CN112159756A - Rapid PCR amplification and CRISPR visual detection device and detection method - Google Patents
Rapid PCR amplification and CRISPR visual detection device and detection method Download PDFInfo
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- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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Abstract
The invention relates to a rapid PCR amplification and CRISPR visualization detection device and a detection method. Visual detection device includes airtight module and heating module, and wherein airtight module includes: the PCR amplification device comprises a lower carrier, an upper cover body, a plurality of PCR amplification chambers arranged on the lower carrier and a plurality of CRISPR reaction chambers with corresponding numbers, wherein the amplification chambers are communicated with the corresponding CRISPR reaction chambers through connecting channels, and the upper cover body is hermetically connected with the lower carrier and seals each chamber and the connecting channels; the heating module is used for heating the PCR amplification chamber and/or the CRISPR reaction chamber. Compared with the prior art, the device has large specific surface area and improves the heat transfer efficiency, thereby greatly shortening the PCR amplification time and further shortening the total detection time to within a few minutes. The invention can realize the amplification of a plurality of PCR systems and the CRISPR visual detection, and integrates the temperature-controllable heating module in the detection device, so that the detection is more convenient and the detection time is shortened.
Description
Technical Field
The invention belongs to the field of nucleic acid amplification and visual detection, and particularly relates to a rapid PCR amplification and CRISPR visual detection device and a detection method.
Background
Since the advent of the Polymerase Chain Reaction (PCR) technology in 1985, the nucleic acid amplification detection technology has been rapidly applied to various fields such as medical treatment, agriculture, and environmental sanitation with its high detection sensitivity. For the detection of nucleic acid amplification products, the visual detection method can be used for judging results by visual observation without instrument and equipment, so that the method has great advantages of being applied to the field and rapid detection of nucleic acids. The existing visual detection method generally adopts high-concentration fluorescent intercalating dyes (SYTO 9, SYBR GREEN I, ethidium bromide, propidium iodide and the like) and nucleic acid intercalating dyes (crystal violet and the like), and the visual detection is realized by the bright fluorescence emitted by the dye intercalating nucleic acid double strands; or adopting calcein, methylene blue, turbidity method, etc., and realizing visual detection by reacting ions in the solution with pyrophosphate which is a by-product of nucleic acid amplification; or adding a pH sensitive reagent into the amplification solution, generating by-product hydrogen ions in the amplification process to cause the pH change of the solution, and observing the color change of the pH sensitive reagent to realize visual detection. The detection target of the above visual detection method is not the amplicon sequence itself, and thus the detection result lacks specificity. The development of specific visual detection methods is essential to enhance the reliability of nucleic acid on-site, rapid and visual detection results.
The CRISPR method has been developed in recent years for its high specificity for target sequence recognition to be applied in the field of nucleic acid detection. CRISPR methods are often combined with nucleic acid amplification because of their low sensitivity of detection alone. However, because the CRISPR system is incompatible with the nucleic acid amplification system or has a different reaction temperature from the nucleic acid amplification system, which causes a great reduction or inactivation of the cleavage efficiency of the CRISPR/Cas enzyme, researchers usually perform nucleic acid amplification and CRISPR detection in two steps, and transfer the amplified product into the CRISPR system for detection after the nucleic acid amplification is completed. The operation is complicated and fussy, and because the yield of the amplicon is high, the problem of amplicon aerosol pollution is easily caused by uncovering after the amplification is finished, so that the subsequent detection result is false positive, and the detection result is unreliable. Therefore, the development of a method and a closed device for integrating nucleic acid amplification and CRISPR detection is necessary for realizing on-site, rapid, visual and specific detection of nucleic acid.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a rapid PCR amplification and CRISPR visualization detection device and a detection method.
The visual detection device and the detection method provided by the invention can realize the amplification of a plurality of PCR systems and the CRISPR visual detection, and the temperature-controllable heating module is integrated in the detection device, so that the detection is more convenient and the detection time is shortened.
The purpose of the invention can be realized by the following technical scheme:
the invention provides a rapid PCR amplification and CRISPR visual detection device, which comprises: the device comprises a closed module and a heating module;
the hermetic module includes: a lower carrier and an upper cover;
the lower carrier is provided with a plurality of PCR amplification chambers and a plurality of CRISPR reaction chambers with the number corresponding to that of the PCR amplification chambers, and the PCR amplification chambers are respectively communicated with the corresponding CRISPR reaction chambers through connecting channels;
the upper cover body is hermetically connected with the lower cover body, so that each PCR amplification chamber, each CRISPR reaction chamber and each connecting channel are sealed;
the heating module is arranged above and/or below the closed module and is used for heating each PCR amplification chamber and/or each CRISPR reaction chamber.
Wherein each of said PCR amplification chambers is for providing a PCR amplification reaction site and each of said CRISPR reaction chambers is for providing a CRISPR reaction site.
In a preferred embodiment of the invention, the lower support is a triangular support, a rectangular support or a disc support, preferably a disc support.
In a preferred embodiment of the present invention, the lower vector is a disc vector, and each of the PCR amplification chambers is uniformly distributed on the same circumference, and each of the CRISPR reaction chambers is uniformly distributed on the same circumference. The lower carrier is set to be a disc carrier, the number of the PCR amplification chambers and the CRISPR reaction chambers which can be set is large, and the subsequent substances in the PCR amplification chamber chambers are convenient to swing to the CRISPR reaction chambers through electric centrifugation or handheld swinging.
