CN210945600U

CN210945600U - Quick PCR reaction chip and quick fluorescence quantitative detector

Info

Publication number: CN210945600U
Application number: CN201921551373.2U
Authority: CN
Inventors: 周志图
Original assignee: Bioteke Corp
Current assignee: Bioteke Corp
Priority date: 2019-09-18
Filing date: 2019-09-18
Publication date: 2020-07-07
Anticipated expiration: 2029-09-18

Abstract

The utility model discloses a quick PCR reaction chip and quick fluorescence quantitative determination appearance, PCR chip include panel and basement membrane, and at least one reaction tank is seted up to the panel basal surface, and the reaction tank does not link up the panel, and the sample application hole has been seted up to the reaction tank both sides and exhaust hole and panel top surface intercommunication, adheres to the basement membrane in the panel bottom surface, and the panel is the printing opacity material. When the PCR chip is arranged on the temperature control module, the bottom film is tightly attached to the temperature control module, the PCR reaction liquid and the temperature control module in the reaction tank above the bottom film are only separated by one layer of bottom film after sample addition, the refrigerating capacity or the heating capacity of the temperature control module can quickly penetrate through the bottom film to be conducted to the PCR reaction liquid, the film is faster than the heat conduction speed of a plastic plate, the heat conduction is more uniform, the rapid temperature rise or cooling of the PCR reaction liquid is realized, and the rapid PCR can be realized. The heating and refrigerating sheet of the rapid fluorescence quantitative detector is horizontally arranged, the PCR chip is horizontally arranged on the heating and refrigerating sheet, the bottom film is tightly attached to the plane of the heating and refrigerating sheet, and the refrigerating capacity or the heating capacity of the heating and refrigerating sheet can be rapidly and uniformly conducted to the PCR reaction liquid.

Description

Quick PCR reaction chip and quick fluorescence quantitative detector

Technical Field

The utility model belongs to the technical field of molecular biology detects technique and specifically relates to a quick PCR reaction chip and quick fluorescence quantitative determination appearance.

Background

The PCR amplifier is also called a PCR gene amplifier, a PCR nucleic acid amplifier, and an instrument for amplifying specific DNA by using PCR (Polymerase chain reaction) technology, and is widely used in medical and biological laboratories, and for diagnosis of infectious diseases, paternity testing, and the like. Based on the principle of three steps of PCR reaction, a PCR amplification instrument needs to be provided with 3 temperature points of denaturation-annealing-extension, in a standard reaction, double-stranded DNA is denatured at 90-95 ℃, then is rapidly cooled to 40-60 ℃, a primer is annealed and combined onto a target sequence, then is rapidly heated to 70-75 ℃, and a primer chain is extended along a template under the action of TaqDNA polymerase, so that the PCR amplification instrument is particularly important for rapid temperature switching and accurate control of reaction liquid.

The fast fluorescent quantitative detector is one PCR amplifier with fluorescent signal collecting system and computerized analysis and processing system, and the fluorescent signal is collected in real time with the fluorescent signal collecting system by means of the primer added during PCR amplification and the principle of combining the primer and fluorescent probe with template specificity and amplification.

The PCR chip is used for a micro PCR reaction system, and Chinese patent CN106661533A discloses a multiple PCR chip and a multiple PCR device comprising the same, wherein the multiple PCR chip is formed by bonding a third plate, a second plate and a third plate which are sequentially arranged from top to bottom, a reaction area is arranged between the second plate and the third plate, an inflow part and an outflow part which are communicated with the internal reaction area are arranged at two ends of the second plate, the third plate covers the reaction area, and a plurality of probes are bonded at the bottom of the third plate at intervals. The reaction area of the PCR chip is large in volume, a large amount of sample liquid needs to be used, and the sample liquid is wasted; the light detecting section detects fluorescence reflected from the reaction region and the probe position, and the fluorescence reflection from the reaction region is not dispersed and concentrated. The heating block needs to heat the first plate first, and the reaction liquid is given to first board transmission heat, and the first plate of heating plastic material needs time, leads to the heating time long, is difficult to accurate control temperature, and PCR overall reaction speed is slower. The PCR chip is formed by bonding and assembling the first plate, the second plate and the third plate, and has a complex integral structure and high manufacturing cost.

