阅读说明：本技术 快速pcr反应芯片以及快速荧光定量检测仪 (quick PCR reaction chip and quick fluorescence quantitative detector ) 是由 周志图 于 2019-09-18 设计创作，主要内容包括：本发明公开了一种快速PCR反应芯片及快速荧光定量检测仪,PCR芯片包括面板与底膜,面板底表面开设至少一处反应池,反应池不贯通面板,反应池两侧开设有加样孔和排气孔与面板顶面连通,在面板底面附着底膜,面板为透光材质。当PCR芯片置于温控模块上,底膜紧贴温控模块,加样后底膜上方反应池中的PCR反应液与温控模块仅间隔一层底膜,温控模块的制冷量或制热量可以快速穿过底膜传导至PCR反应液,薄膜相比塑料板导热速度更快,导热更均匀,实现PCR反应液快速升温或降温,可以实现快速PCR。快速荧光定量检测仪的加热制冷片水平放置,PCR芯片水平放置在加热制冷片上,底膜紧贴加热制冷片的平面,加热制冷片的制冷量或制热量可以快速均匀传导至PCR反应液。(The invention discloses a rapid PCR reaction chip and a rapid fluorescence quantitative detector, the PCR chip comprises a panel and a bottom film, at least one reaction tank is arranged on the bottom surface of the panel, the reaction tank does not penetrate through the panel, sample adding holes and exhaust holes are arranged on two sides of the reaction tank and communicated with the top surface of the panel, the bottom surface of the panel is attached with the bottom film, and the panel is made of a light-transmitting 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.)

1. A rapid PCR reaction chip is characterized in that: PCR chip (2) are including panel (6) and basement membrane (13), and at least one reaction tank (9) is seted up to panel (6) basal surface, and reaction tank (9) do not link up panel (6), and application of sample hole (10) and exhaust hole (7) and panel (6) top surface intercommunication have been seted up to reaction tank (9) both sides, and basement membrane (13) are adhered to at panel (6) bottom surface, and panel (6) are the printing opacity material.

2. The rapid PCR reaction chip according to 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 according to 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 according to 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 according to claim 1, wherein the thickness of the base film (13) is 0.02 ~ 0.3.3 mm.

8. The rapid PCR reaction chip according to claim 1, wherein: the material of the panel (6) is selected from one or a plurality of silicon, glass, polymethyl methacrylate, polycarbonate, polystyrene, polyethylene terephthalate, polyethylene naphthalate, cyclic olefin copolymer or epoxy resin; the bottom film (13) is made of transparent material and is selected from one or a combination 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).

Technical Field

The invention relates to the technical field of molecular biology detection, in particular to a rapid PCR reaction chip and a rapid fluorescence quantitative detector.

background

Based on the three-step PCR reaction principle, the PCR amplification instrument needs to set 3 temperature points of denaturation-annealing-extension, double-stranded DNA is denatured at 90 ~ 95 ℃ in the standard reaction, then the double-stranded DNA is rapidly cooled to 40 ~ 60 ℃, a primer is annealed and combined onto a target sequence, then the temperature is rapidly raised to 70 ~ 75 ℃, and the primer chain is extended along a template under the action of TaqDNA Polymerase, so the PCR amplification instrument is particularly important for the rapid temperature switching and the precise control of the 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.

Disclosure of Invention

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 technical scheme and the beneficial effects adopted by the invention are 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. According to the invention, the bottom film is attached to the bottom surface of the panel, 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 in the reaction tank above the bottom film is separated from the temperature control module by only one layer of bottom film after sample addition, the refrigerating capacity or the heating capacity of the temperature control module can be quickly transmitted to the PCR reaction liquid through the bottom film, the film has higher heat conduction speed and more uniform heat conduction compared with a plastic plate, the PCR reaction liquid is quickly heated or cooled (the heating and cooling speed of 8-15 ℃/second can be realized), the reaction speed is greatly increased, the reaction time of 40 cycles only needs about 20 minutes, and the quick PCR can. 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 sealing light-shielding film is attached to the upper surface of the rapid PCR reaction chip and is used for preventing reaction liquid in the reaction tank from volatilizing into outside air during heating and reaction, so that a detection result is influenced, and the outside air has nucleic acid to pollute a reaction system of next reaction and cause 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 flow channel is arranged on the panel, does not penetrate through the panel, has better sealing performance, prevents pollutants from falling into the flow channel from the upper part, can automatically enter the reaction pool through capillary action, and is more convenient to sample.

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 sample adding hole is close to the reaction tank and the width of the sample adding flow channel is wide, so that PCR reaction liquid can quickly flow into the reaction tank for reaction through capillary action after entering the sample adding hole.

The orifice of the sample adding hole is funnel-shaped; the reaction tank is a circular counter bore. The diameter of the sample adding hole on the upper surface of the panel is larger than that of the exhaust hole, the diameter of the sample adding hole is larger, and the sample adding hole is funnel-shaped, so that sample introduction is facilitated. The reaction tank adopts the circular counter bore, the tested sample is concentrated in the circular reaction tank, the fluorescence signal is most concentrated, and the fluorescence signal can be efficiently collected.

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 panel of the rapid PCR reaction chip adopts high-permeability high polymer such as silicon, glass, polymethyl methacrylate, polycarbonate, polystyrene, polyethylene terephthalate, polyethylene naphthalate or cycloolefin copolymer and epoxy resin, can be produced in batches, and has the advantages of low cost, easiness in processing and low requirement on a forming process.

The bottom film is made of transparent material and is selected from one or more of PVC, PE, PP or PS. The bottom film of the rapid PCR reaction chip adopts a light-transmitting material which can be PVC, PE, PP, PS and the like, the light-transmitting material cannot interfere a light path, and films with other colors such as a black film may have mixed colors which interfere the light path and influence the fluorescence detection 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 sealing light-resistant film is arranged in the groove of the panel, so that the sealing light-resistant film can be prevented from being scratched and falling off in the experimental process and losing the sealing 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 rapid fluorescence quantitative detector is provided with the light source and the lighting part above the reaction pool of the rapid PCR reaction chip, adopts a mode of directly collecting fluorescence from the top, has high fluorescence collecting speed and avoids the problem of signal attenuation caused by fluorescence attenuation.

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 FIG. 1, a fluorescence collection module 1, a PCR chip 2 and a temperature control module 3 are sequentially arranged in a shell of the rapid fluorescence quantitative detector from top to bottom, a display screen 5 is arranged on the upper surface of the shell facing outwards, and the display screen 5 can display the progress and 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).