In a preferred embodiment of the invention, the heating module comprises a CRISPR heating module and a PCR heating module;
the CRISPR heating module is arranged above and/or below the CRISPR reaction chamber;
the PCR heating module is arranged above and/or below the PCR reaction chamber;
the PCR heating module is used for providing temperature reaction conditions for PCR amplification reaction, and the CRISPR heating module is used for providing temperature reaction conditions for CRISPR reaction.
For example, the multiple PCR chambers are arranged in the same circumference, the multiple CRISPR chambers are arranged in the same circumference and inside the circumference where the multiple PCR chambers are located, and different heating modules are arranged at corresponding positions to heat the PCR reaction and the CRISPR reaction, respectively, so that efficiency can be improved.
In a preferred embodiment of the present invention, each of the PCR amplification chambers is near the edge of the lower carrier or near the center of the lower carrier;
each CRISPR reaction chamber is arranged opposite to each PCR amplification chamber and is arranged close to the center of the lower carrier or close to the edge of the lower carrier.
Specifically, the edges or the centers of the carriers are arranged in the PCR amplification chamber and the CRISPR reaction chamber, so that the PCR amplification chamber and the CRISPR are kept at a certain distance, the temperature is not influenced during heating, and the reaction in each chamber is also not influenced.
In a preferred embodiment of the invention, the heating module further comprises a carrying housing;
the CRISPR heating module is arranged in the bearing shell;
each PCR heating module is sequentially arranged in the bearing shell;
and the bearing shell is rotatably connected with the closed module, so that the closed module can rotate relative to the bearing shell, and the PCR amplification chamber passes through each PCR heating module for multiple times to perform PCR amplification reaction.
The PCR temperature-variable amplification reaction is generally a multi-temperature reaction program, for example, if a three-temperature reaction program is adopted, each cycle of PCR amplification comprises three steps of denaturation, annealing and extension, wherein the denaturation temperature is 89-98 ℃, the annealing temperature is 40-65 ℃ and the extension temperature is 65-72 ℃, the detection device is provided with not less than three PCR heating modules, preferably three PCR heating modules, and the three modules are respectively set with the corresponding denaturation, annealing and extension temperatures, so that the PCR amplification reaction can be rapidly completed only by the sealing module passing through the three heating modules for multiple times, the operation steps are greatly simplified, and the whole detection time is shortened.
In the present invention, it is preferable that the heating module is fixed and the sealing module rotates relative to the heating module, but it is needless to say that the sealing module is not moved and the heating module rotates.
In a preferred embodiment of the present invention, the heating module comprises a CRISPR heating module and three PCR heating modules, and a three-temperature reaction procedure is suitable for the PCR amplification procedure, that is, the PCR amplification procedure comprises three steps of denaturation, annealing and extension.
In a preferred embodiment of the present invention, the heating module comprises a CRISPR heating module and two PCR heating modules, and the PCR amplification procedure is a dual temperature reaction procedure, i.e. the PCR amplification procedure comprises two steps of denaturation and annealing extension.
In a preferred embodiment of the present invention, each of the PCR amplification chambers is close to the edge of the lower carrier and is used for providing a PCR amplification reaction site;
each of the CRISPR reaction chambers is proximal to the lower carrier center and is used to provide a CRISPR reaction site.
In a preferred embodiment of the present invention, each of the PCR amplification chambers is near the center of the lower carrier and is used for providing a PCR amplification reaction site;
each of the CRISPR reaction chambers is adjacent to the lower vector edge and both serve to provide a CRISPR reaction site.
In a preferred embodiment of the present invention, the method further comprises: the temperature control module and the driving module;
the temperature control module is used for controlling the temperature of the heating module;
the bearing shell is provided with a rotating shaft in a penetrating way, and the top end of the rotating shaft is connected with the lower carrier;
the output end of the driving module is connected with the bottom end of the rotating shaft.
The temperature control module can adopt the existing temperature controller, can carry out accurate accuse temperature to the heating module, adds drive module and makes efficiency more increase, and the test result is more accurate.
In a preferred embodiment of the present invention, the upper cover is a sealing film attached to the upper side of the lower cover and sealing each of the PCR amplification chambers, each of the CRISPR reaction chambers, and each of the connecting channels.
Preferably, the upper cover body adopts a transparent heat-resistant sealing film, so that the use and the observation of detection results are convenient.
In a preferred embodiment, the sealing film is made of a material with good hydrophobicity and transparency, preferably polystyrene.
In a preferred embodiment of the present invention, the lower carrier and the upper cover are connected by a screw, a snap or a snap connection.
Preferably, the upper cover body is a transparent heat-resistant hard shell, preferably heat-resistant glass or heat-resistant ceramic.
In a preferred embodiment of the present invention, the lower carrier is connected to the upper cover by a screw thread, the lower carrier is provided with an internal screw thread, the upper cover is provided with an external screw thread, and a sealing gasket is disposed in the internal screw thread.
In a preferred embodiment of the invention, both the PCR amplification chamber and the CRISPR reaction chamber are cylindrical.
The diameter of the lower carrier is 5-20 cm, and the depths of the PCR amplification chamber and the CRISPR reaction chamber are 0.1-10 mm;
the cross-sectional diameters of the PCR amplification chamber and the CRISPR reaction chamber are 1mm-20mm,
the connecting channel is a cuboid and is 0.1mm-3mm in width.