SUMMERY OF THE UTILITY MODEL

The applicant provides a rapid PCR reaction chip and a rapid fluorescence quantitative detector with reasonable structure aiming at the defects of large reaction liquid consumption, non-concentrated fluorescence reflection, low and inaccurate heating speed, complex structure and the like of the PCR device in the prior production, and the PCR chip has high heat conduction speed, simple structure, easy processing, high reaction speed of the detector and concentrated and easy acquisition of fluorescence signals.

The utility model discloses technical scheme and beneficial effect who adopts as follows:

the utility model provides a quick PCR reaction chip, the PCR chip includes panel and basement membrane, and at least one reaction tank is seted up to the panel basal surface, and the reaction tank does not link up the panel, and the reaction tank both sides have been seted up and have been added the sample hole and exhaust hole and panel top surface intercommunication, and basement membrane is adhered to the panel bottom surface, and the panel is the printing opacity material. The utility model discloses adhere to the basement membrane in the panel bottom surface, on temperature control module was arranged in to the PCR chip, temperature control module was hugged closely to the basement membrane, only interval one deck basement membrane of PCR reaction liquid and temperature control module in the basement membrane top reaction tank behind the application of sample, temperature control module's refrigerating output or heating capacity can pass basement membrane conduction to PCR reaction liquid fast, the plastic slab heat conduction speed is faster compared to the film, it is more even to heat conduction, realize PCR reaction liquid rapid heating up or cooling down (can realize 8-15 ℃/second lift warm-up speed), the reaction rate is accelerated greatly, 40 the reaction time of circulation only need about 20 minutes, can realize quick PCR. In addition, the reaction tank arranged on the bottom surface of the panel does not penetrate through the panel of the PCR chip, so that the reaction tank is an accommodating space shielded from the upper part, and pollutants are prevented from falling into the reaction tank from the upper part; the panel only uses a holistic panel to realize setting up of sample liquid storage space and the closure of reaction tank top promptly itself, overall structure is simple, directly adopt the mould shaping can, easy manufacturing, cost greatly reduced.

As a further improvement of the above technical solution:

and a sealing light shielding film is attached to the upper surface of the PCR chip panel and shields the sample adding hole and the exhaust hole to expose the reaction pool area. The utility model discloses there is the light-shielding film that seals at the upper surface attachment of quick PCR reaction chip, seals the light-shielding film and is arranged in preventing that the reaction liquid in the reaction tank volatilizees during heating and reaction and gets into the outside air, has both influenced the testing result, also can make the outside air have nucleic acid, pollutes the reaction system of next reaction, causes the laboratory pollution.

Strip-shaped flow channels are arranged on two sides of the reaction tank on the bottom surface of the panel, the flow channels do not penetrate through the panel, and sample adding holes and exhaust holes are formed in two ends of each flow channel. The utility model discloses set up the runner on the panel, the runner does not link up the panel, has better seal, prevents that the pollutant from falling into the runner from the top in, runner itself also can realize PCR reaction liquid through the automatic purpose that gets into the reaction tank of capillary, it is more convenient to advance the appearance.

The flow channels on the bottom surface of the panel are arranged in parallel, and the reaction tanks on the adjacent flow channels are arranged in a staggered manner. A plurality of rectangular shape runner parallel arrangement, the reaction tank on the adjacent runner is staggeredly arranged, can arrange more reaction tanks in making unit area on the one hand, make full use of the finite space of panel, and on the other hand, adjacent reaction tank interval is far away, and the fluorescence mutual interference that prevents adjacent reaction tank when fluorescence detects leads to detects the error.

The length of the sample adding flow channel between the sample adding hole and the reaction cell is less than that of the exhaust flow channel between the exhaust hole and the reaction cell, and the width d1 of the sample adding flow channel is greater than that d2 of the exhaust flow channel. The utility model discloses the application of sample hole is near and application of sample runner width broad from the reaction cell for PCR reaction liquid can flow in the reaction cell through capillary action fast and react after getting into the application of sample hole.

