improve the temperature change rate of the metal heating block, and the limit is low, and if the metal heating block is supposed to rise/fall the temperature by 100 degrees per second, the accurate temperature control is almost impossible. Moreover, even if the rate of temperature change of the metal heating block is increased, it is a great challenge to maintain the uniformity of the temperature thereof.

In addition, the existing PCR instrument is basically provided with a metal heating module with the lower part for controlling the temperature, and a hot cover with a hole, a light source and a detection device on the upper part. The matched consumable material is a pipe or a perforated plate with a conical bottom, a cover is covered after a reaction system is added, or a colorless and transparent sealing plate film is pasted on the upper surface of the reaction system, and then the reaction system is placed on a metal heating module for reaction. Exciting light emitted by a light source positioned above the thermal cover irradiates the reaction system through the filter and the hole on the thermal cover, and an excited fluorescence signal is received and detected by the photoelectric tube and the like through the hole on the thermal cover, the filter and the lens. The problem with this configuration of the system is that the thermal cover must be perforated to allow passage of excitation light and fluorescence. Therefore, the tube cover or the sealing plate film is required to be clean and have good transmittance, and meanwhile, the opening is required to be formed in the heat cover so as to ensure that the fluorescent signal is not influenced; if the sealing of the tube cap or the sealing plate membrane is not good enough, water in the reaction system overflows from the sealing position after evaporation, and finally detection failure is caused. In addition, the detection from the top also requires that no air bubbles can exist in the PCR reaction system, otherwise the air bubbles float on the reaction system or break during the reaction process, which can cause inaccurate detection results.

How to solve the technical problems is a matter which needs to be solved urgently by the technical personnel in the field.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a rapid PCR instrument.

In order to achieve the purpose, the invention adopts the technical scheme that: a rapid PCR instrument comprises a group of heating modules which can be movably arranged in the vertical direction in a PCR instrument device body, and a first heating module and a second heating module which are parallel to each other, wherein the two heating modules provide different reaction temperatures for a PCR reaction tube, and the first heating module is used for providing the reaction temperature for the PCR reaction tube in a template denaturation stage; the second heating module is used for providing reaction temperature for the PCR reaction tube in the renaturation and extension stage, holes for the PCR reaction tube to insert and pass through are formed in the first heating module and the second heating module, the diameter of each hole is larger than that of the PCR reaction tube, the holes in the first heating module and the holes in the second heating module correspond to each other in position and number one by one, the bottom of the PCR reaction tube is colorless and transparent, and the PCR instrument further comprises a detection module arranged below the PCR reaction tube and a heat cover arranged above the PCR reaction tube and used for compressing a tube cover of the PCR reaction tube.

In a specific embodiment, sealing rings made of flexible heat conduction materials are installed in the holes on the first heating module and the second heating module.

As a specific embodiment, the sealing ring is made of a graphite material.

In a specific embodiment, the first heating module is located above the second heating module, and the first heating module provides a higher reaction temperature for the PCR reaction tube than the second heating module provides for the PCR reaction tube.

As a specific embodiment, the upper pipe diameter of the PCR reaction pipe is smaller than the lower pipe diameter.

As a specific implementation manner, a support for installing the first heating module and the second heating module is arranged in the PCR instrument device body, the support includes side plates located on two sides of the first heating module and the second heating module in the length direction, a sliding groove extending along the vertical direction is formed in the side plates, and two ends of the first heating module and the second heating module in the length direction are inserted into the sliding grooves on the two sides and can be slidably arranged along the length extending direction of the sliding groove.

In a specific embodiment, a driving motor for driving the first heating module and the second heating module to ascend and descend along the channel direction of the chute is further arranged in the PCR instrument body.

In a specific embodiment, the first heating module, the second heating module and the hot cover are all metal blocks, and the temperature of the hot cover is not lower than 100 ℃.

As a specific implementation manner, the detection module includes an excitation light source, a filter and a CCD camera, light emitted from the excitation light source is irradiated to the reaction system in the PCR reaction tube from the bottom of the PCR reaction tube, and the excited fluorescence is received and analyzed by the CCD camera via the filter.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages: the rapid PCR instrument of the invention adopts the upper and lower independent heating modules to heat the PCR reaction tube, controls the temperature of the PCR reaction system by controlling the temperature of the two heating modules and the contact time between the heating modules and the PCR reaction tube, so as to achieve the purposes of rapidly changing the temperature of the reaction system and shortening the reaction time, and simultaneously improve the uniformity of the temperatures of different reaction systems.

Drawings

FIG. 1 is a schematic structural diagram of the rapid PCR apparatus;

wherein 1, a first heating module; 2. a second heating module; 3. a PCR reaction tube; 4. a hot lid; 5. a detection module; 6. a hole; 7. a seal ring; 8. a side plate; 9. the motor is driven.