In a preferred embodiment, the diameter of the lower carrier is 8-15 cm, and the depths of the PCR amplification chamber and the CRISPR reaction chamber are 0.5mm-5mm, preferably 5 mm;
preferably, the cross-sectional diameters of the PCR amplification chamber and the CRISPR reaction chamber are 5mm-10mm, the connecting channel is a cuboid, and the width of the connecting channel is preferably 0.5mm-1mm, preferably 1 mm.
The second part of the invention provides a PCR amplification and CRISPR visual detection method which is carried out based on the rapid PCR amplification and CRISPR visual detection device and comprises a PCR amplification system and a CRISPR visual system;
S101、
in the PCR system, the PCR reaction solution is prepared,
the primer concentration is provided in a range of not less than the primer concentration in the standard PCR and not more than ten times the primer concentration in the standard PCR, preferably, the primer concentration is 0.4uM to 4 uM;
the length of the template is 50 bp-500 bp, preferably 50 bp-300 bp, more preferably 50 bp-200 bp;
the concentration of polymerase is provided as 5 times or more the concentration in standard PCR, preferably the amount of polymerase added is 5-20 units;
the polymerase is preferably rapid PCR polymerase or a premixed reaction solution of rapid PCR polymerase, including but not limited to Takara SpeedSTAR polymerase, Takara Taq HS polymerase, Takara Ex Taq polymerase, Takara Z-Taq polymerase, Kapaliosystems KAPA2G rapid polymerase and premixed reaction solution containing the above enzymes;
S102、
selecting a three-temperature reaction program for PCR, and adopting a detection device provided with three PCR heating modules; each cycle of PCR amplification comprises three steps of denaturation, annealing and extension, wherein the denaturation temperature is 89-98 ℃, the annealing temperature is 40-65 ℃, and the extension temperature is 65-72 ℃;
the time length of each cycle is 1-60 s, preferably 1-30 s, and more preferably 3-10 s;
or selecting a double-temperature program for PCR, and adopting a detection device provided with two PCR heating modules; each cycle of PCR amplification comprises two steps of denaturation and annealing extension, wherein the denaturation temperature is 89-98 ℃, and the annealing extension temperature is 40-72 ℃; the time length of each cycle is 1-60 s, preferably 1-30 s, and more preferably 2-10 s;
S103、
in the CRISPR visualization system, the CRISPR is a micro-nano structure,
the higher the working concentration of the CRISPR/Cas enzyme, the faster the probe cleavage speed, and the shorter the reaction time required for visual detection. The working concentration of the CRISPR/Cas enzyme is 0.01 uM-1 uM, preferably 0.05 uM-0.5 uM, and more preferably 0.05 uM-0.25 uM;
the working concentration of the gRNA is 0.1 uM-2 uM, and preferably 0.5-1.5 uM;
the working concentration ratio of CRISPR/Cas enzyme to gRNA was 1: 1-1: 50, preferably 1: 1-1: 10;
the higher the concentration of the fluorescence quenching probe, the shorter the reaction time required for visual detection. The working concentration of the fluorescence quenching probe is 0.1 uM-5 uM, preferably 0.2 uM-2.5 uM;
the working concentration of magnesium ions affects the working efficiency of CRISPR/Cas enzymes. The working concentration of magnesium ions is 8 mM-15 mM, preferably 10 mM-12 mM;
S104、
the optimal temperature of the CRISPR visualization reaction is 35-37 ℃ at 25-40 ℃;
the time length of the CRISPR visualization reaction temperature is 1-30 min, preferably 1-10 min, and more preferably 1-5 min;
during or after the reaction of the CRISPR visualization system is finished, ultraviolet light or blue light is adopted for excitation so as to realize visualization interpretation and photographing record of the fluorescence result of the sample.
In a preferred embodiment of the invention, when the amplification product is used as a template for CRISPR visualization detection in step S101 and step S102, the volume of the amplification product and the CRISPR visualization system is 1: 20-2: 1.
Compared with the prior art, the beneficial effects of the invention are embodied in the following aspects:
1) the invention is provided with a plurality of PCR amplification chambers and CRISPR reaction chambers, can realize the amplification of a plurality of PCR systems and the visual detection of CRISPR, and integrates a temperature-controllable heating module in the detection device, so that the detection is more convenient and the detection time is shortened.
2) The detection device is also provided with a driving module, a heating module is arranged in a bearing shell of the detection device, a rotating shaft penetrates through the bearing shell, the top end of the rotating shaft is connected with a lower carrier of the closed module, the bottom end of the rotating shaft is connected with the output of the driving module, and the driving module drives the rotating shaft to rotate, so that the lower carrier can rotate around the rotating shaft at a certain speed, namely, a PCR amplification chamber in the lower carrier circularly passes through the heating module in the bearing shell, and the three-temperature or two-temperature rapid PCR amplification of a plurality of cycles is completed.
3) After the reaction is finished, the result is observed by naked eyes under the irradiation of a portable ultraviolet lamp or a blue light lamp, and the fluorescence result is recorded by taking a picture by a mobile phone. Wherein the positive sample shows fluorescence which is visible to the naked eye in the CRISPR reaction chamber, and the negative sample generates no fluorescence.
4) The transparent hard cover body is arranged, so that the result can be observed by naked eyes conveniently.
5) The device has large specific surface area, and can greatly improve the heat transfer efficiency, thereby greatly shortening the PCR amplification time and further shortening the total detection time to within minutes.