The orifice of the sample adding hole is funnel-shaped; the reaction tank is a circular counter bore. The utility model discloses a diameter of the application of sample hole mouth department of panel upper surface is greater than the diameter of exhaust hole mouth department, and the diameter of application of sample hole mouth department is great, and the application of sample hole mouth department is the infundibulate, conveniently advances the appearance. The utility model discloses a reaction tank adopts circular counter bore, and the sample of being surveyed is concentrated in circular reaction tank, and fluorescence signal is the most concentrated, can high-efficiently collect fluorescence signal.

The thickness of the basement membrane is 0.02-0.3 mm. The basement membrane thickness is thin, and heat conduction speed is faster, and the heat conduction is more even.

The material of the panel is selected from one or more of silicon, glass, polymethyl methacrylate, polycarbonate, polystyrene, polyethylene terephthalate, polyethylene naphthalate, cyclic olefin copolymer or epoxy resin. The utility model discloses a panel of quick PCR reaction chip adopts high permeability high polymer such as silicon, glass, polymethyl methacrylate, polycarbonate, polystyrene, polyethylene terephthalate, polyethylene naphthalate or cyclic olefin copolymer and epoxy, but batch production has low cost, easily processing, advantage that the shaping technological requirement is low.

The bottom film is made of transparent material and is selected from one or more of PVC, PE, PP or PS. The utility model discloses the basement membrane of quick PCR reaction chip adopts the printing opacity material, can be PVC, PE, PP and PS etc. and the printing opacity material can not produce the interference to the light path, and the membrane of other colours probably has the variegated like the black membrane, and the light path can be disturbed to the variegated, influences fluorescence detection numerical value.

The sealing light shielding film is adhered in the groove of the panel by ultraviolet glue, epoxy resin glue, phenolic resin glue or silicon rubber. The utility model discloses an in the recess of panel is arranged in to the light-resistant membrane that seals, can prevent to cut to the scratch in the experimentation and drop, lose sealed effect.

A rapid fluorescence quantitative detector using the rapid PCR reaction chip is characterized in that a fluorescence acquisition module, a PCR chip and a temperature control module are sequentially arranged in a shell, the PCR chip is arranged on a heating and refrigerating sheet of the temperature control module, and a bottom film is in contact with the heating and refrigerating sheet; the light source and the lighting component of the fluorescence acquisition module are positioned above the reaction pool of the PCR chip. The utility model discloses quick fluorescence quantitative determination appearance sets up light source and daylighting part above the reaction tank of quick PCR reaction chip, adopts the mode that the top directly gathered fluorescence, gathers that fluorescence is fast, avoid the weak problem that causes the signal to weaken of fluorescence.

As a further improvement of the above technical solution:

the heating and refrigerating piece is horizontally arranged, the PCR chip is horizontally arranged on the heating and refrigerating piece, and the bottom film is tightly attached to the plane of the heating and refrigerating piece. The bottom film is tightly attached to the heating and refrigerating sheet, and the refrigerating capacity or heating capacity of the heating and refrigerating sheet can be quickly and uniformly transferred to the PCR reaction liquid.

Drawings

FIG. 1 is a schematic diagram of the internal structure of the PCR amplification apparatus of the present invention.

Fig. 2 is an enlarged view of a portion a in fig. 1.

FIG. 3 is a bottom view of the PCR chip of the present invention (without a base film attached).

Fig. 4 is a cross-sectional view at B-B in fig. 3 (a position where the base film 13 is added is shown).

FIG. 5 is a plan view of the sealing light shielding film 14 attached to the PCR chip.

FIG. 6 is a diagram of a PCR chip without flow channels according to the second embodiment.

In the figure: 1. a fluorescence collection module; 2. a PCR chip; 3. a temperature control module; 4. a groove; 5. a display screen; 6. a panel; 7. an exhaust hole; 8. a sample addition flow channel; 9. a reaction tank; 10. a sample application hole; 11. a lighting member; 12. a light source; 13. a base film; 14. sealing a light shielding film; 15. heating the refrigeration sheet; 16. a heat sink base; 17. an exhaust flow passage.

Detailed Description

The following describes embodiments of the present invention with reference to the drawings.

As shown in figure 1, the utility model discloses quick fluorescence quantitative determination appearance's the inside from the top down of shell sets gradually fluorescence collection module 1, PCR chip 2 and temperature control module 3, and the shell upper surface is equipped with display screen 5 outwards, and display screen 5 can show progress, the real-time temperature of PCR reaction.