Detailed Description

The technical solution of the present invention is further explained with reference to the accompanying drawings and specific embodiments.

A rapid PCR instrument is shown in figure 1 and comprises a group of heating modules which can be movably arranged in the PCR instrument body along the up-down direction, wherein the heating modules comprise a first heating module 1 and a second heating module 2 which are parallel to each other, the two heating modules provide different reaction temperatures for a PCR reaction tube 3, and the first heating module 1 is used for providing the reaction temperatures for the PCR reaction tube 3 in a template denaturation stage; the second heating module 2 is used for providing reaction temperature for the PCR reaction tube 3 in renaturation and extension stage, all offer on first heating module 1 and the second heating module 2 and supply the PCR reaction tube 3 to insert the hole 6 of establishing the passing through, the diameter of this hole 6 is greater than the diameter of PCR reaction tube 3, hole 6 on first heating module 1 and the hole 6 on the second heating module 2 are in position and the one-to-one in quantity, the bottom of PCR reaction tube 3 is colorless transparent, the PCR appearance is still including setting up detection module 5 and setting in PCR reaction tube 3 top in PCR reaction tube 3 below PCR reaction tube 3, be used for compressing tightly the hot lid 4 on the PCR reaction tube 3 tube lid. The first heating module 1, the second heating module 2 and the heat cover 4 are all made of metal blocks.

In this example, the detection module 5 is located below the PCR reaction tube 3, and the bottom of the PCR reaction tube 3 is colorless and transparent. Here, the detection module 5 includes an excitation light source, a filter, and a CCD camera, and the laser sheet is disposed in the light outgoing direction of the excitation light source. The excitation light source may be a composite light source (such as a halogen lamp, a xenon lamp, or a white light LED), or may be one or more monochromatic light sources, in the detection process, the excitation light in the detection module 5 (if the light source is the composite light source, the light passes through the optical filter before reaching the PCR tube, if the light source is the monochromatic light source, the optical filter is not needed) is irradiated from below the PCR reaction tube 3, and the excited fluorescence is also transmitted through the bottom of the PCR reaction tube 3, and is received by the CCD camera (the optical filter may be added in front of the CCD camera to filter the stray light other than the fluorescence signal, for example, the reflected excitation light) and analyzed. Here, since the optical signal is transmitted from the bottom of the tube and the bubble is usually located on the liquid surface, the detection method can completely avoid the interference of the bubble, and the thermal cover 4 is pressed against the tube cover of the PCR reaction tube 3, the temperature of the thermal cover 4 is not lower than 100 ℃, and is set to be 105 ℃ to 110 ℃, so that the temperature of the tube cover of the PCR reaction tube 3 is not lower than 100 ℃, and the water is effectively prevented from evaporating from the hot reaction system and condensing on the tube cover. And here, since the optical signal does not need to pass through the cap, the color of the cap is not limited. Meanwhile, since the detection module 5 is located below the PCR reaction tube 3, the thermal cover 4 in this embodiment is a one-piece metal plate without opening holes.

Here, the sealing rings 7 made of flexible heat conducting material are installed in the holes 6 of the first heating module 1 and the second heating module 2. In particular, the sealing ring 7 is made of graphite material. By arranging the sealing ring 7, heat transfer is facilitated when the heating module heats the PCR reaction tube 3. The inner ring of the sealing ring 7 is a smooth surface so as to reduce the frictional resistance between the heating module and the PCR reaction tube 3 in the lifting process. The hole 6 of the heating module is larger than the diameter of the PCR reaction tube 3 so as to facilitate the lifting of the heating module, and the sealing ring 7 is arranged so as to facilitate the heat transfer when the PCR reaction tube 3 is heated and reduce the heat loss. In addition, there may be a plurality of holes 6, so that PCR reactions can be simultaneously performed on a plurality of PCR reaction tubes 3.

In this example, the first heating module 1 is located above the second heating module 2, the first heating module 1 and the second heating module 2 have independent temperature control devices, and the reaction temperature provided by the first heating module 1 to the PCR reaction tube 3 is higher than the reaction temperature provided by the second heating module 2 to the PCR reaction tube 3. The upper part of the PCR reaction tube 3 has a small tube diameter and the lower part has a large tube diameter. Through the setting mode, when the second heating module 2 heats the PCR reaction tube 3, the first heating module 1 is not contacted with the PCR reaction tube 3, and the PCR reaction system is not influenced.