6) The method is a one-step method operation, only needs to open the cover during sample adding, and then amplification and detection are carried out in a closed device, so that the problems of multi-step operation and amplicon aerosol pollution caused by cover opening after amplification can be effectively solved.
Drawings
Fig. 1 is a top view of a visual inspection apparatus according to embodiment 1 of the present invention;
fig. 2 is a cross-sectional view of a visual inspection apparatus according to embodiment 1 of the present invention;
FIG. 3 is a top view of a detection apparatus having a set of heating modules according to embodiment 1 of the present invention;
FIG. 4 is a sectional view of a detecting device provided with a set of heating modules according to embodiment 1 of the present invention;
fig. 5 is a cross-sectional view of a detection apparatus provided with two sets of heating modules according to embodiment 1 of the present invention;
FIG. 6 is a top view of a heating module of the detecting device in embodiment 2 of the present invention;
fig. 7 is a top view of a visual inspection apparatus according to embodiment 3 of the present invention;
fig. 8 is a cross-sectional view of a visual inspection device according to embodiment 3 of the present invention;
FIG. 9 is a top view of a detecting device with a set of heating modules according to embodiment 3 of the present invention;
FIG. 10 is a sectional view showing a detection apparatus provided with a set of heating modules in example 3 of the present invention;
fig. 11 is a sectional view of a detecting device provided with two sets of heating modules according to embodiment 3 of the present invention;
FIG. 12 is a top view of a detecting device with a set of heating modules according to embodiment 4 of the present invention;
fig. 13 is a sectional view showing a detection apparatus provided with a set of heating modules according to embodiment 4 of the present invention;
fig. 14 is a sectional view of a detecting device provided with two sets of heating modules according to embodiment 4 of the present invention;
FIG. 15 shows the real-time temperature variation of a sample during rapid PCR thermal cycling according to the present invention;
FIG. 16 is a graph showing data on the temperature ramp rate of a sample during rapid PCR thermal cycling in accordance with the present invention;
FIG. 17 shows the interpretation of the test results in example 5 of the present invention; with distinguishable fluorescence producing a positive result and with no distinguishable fluorescence producing a negative result.
Reference numbers of the drawings:
1. a sealing module; 11. a lower carrier; 12. an upper cover body; 13. a PCR amplification chamber; 14. a CRISPR reaction chamber; 15. a connecting channel; 2. a heating module; 21. a CRISPR heating module; 22. a denaturation PCR heating module; 23. annealing the PCR heating module; 24. extending the PCR heating module; 25. annealing and extending the PCR heating module; 3. a load bearing housing; 31. a rotating shaft.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Referring to fig. 1, 2, 3, 4, and 5, the drawings are schematic views and do not represent actual dimensions.
The PCR amplification and CRISPR visualization detection device comprises: a closed module 1 and a heating module 2. The hermetic module 1 includes: lower carrier 11, upper cover 12, a plurality of PCR amplification chambers 13, a plurality of CRISPR reaction chambers 14 corresponding in number to the plurality of PCR amplification chambers 13, and a plurality of connecting channels 15 communicating each PCR amplification chamber 13 and each CRISPR reaction chamber 14. That is, one PCR amplification chamber 13 corresponds to one CRISPR reaction chamber 14, and the two chambers are connected by a connecting channel 15, which is capable of blocking the mixing of the substances in the PCR amplification chamber 13 and the CRISPR reaction chamber 14 without external force, and when external force (such as shaking or centrifugation) is applied, the substances in the PCR amplification chamber 13 can enter the CRISPR reaction chamber to continue the reaction.
The number of amplification chambers 13 and CRISPR reaction chambers 14 is designed according to the size of the lower vector and the experimental requirements, and the number of the two chambers is the same and 6 in this embodiment.
In this embodiment, the lower carrier 11 is a disc-shaped carrier, and each of the PCR amplification chambers 13 is disposed near the edge of the disc-shaped carrier and uniformly disposed on the same circumference, the depth of the PCR amplification chamber 13 is 0.5mm, and the diameter of the cross section is 5 mm; each CRISPR reaction chamber 14 is arranged close to the center of the disc-shaped carrier and is uniformly arranged on the same circumference, the depth of each CRISPR reaction chamber 14 is 0.5mm, and the diameter of the cross section is 5 mm; the connecting channel is a cuboid, and the width of the connecting channel is 0.5 mm. In practical application, after the PCR amplification chamber 13 and the CRISPR reaction chamber 14 are loaded, the two chambers and the connecting channel 15 are sealed by the upper cover 12. In this embodiment, the upper cover 12 is a polystyrene sealing film.
In the embodiment, the disc-shaped carrier is adopted, so that substances in the PCR amplification chamber can conveniently enter the CRISPR reaction chamber and are mixed with the CRISPR reagent in a swinging or centrifugal mode, and the device can keep good tightness under the conditions of high temperature and room temperature by sealing the polystyrene sealing film.
The heating module 2 comprises a CRISPR heating module 21 and three PCR heating modules, namely a denaturation PCR heating module 22, an annealing PCR heating module 23 and an extension PCR heating module 24, wherein the CRISPR heating module 21 is circular, the denaturation PCR heating module 22, the annealing PCR heating module 23 and the extension PCR heating module 24 are in fan-shaped shapes and are arranged adjacently, the heights of the heating modules are the same, namely, the heating modules are on the same plane, and the heating modules are respectively controlled by a temperature control device.