As shown in fig. 1 and 2, the fluorescence collection module 1 is located above the temperature control module 3, and a light collection component 11 and a light source 12 connected to a power supply are disposed on a bottom surface of the fluorescence collection module, the light source 12 projects onto a sample to be tested of the PCR chip 2 through a light path and can excite fluorescence, and the light collection component 11 is used for collecting the fluorescence excited by the sample to be tested. An optical filter (not shown in the figure) capable of filtering fluorescence excited by the sample to be detected is arranged between the lighting component 11 and the sample to be detected, and the optical filter is used for realizing single-channel, double-channel or multi-channel fluorescence detection. The light source 12 is a halogen lamp, a xenon lamp, a light emitting diode or LED light with different fixed wavelengths, and the number of the light sources is one or more; the light-collecting part 11 is a CCD (charge coupled device), a CMOS (complementary metal oxide semiconductor), or a PD (photodiode).

The first embodiment is as follows:

as shown in FIG. 3, the PCR chip 2 comprises a flat rectangular face plate 6 and a bottom membrane 13 covering the bottom of the face plate 6, wherein a plurality of elongated flow channels with the same length are arranged in parallel on the bottom surface of the middle part of the face plate 6 along the direction of the short side of the rectangle, a round counter bore reaction tank 9 is arranged in the middle of the flow channels, and the flow channels and the reaction tank 9 do not penetrate through the whole thickness of the face plate 6. As shown in fig. 4, the bottom film 13 is adhered to the bottom of the face plate 6 so that the flow path and the reaction cell 9 form a closed accommodation space. The reaction tanks 9 on the adjacent flow channels are arranged in a staggered mode, on one hand, more reaction tanks 9 can be arranged in unit area, the limited space of the panel 6 is fully utilized, on the other hand, the interval distance between the adjacent reaction tanks 9 is long, and detection errors caused by mutual interference of fluorescence of the adjacent reaction tanks 9 during fluorescence detection are prevented. The two end parts of the flow channel are respectively provided with a vertical sample adding hole 10 and an exhaust hole 7 which are communicated with the top surface of the panel 6, the sample adding hole 10 is close to the reaction cell 9, the exhaust hole 7 is far from the reaction cell 9, the width d1 of the sample adding flow channel 8 between the sample adding hole 10 and the reaction cell 9 is larger than the width d2 of the exhaust flow channel 17 between the exhaust hole 7 and the reaction cell 9, and the diameter of the reaction cell 9 is larger than the width d1 of the sample adding flow channel 8. The sample adding hole 10 is close to the reaction cell 9, and the width of the sample adding flow channel 8 between the sample adding hole 10 and the reaction cell 9 is wide, so that PCR reaction liquid can quickly flow into the reaction cell 9 for filling through capillary action after entering the sample adding hole 10. As shown in FIG. 3, the diameter of the orifice of the sample adding hole 10 on the upper surface of the panel 6 is larger than that of the orifice of the exhaust hole 7, and as shown in FIG. 4, the orifice of the sample adding hole 10 is funnel-shaped, so that sample introduction is facilitated.

The volume of the reaction tank 9 is 1-20 mu L, the amount of the samples to be used is effectively saved, the waste of the samples is avoided, the volume of the samples to be used is small, the temperature rising and falling speed is high, the reaction time is greatly shortened, and the temperature can be accurately controlled. The panel 6 is made of transparent, heat-conductive and light-transmitting materials, such as silicon, glass, polymethyl methacrylate, polycarbonate, polystyrene, polyethylene terephthalate, polyethylene naphthalate, cyclic olefin copolymer, epoxy resin, and the like. The thickness of the basement membrane 13 is 0.02-0.3 mm. The bottom film 13 is made of a transparent material, the bottom film 13 is made of a high polymer material, the transparent material comprises PVC (polyvinyl chloride), PE (polyethylene), PP (polypropylene), PS (polystyrene) and the like, the light path cannot be interfered by the transparent material, and films of other colors such as a black film may have mixed colors which interfere the light path and influence the fluorescence detection value. The bottom film 13 can be adhered to the bottom surface of the panel 6 by using adhesive such as ultraviolet glue, epoxy resin glue, phenolic resin glue or silicone rubber, so as to seal the bottom surface of the panel 6 and form independent reaction tanks 9.