When the PCR reaction tube is used specifically, after the PCR reaction tube 3 passes through the holes 6 on the first heating module 1 and the second heating module 2, the PCR reaction tube is fixed, the heat cover 4 is pressed on the tube cover of the PCR reaction tube 3, different reaction temperatures are set for the first heating module 1 and the second heating module 2 respectively, and if the reaction temperature provided by the first heating module 1 is 95 ℃, the reaction temperature provided by the second heating module 2 is 60 ℃. Then, setting the contact time of the two heating modules and the PCR reaction tube 3 as required to regulate and control the temperature of the PCR reaction system, and adjusting the position of the first heating module 1 to the reaction system during the denaturation stage to heat the PCR reaction tube 3; in the renaturation stage and the extension stage, the position of the second heating module 2 is adjusted to the reaction system to cool the reaction system, and the purposes of quickly changing the temperature of the reaction system and shortening the time required by the reaction can be achieved by adopting the mode.

In this example, a support for installing the first heating module 1 and the second heating module 2 is arranged in the PCR instrument body, the support includes side plates 8 located on two sides of the first heating module 1 and the second heating module 2 in the length direction, a chute extending in the vertical direction is formed in the side plates 8, and two ends of the first heating module 1 and the second heating module 2 in the length direction are inserted into the chutes on two sides and can slide along the length extending direction of the chute. Here, a driving motor 9 for driving the first heating module and the second heating module to ascend and descend along the channel direction of the chute is further arranged in the PCR instrument body. Here, there are two sets of driving motors 9 for driving the first and second heating modules 1 and 2 to ascend and descend in the track direction of the chute, respectively. Specifically, there are two driving motors 9 in each group, which are located at two ends of the heating module in the length direction, respectively, the two driving motors 9 are synchronous driving motors 9, and output ends of the two driving motors 9 are connected to two ends of the heating module, respectively. The lifting action of the heating module can be completed within 0.1 second by the driving motor 9, so that the working time is greatly saved.

In addition, the distance between the first heating module 1 and the second heating module 2 is basically fixed, the driving motors 9 can also be a group, and the two heating modules are driven to synchronously lift and fall by the two driving motors 9 positioned at the two sides of the heating modules.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A rapid PCR instrument is characterized by comprising a group of heating modules which can be movably arranged in the PCR instrument body along the vertical direction, a first heating module and a second heating module which are parallel to each other, wherein the two heating modules provide different reaction temperatures for a PCR reaction tube, and the first heating module is used for providing the reaction temperature for the PCR reaction tube in a template denaturation stage; the second heating module is used for providing reaction temperature for the PCR reaction tube in the renaturation and extension stage, holes for the PCR reaction tube to insert and establish are formed in the first heating module and the second heating module, the diameter of each hole is larger than that of the PCR reaction tube, the holes in the first heating module correspond to the holes in the second heating module in position and quantity one by one, the bottom of the PCR reaction tube is colorless and transparent, and the PCR instrument further comprises a detection module arranged below the PCR reaction tube and a heat cover arranged above the PCR reaction tube and used for compressing the tube cover of the PCR reaction tube.

2. The rapid PCR instrument of claim 1, wherein the first and second heating modules have sealing rings made of flexible heat-conducting material installed in the holes.

3. The multi-functional PCR instrument according to claim 2, wherein the sealing ring is made of graphite material.

4. The rapid PCR instrument of claim 1, wherein the first heating module is located above the second heating module, and the first heating module provides a higher reaction temperature for the PCR reaction tubes than the second heating module provides for the PCR reaction tubes.

5. The rapid PCR apparatus according to claim 4, wherein the upper tube diameter of the PCR reaction tube is smaller than the lower tube diameter.

6. The rapid PCR instrument of claim 1, wherein a support for mounting the first heating module and the second heating module is arranged in the PCR instrument device body, the support comprises side plates located on two sides of the first heating module and the second heating module in the length direction, a sliding groove extending in the vertical direction is formed in each side plate, and two ends of the first heating module and the second heating module in the length direction are inserted into the sliding grooves on the two sides and can be slidably arranged in the length extending direction of the sliding groove.

7. The rapid PCR instrument according to claim 6, wherein a driving motor for driving the first heating module and the second heating module to move up and down along the channel direction of the chute is further arranged in the PCR instrument device body.

8. The rapid PCR instrument according to claim 1, wherein the first heating module, the second heating module and the thermal cover are all metal blocks, and the temperature of the thermal cover is not lower than 100 ℃.

9. The rapid PCR instrument of claim 1, wherein the detection module comprises an excitation light source, a filter and a CCD camera, light emitted from the excitation light source is irradiated to the reaction system in the PCR reaction tube from the bottom of the PCR reaction tube, and the excited fluorescence is received and analyzed by the CCD camera via the filter.