When in use, the utility model is used,
1) after the sample is added to the PCR amplification chamber 13 and the CRISPR reaction chamber 14, the two chambers and the connecting channel 15 are sealed by an upper cover body 12;
2) the closed module 1 is arranged above the heating module 2, wherein each PCR amplification chamber 13 is positioned in a PCR heating module area (a denaturation PCR heating module 22, an annealing PCR heating module 23 and an extension PCR heating module 24), and each PCR heating module is set to be suitable for heating at the temperature of 90-98 ℃, 55-65 ℃ and 72 ℃. And rotating the closed module 1 around the central axis of the heating module 2 at a certain speed for 35-45 circles in the clockwise direction to complete 35-45 cycles of three-temperature rapid PCR amplification. The heating module 2 may be disposed on a single side of the sealed module 1 (as shown in fig. 4), or disposed on both sides of the sealed module 1 (as shown in fig. 5).
3) And (3) taking out the closed module 1, and enabling the amplification solution in the PCR reaction chamber 13 to enter the CRISPR reaction chamber 14 to be mixed with the CRISPR reagent in a swinging or centrifugal mode.
4) Opening the CRISPR heating module, controlling the appropriate temperature to be generally 37 ℃, placing the closed module 1 above the heating module 2, standing for 2-10 min, and carrying out CRISPR reaction on the mixed reagent in a CRISPR reaction chamber;
5) and after the reaction is finished, observing the result by naked eyes under the irradiation of a portable ultraviolet lamp or a blue light lamp, and photographing by using a mobile phone to record the fluorescence result. Wherein the positive sample exhibits macroscopic fluorescence in the CRISPR reaction chamber 14 and the negative sample produces no fluorescence.
Example 2
The schematic view of the closed module 1 of example 2 is shown in fig. 1 and fig. 2, and is different from that of example 1 in that the depth of the PCR amplification chamber 13 is 2mm, and the cross-sectional diameter is 10 mm; the CRISPR reaction chamber 14 is 3mm deep and 10mm in cross-sectional diameter. The connecting channel is a cuboid, and the width of the connecting channel is 0.6 mm. In this embodiment, the upper cover 12 is a transparent hard high temperature resistant cover, and the joint between the cover and the heating module is provided with an external thread.
Referring to fig. 6, the heating module 2 includes a CRISPR heating module 21 and two PCR heating modules, which are a denaturing PCR heating module 22 and an annealing and extending PCR heating module 25, respectively, where the CRISPR heating module 21 is a circular ring, the denaturing PCR heating module 22 and the annealing and extending PCR heating module 25 are semicircular and are adjacently disposed, heights of the heating modules are the same, that is, the heating modules are on the same plane, and the heating modules are controlled by a temperature control device, respectively.
The detection device further comprises a bearing shell 3, the CRISPR heating module 21 and each PCR heating module (the denaturation PCR heating module 22 and the annealing and extension PCR heating module 25) are arranged in the bearing shell 3, the bearing shell is provided with a rotating shaft 31, the lower carrier 11 is connected to the upper end of the rotating shaft 31 and can rotate along the rotating shaft 31, so that the sealing module 1 can rotate relative to the heating module 2, and the PCR amplification reaction in the PCR amplification chamber 13 or the CRISPR reaction chamber 14 can carry out multiple amplification reactions through each PCR heating module for multiple times.
The carrier housing 3 is provided with an internal thread and with a sealing gasket, the upper cover body is threadably connectable with the carrier housing 3, and when tightened, seals the respective PCR amplification chamber 13, CRISPR reaction chamber 14 and connecting channel 15.
When in use, the utility model is used,
1) after the sample is added to the PCR amplification chamber 13 and the CRISPR reaction chamber 14, the upper cover body is in threaded connection with the bearing shell 3, and the two chambers and the connecting channel 15 are sealed;
2) the closed module 1 is placed above the heating module 2, wherein each PCR amplification chamber 13 is located in a PCR heating module area (a denaturation PCR heating module 22 and an annealing and extension PCR heating module 25), and each PCR heating module is set to be suitable for heating at temperatures of generally 90-98 ℃ and 55-65 ℃. And rotating the closed module 1 around the central axis of the heating module 2 at a certain speed for 35-45 circles in the clockwise direction to complete 35-45 cycles of three-temperature rapid PCR amplification.
The other steps are similar to example 1.
In a possible embodiment, the PCR amplification and CRISPR visual detection apparatus further comprises: a drive module (not shown). The rotating shaft 31 is inserted into the bearing housing 3, the top end of the rotating shaft 31 is connected with the lower carrier 11, the output of the driving module is connected with the bottom end of the rotating shaft 31, and the driving module can adopt common driving devices such as a motor.