As shown in fig. 5, seal the film of light shielding film 14 for the rectangular light-tight material, thickness is 30-300 μm, as shown in fig. 4, set up the rectangle recess 4 that is greater than the reaction zone in the front of panel 6, seal the peripheral dimension and the recess 4 phase-match of light shielding film 14, accomplish PCR reaction liquid and advance the appearance back, seal light shielding film 14 and laminate in recess 4 through the gum under normal atmospheric temperature, at this moment, as shown in fig. 5, seal light shielding film 14 and shield and seal the whole sample adding hole 10 and the exhaust hole 7 in both sides, expose middle part reaction tank 9 region. The sealing light shielding film 14 is used for preventing the reaction liquid in the reaction tank 9 from volatilizing into the outside air during heating and reaction, which affects the detection result, and also causes the outside air to have nucleic acid to pollute the reaction system of the next reaction, and causes laboratory pollution. The material of the sealing light shielding film 14 is polyvinyl chloride, polycarbonate, polyethylene terephthalate, polyethylene naphthalate and the like; the sealing light shielding film 14 is bonded by a normal temperature or non-normal temperature bonding method, and the bonding glue is ultraviolet glue, epoxy resin glue, phenolic resin glue or silicon rubber.

As shown in fig. 1 and 2, the flat rectangular parallelepiped heating/cooling plate 15 of the temperature control module 3 is horizontally fixed on the heat sink base 16, the lower portion of the heat sink base 16 has a plurality of heat dissipation ribs, the PCR chip 2 is horizontally placed on the heating/cooling plate 15, the bottom film 13 is tightly attached to the plane of the heating/cooling plate 15, and the heating/cooling plate 15 adjusts the temperature inside the reaction tank 9 of the PCR chip 2 through heating or cooling functions. The temperature control module 3 is connected with a temperature control software system capable of setting temperature parameters. The plane of heating refrigeration piece 15 is hugged closely to basement membrane 13, the PCR reaction liquid of basement membrane 13 top is only separated by one deck basement membrane 13 with temperature control module 3 after the application of sample, the refrigerating output or the heating output of temperature control module 3 can pass basement membrane 13 fast and conduct to PCR reaction liquid, the film is faster than plastic slab heat conduction speed, realize PCR reaction liquid rapid heating up or cooling down (can realize 8-15 ℃/second rise and fall warm speed), the reaction rate has been accelerated greatly, the temperature control precision has been improved, 40 endless reaction times only need about 20 minutes, can realize quick PCR.

The utility model discloses during the use, carry out following step:

(1) adding PCR reaction liquid into a sample adding hole 10 of a PCR chip 2 by using a pipette, wherein the reaction liquid comprises template DNA, a primer, Taq enzyme, buffer solution and the like, and the PCR reaction liquid flows into a reaction tank 9 through capillary action due to the existence of an exhaust hole 7, and then bonding a sealing light shielding film 14 in a groove 4 of the PCR chip 2;

(2) placing the PCR chip 2 added with the sample on a heat radiating fin base 16 of the heating and refrigerating fin 15;

(3) starting a temperature control software system, and setting the reaction temperature required by the PCR chip 2;

(4) after the reaction is finished, the fluorescence collection module 1 is started, the light source 12 excites fluorescence and projects the fluorescence onto the reaction pool 9 of the PCR chip 2, the excited fluorescence of the reaction liquid is projected onto the lighting part 11 to form spectral imaging and is collected by the lighting part 11, and the collected fluorescence is analyzed and positioned by software to obtain a light intensity value.

Example two:

as shown in fig. 6, in this embodiment, no flow channel is provided, and the sample adding hole 10 and the exhaust hole 7 are directly formed at two opposite ends of the reaction cell 9, so that the purpose of sample introduction and exhaust of the reaction solution can be achieved, and after sample introduction, the sealing light shielding film 14 is attached to the upper surface of the panel 6 to shield the sample adding hole 10 and the exhaust hole 7.

The above description is illustrative of the present invention and is not intended to limit the present invention, and the present invention may be modified in any manner without departing from the spirit of the present invention. When the utility model discloses a when reaction tank 9 adopted circular counter bore, the sample of being surveyed is concentrated in circular reaction tank 9, and fluorescence signal is most concentrated, can collect fluorescence signal by the high efficiency, and reaction tank 9 also can adopt the counter bore reaction tank 9 that the cross section is any other shapes such as triangle-shaped, rectangle, square certainly, and the sample of being surveyed in the reaction tank 9 can launch fluorescence signal equally. The light source 12 may be disposed at the side of the reaction cell 9. The flow passage may be provided in a single piece.

The sample adding hole 10 and the exhaust hole 7 can also be blind holes which are arranged from the upper surface of the panel 6 to the bottom and do not penetrate through the panel 6, the hole openings are communicated with the top surface of the panel 6, the hole bottoms are communicated with the reaction tank 9, and the purpose of feeding the reaction liquid can be achieved.

Claims

1. A rapid PCR reaction chip is characterized in that: the PCR chip (2) comprises a panel (6) and a bottom film (13), at least one reaction tank (9) is arranged on the bottom surface of the panel (6), the reaction tank (9) does not penetrate through the panel (6), sample adding holes (10) and exhaust holes (7) are formed in two sides of the reaction tank (9) and communicated with the top surface of the panel (6), the bottom film (13) is attached to the bottom surface of the panel (6), and the panel (6) is made of a light-transmitting material.

2. The rapid PCR reaction chip of claim 1, wherein: a sealing light shielding film (14) is attached to the upper surface of a panel (6) of the PCR chip (2), and the sealing light shielding film (14) shields the sample adding hole (10) and the exhaust hole (7) to expose the area of the reaction tank (9).

3. The rapid PCR reaction chip of claim 1, wherein: strip-shaped flow channels are arranged on two sides of the reaction tank (9) on the bottom surface of the panel (6), the flow channels do not penetrate through the panel (6), and the two ends of each flow channel are provided with a sample adding hole (10) and an exhaust hole (7).

4. The rapid PCR reaction chip according to claim 3, wherein: the flow channels on the bottom surface of the panel (6) are arranged in parallel, and the reaction tanks (9) on the adjacent flow channels are arranged in a staggered way.

5. The rapid PCR reaction chip according to claim 3, wherein: the length of the sample adding flow channel (8) between the sample adding hole (10) and the reaction cell (9) is less than the length of the exhaust flow channel (17) between the exhaust hole (7) and the reaction cell (9), and the width d1 of the sample adding flow channel (8) is greater than the width d2 of the exhaust flow channel (17).

6. The rapid PCR reaction chip of claim 1, wherein: the orifice of the sampling hole (10) is funnel-shaped; the reaction tank (9) is a circular counter bore.

7. The rapid PCR reaction chip of claim 1, wherein: the thickness of the bottom film (13) is 0.02-0.3 mm.

8. The rapid PCR reaction chip of claim 1, wherein: the material of the panel (6) is silicon, glass, polymethyl methacrylate, polycarbonate, polystyrene, polyethylene terephthalate, polyethylene naphthalate, cyclic olefin copolymer or epoxy resin; the bottom film (13) is made of a light-transmitting material, and can be made of PVC, PE, PP or PS; the sealing light shielding film (14) is bonded in the groove (4) of the panel (6) by adopting ultraviolet glue, epoxy resin glue, phenolic resin glue or silicon rubber.

9. A rapid fluorescence quantitative detector using the rapid PCR reaction chip of claim 1, characterized in that: a fluorescence acquisition module (1), a PCR chip (2) and a temperature control module (3) are sequentially arranged in the shell, the PCR chip (2) is arranged on a heating and refrigerating sheet (15) of the temperature control module (3), and a base film (13) is in contact with the heating and refrigerating sheet (15); the light source (12) and the lighting component (11) of the fluorescence collection module (1) are positioned above the reaction pool (9) of the PCR chip (2).

10. The fluorescence quantitative detector according to claim 9, characterized in that: the heating and refrigerating sheet (15) is horizontally arranged, the PCR chip (2) is horizontally arranged on the heating and refrigerating sheet (15), and the bottom film (13) is tightly attached to the plane of the heating and refrigerating sheet (15).