The detection device in this embodiment is subjected to thermal cycle evaluation, after the samples are added to the PCR amplification chamber 13 and the CRISPR reaction chamber 14, the two chambers and the connecting channel 15 are sealed by the upper cover body 12, the denatured PCR heating module 22 and the annealing and extension PCR heating module 25 are controlled by the temperature control module to be heated to 95 ℃ and 60 ℃ respectively, and then the sealed sample is placed on the device to detect the real-time temperature change of the sample in one of the PCR amplification chambers, as shown in fig. 15, the temperature of the sample can be raised to more than 90 ℃ within 1s, the temperature of the sample can be raised to the platform period of temperature within 2s, the denatured PCR heating module is rotated to enter the extension PCR heating module after being kept for 5s, the temperature of the sample can be lowered to 60 ℃ within 1s, and the temperature of the sample can be lowered to the platform period of temperature within 2 s. The data in fig. 15 is processed (subjected to derivative) to obtain a temperature change rate curve shown in fig. 16, as shown in fig. 16, it can be seen that the maximum temperature rise rate can reach 90 ℃/s, the maximum temperature fall rate can reach 60 ℃/s, and the extremely fast heat conduction rate ensures the feasibility of fast thermal cycling of the sample. At present, the fastest PCR instrument in the market is xxpress, and the temperature rising and falling speed is only 10 ℃/s.
Example 3
Example 3 is different from example 1 in that, as shown in fig. 7 and 8, each PCR amplification chamber 13 is arranged close to the center of the disc-shaped carrier and uniformly arranged on the same circumference, the depth of the PCR amplification chamber 13 is 5mm, and the diameter of the cross section is 10 mm; each CRISPR reaction chamber 14 is arranged close to the edge of the disc-shaped carrier and is uniformly arranged on the same circumference, the depth of each CRISPR reaction chamber 14 is 5mm, and the diameter of the cross section of each CRISPR reaction chamber is 10 mm. In practical application, after the sample is added to the PCR amplification chamber 13 and the CRISPR reaction chamber 14, the two chambers and the connecting channel 15 are sealed by the upper cover body 12, and the connecting channel is a cuboid and 1mm in width. In this embodiment, the upper cover 12 is a polystyrene sealing film.
As shown in fig. 9, 10, and 11, the heating module 2 includes a CRISPR heating module 21 and three PCR heating modules, which are a denaturing PCR heating module 22, an annealing PCR heating module 23, and an extension PCR heating module 24, respectively, where the CRISPR heating module 21 is circular, the denaturing PCR heating module 22, the annealing PCR heating module 23, and the extension PCR heating module 24 are all in a fan shape and are arranged adjacently, the heights of the heating modules are the same, i.e., on the same plane, and the heating modules are controlled by temperature control devices, respectively.
The other steps are similar to example 1. The heating module 2 may be disposed on a single side of the sealed module 1 (as shown in fig. 10), or disposed on both sides of the sealed module 1 (as shown in fig. 11).
Example 4
Example 4 differs from example 2 in that the depth of the PCR amplification chamber 13 is 4mm and the cross-sectional diameter is 8 mm; the CRISPR reaction chamber 14 is 4mm deep and 8mm in cross-sectional diameter. The connecting channel is a cuboid, and the width of the connecting channel is 0.7 mm.
As shown in fig. 12, 13 and 14, the heating module 2 includes a CRISPR heating module 21 and two PCR heating modules, which are a denaturing PCR heating module 22 and an annealing and extending PCR heating module 25, respectively, where the CRISPR heating module 21 is a circular ring, the denaturing PCR heating module 22 and the annealing and extending PCR heating module 25 are semicircular and are adjacently disposed, the heights of the heating modules are the same, that is, the heating modules are on the same plane, and the heating modules are controlled by a temperature control device, respectively.
The upper cover 12 in this embodiment can be a polystyrene sealing film in embodiment 1 or a hard high temperature transparent cover in embodiment 2.
The other operation steps are similar to those of example 1 or example 2. The heating module 2 may be disposed on a single side of the sealed module 1 (as shown in fig. 13), or disposed on both sides of the sealed module 1 (as shown in fig. 14).
Example 5
The gene of E.coli (ATCC25922) was detected by the method described in example 1, and E.coli (ATCC25922) was obtained from the center of the bacterial bank.
Preparation of reagents before reaction:
preparing 10 mu L of rapid PCR reaction system containing Taq HS polymerase 12.5unit, Tris-HCl10mM, KCl 50mM and MgCl21.5mM, 0.2mM each of dNTPs, and 0.4. mu.M each of primer concentration (purchased from Takara, Inc., cat # R007A).
Preparing 10 mu L CRISPR detection system containing Tris-HCl10mM, NaCl 50mM and MgCl210mM, 100. mu.g/mL BSA, Cas12a 0.75. mu.M, gRNA 1.5. mu.M, ssDNA fluorescence quenching probe 2.5. mu.M, RNase inhibitor 4 unit. (Cas12a from NEB, cat # M0653).
The primer sequences used were:
F：5'CCGAACGGAGGGCAGATTAGCAC 3'
R：5'TGGGCAGGAGGGTAACACCAGAT 3'
amplified template sequence:
CCGAACGGAGGGCAGATTAGCACACTTTTTCAACATCATTGTGCTCAACAATGTGCTCCTGCTAAACCATAATTCTTTTTATCAGATGGAATATCTGTCACATTGCTTTTCAACGATAGCTTCCTGGGAGAGATTTTTTCTTATTATTCCTCCCCATCTGGTGTTACCCTCCTGCCCA(GenBank:ASHD01000027.1)
gRNA sequence:
UAAUUUCUACUAAGUGUAGAU-GCAGGAGCACAUUGUUGAGCA
single-stranded DNA fluorescent probe sequence:
5’6-FAM-TTTTTTTTTTTT-3’BHQ
and respectively adding the rapid PCR reaction system and the CRISPR detection system into the PCR amplification chamber 13 and the CRISPR reaction chamber 14 of the closed module 1, and sealing the closed module 1 by adopting a sealing film.
The rapid PCR reaction and CRISPR reaction were performed using the embodiment of example 1. Wherein the amplification procedure of the rapid PCR is as follows: the 95 ℃ hot start is carried out for 30s, 35 cycles are carried out, each cycle comprises reaction at 95 ℃ for 3s, reaction at 58 ℃ for 3s and reaction at 72 ℃ for 3s, and the total time for amplification is 6 min. The reaction program of CRISPR is 37 ℃ reaction for 3 min.
The results were observed under blue light after the reaction was complete. As shown in FIG. 17, the positive samples containing E.coli were generated with fluorescence, and the negative samples containing no E.coli were generated without fluorescence.
The visual detection device has large specific surface area, and can greatly improve the heat transfer efficiency, thereby greatly shortening the PCR amplification time, and further shortening the total detection time to within a few minutes.
Example 6
The gene of E.coli (ATCC25922) was detected by the method described in example 2.
The rapid PCR amplification system and the CRISPR detection system are the same as those in example 5. Wherein the amplification procedure of the rapid PCR is as follows: the 95 ℃ hot start is carried out for 30s, 35 cycles are carried out, each cycle comprises reaction at 95 ℃ for 3s and reaction at 58 ℃ for 3s, and the total amplification time is 4 min. The reaction program of CRISPR is 37 ℃ reaction for 3 min.
And (3) detection results: samples containing E.coli showed bright fluorescence and samples without E.coli showed no fluorescence.
Example 7
The gene of E.coli (ATCC25922) was detected by the method described in example 3.
The rapid PCR amplification system, the CRISPR detection system and the reaction procedure are the same as in example 5.
And (3) detection results: samples containing E.coli showed bright fluorescence and samples without E.coli showed no fluorescence.
Example 8
The gene of E.coli (ATCC25922) was detected by the method described in example 4.
The rapid PCR amplification system, the CRISPR detection system and the reaction procedure are the same as in example 6.
And (3) detection results: samples containing E.coli showed bright fluorescence and samples without E.coli showed no fluorescence.
Example 9
The gene of E.coli (ATCC25922) was detected by the method described in example 1.
The amplification system of the rapid PCR is 20 mu L, the CRISPR detection system is 10 mu L, and the volume ratio of the amplification system to the CRISPR detection system is 2: 1. the rest of the process was the same as in example 5.
And (3) detection results: samples containing E.coli showed bright fluorescence and samples without E.coli showed no fluorescence.
Example 10
The gene of E.coli (ATCC25922) was detected by the method described in example 1.
The amplification system of the rapid PCR is 5 mu L, the CRISPR detection system is 50 mu L, and the volume ratio of the two is 1: 10. the rest of the process was the same as in example 5.
And (3) detection results: samples containing E.coli showed bright fluorescence and samples without E.coli showed no fluorescence.
Example 11
The gene of E.coli (ATCC25922) was detected by the method described in example 1.
The primer concentration in the rapid PCR amplification system was 4. mu.M, and the rest was the same as in example 5.
And (3) detection results: samples containing E.coli showed bright fluorescence and samples without E.coli showed no fluorescence.
Example 12
The gene of E.coli (ATCC25922) was detected by the method described in example 1.
The working concentration of the CRISPR/Cas enzyme in the CRISPR detection system is 0.05 mu M, and the rest is the same as that in example 5.
And (3) detection results: samples containing E.coli showed bright fluorescence and samples without E.coli showed no fluorescence.
Example 13
The gene of E.coli (ATCC25922) was detected by the method described in example 1.
The concentration of the fluorescence quenching probe in the CRISPR detection system is 0.2. mu.M, and the rest is the same as that in example 5.
And (3) detection results: samples containing E.coli showed bright fluorescence and samples without E.coli showed no fluorescence.
Example 14
The gene of E.coli (ATCC25922) was detected by the method described in example 1.
The working concentration of gRNA in CRISPR assay was 0.5 μ M, the rest was the same as in example 5.
And (3) detection results: samples containing E.coli showed bright fluorescence and samples without E.coli showed no fluorescence.
Example 15
The gene of E.coli (ATCC25922) was detected by the method described in example 1.
After the rapid PCR system and the CRISPR detection system are mixed, the final concentration of magnesium ions in the system is 15mM, and the rest parts are the same as in example 5.
And (3) detection results: samples containing E.coli showed bright fluorescence and samples without E.coli showed no fluorescence.
Example 16
The gene of E.coli (ATCC25922) was detected by the method described in example 1.
The excitation light source used was an ultraviolet lamp, and the rest was the same as in example 5.
And (3) detection results: samples containing E.coli showed bright fluorescence and samples without E.coli showed no fluorescence.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the above-described embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A rapid PCR amplification and CRISPR visualization detection device is characterized by comprising: the device comprises a closed module (1) and a heating module (2);
the containment module (1) comprises: a lower carrier (11) and an upper cover (12);
the lower carrier (11) is provided with a plurality of PCR amplification chambers (13) and a plurality of CRISPR reaction chambers (14) with the number corresponding to that of the PCR amplification chambers (13), and the PCR amplification chambers (13) are respectively communicated with the corresponding CRISPR reaction chambers (14) through connecting channels (15);
the upper cover body (12) is connected with the lower carrier (11) in a sealing way, so that each PCR amplification chamber (13), each CRISPR reaction chamber (14) and each connecting channel (15) are sealed;
the heating module (2) is arranged above and/or below the closed module (1) and is used for heating each PCR amplification chamber (13) and/or each CRISPR reaction chamber (14).
2. The rapid PCR amplification and CRISPR visualization device according to claim 1, wherein said lower vector (11) is a disc vector;
and the PCR amplification chambers (13) are uniformly distributed on the same circumference, and the CRISPR reaction chambers (14) are uniformly distributed on the same circumference.
3. The rapid PCR amplification and CRISPR visualization detection apparatus according to claim 1, wherein said heating module (2) comprises a CRISPR heating module (21) and a PCR heating module;
the CRISPR heating module (21) is arranged above and/or below the CRISPR reaction chamber (14);
the PCR heating module is arranged above and/or below the PCR reaction chamber (13);
the PCR heating module is used for providing temperature reaction conditions for PCR amplification reaction, and the CRISPR heating module (21) is used for providing temperature reaction conditions for CRISPR reaction.
4. The rapid PCR amplification and CRISPR visualization device according to claim 3, wherein said heating module (2) further comprises a carrying housing (3);
the CRISPR heating module (21) is arranged in the bearing shell (3);
the PCR heating modules are sequentially arranged in the bearing shell (3);
and the bearing shell (3) is rotatably connected with the closed module (1), so that the closed module (1) and the bearing shell (3) can rotate relatively, and the PCR amplification chamber (13) passes through each PCR heating module for multiple times to perform PCR amplification reaction.
5. The rapid PCR amplification and CRISPR visualization detection apparatus according to claim 4, further comprising: the temperature control module and the driving module;
the temperature control module is used for controlling the temperature of the heating module (2);
a rotating shaft (31) penetrates through the bearing shell (3), and the top end of the rotating shaft (31) is connected with the lower carrier (11);
the output end of the driving module is connected with the bottom end of the rotating shaft (31).
6. The rapid PCR amplification and CRISPR visualization apparatus according to any of claims 1-5, wherein the upper cover (12) is a sealing film, which is attached to the upper side of the lower carrier (11) and seals each PCR amplification chamber (13), each CRISPR reaction chamber (14) and each connecting channel (15).
7. The rapid PCR amplification and CRISPR visualization apparatus according to any of claims 1-5, wherein the lower carrier (11) and the upper cover (12) are connected by a screw, a snap or a snap.
8. The rapid PCR amplification and CRISPR visualization device according to any of claims 1-5, wherein said PCR amplification chamber (13) and said CRISPR reaction chamber (14) are both cylindrical.
9. The rapid PCR amplification and CRISPR visualization detection device according to claim 8, wherein the diameter of the lower carrier (11) is 5-20 cm, preferably 8-15 cm;
the depth of the PCR amplification chamber (13) and the CRISPR reaction chamber (14) is 0.1mm-10mm, preferably 0.5mm-5 mm;
the cross-sectional diameters of the PCR amplification chamber (13) and the CRISPR reaction chamber (14) are 1mm-20mm, preferably 5mm-10 mm;
the connecting channel (15) is a cuboid, and the width of the connecting channel is 0.1mm-3mm, preferably 0.5mm-1 mm.
10. A PCR amplification and CRISPR visualization detection method, which is carried out based on the rapid PCR amplification and CRISPR visualization detection device of any one of claims 1-5, and is characterized by comprising a PCR amplification system and a CRISPR visualization system;
in the PCR system, the PCR reaction solution is prepared,
the concentration of the primer is 0.4 uM-4 uM;
the length of the template is 50 bp-500 bp, preferably 50 bp-200 bp;
the addition amount of the polymerase is 5-20 units,
the polymerase is selected from one or more of Takara speedSTAR polymerase, Takara Taq HS polymerase, Takara Ex Taq polymerase, Takara Z-Taq polymerase or kapubiosystem KAPA2G rapid polymerase, or a premixed reaction solution containing the above enzymes;
selecting a three-temperature reaction program for PCR, and adopting a detection device provided with three PCR heating modules; each cycle of PCR amplification comprises three steps of denaturation, annealing and extension, wherein the denaturation temperature is 89-98 ℃, the annealing temperature is 40-65 ℃, and the extension temperature is 65-72 ℃;
the time length of each cycle is 1-60 s, preferably 1-30 s, and more preferably 3-10 s;
in the CRISPR visualization system, the CRISPR is a micro-nano structure,
the working concentration of the CRISPR/Cas enzyme is 0.01 uM-1 uM, preferably 0.05 uM-0.5 uM, and more preferably 0.05 uM-0.25 uM;
the working concentration of the gRNA is 0.1 uM-2 uM, and preferably 0.5-1.5 uM;
the working concentration ratio of CRISPR/Cas enzyme to gRNA was 1: 1-1: 50, preferably 1: 1-1: 10;
the working concentration of the fluorescence quenching probe is 0.1 uM-5 uM, preferably 0.2 uM-2.5 uM;
the working concentration of magnesium ions is 8 mM-15 mM, preferably 10 mM-12 mM;
the optimal temperature of the CRISPR visualization reaction is 35-37 ℃ at 25-40 ℃;
the time length of the CRISPR visualization reaction temperature is 1-30 min, preferably 1-10 min, and more preferably 1-5 min;
during or after the reaction of the CRISPR visualization system is finished, ultraviolet light or blue light is adopted for excitation so as to realize visualization interpretation and photographing record of the fluorescence result of the sample.
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