WO2021093775A1 - 反应管、反应管阵列、控制参与反应的样品体积的方法及其应用 - Google Patents
反应管、反应管阵列、控制参与反应的样品体积的方法及其应用 Download PDFInfo
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- WO2021093775A1 WO2021093775A1 PCT/CN2020/128153 CN2020128153W WO2021093775A1 WO 2021093775 A1 WO2021093775 A1 WO 2021093775A1 CN 2020128153 W CN2020128153 W CN 2020128153W WO 2021093775 A1 WO2021093775 A1 WO 2021093775A1
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- C—CHEMISTRY; METALLURGY
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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Definitions
- the application relates to the field of reaction vessels, and in particular to a reaction tube.
- Small-volume liquid phase detection (the total liquid volume is 0.5-1000 ⁇ l) is commonly used in various inspection and testing fields such as biomedicine, food science, environmental science, inspection and quarantine, and forensic identification.
- Common reaction vessels used for small-volume liquid phase detection include microcentrifuge tubes, cascade tubes, multi-well plates, etc.
- luminescence detection, fluorescence detection or absorption detection sample cells made of quartz, glass, plastic, etc. are also included.
- the components other than the sample to be tested are generally made into detection reagents, and a certain volume of the detection reagent and a certain volume of the sample solution to be tested are added to the reaction container, and then the two are placed in the reaction container. Mix well.
- the volume of sample solution or testing reagent added to the reaction tube is only on the order of 0.1-100 ⁇ l, and there is a high requirement for liquid volume accuracy. Therefore, operators generally need to use micropipettes and micropipettes. Sampler and other equipment for accurate pipetting operations. This type of operation requires high operating skills and experimental tools for experimental personnel, and is not convenient for experiments in the field or other non-scientific laboratory environments, and is also not conducive to operations by personnel without professional training.
- an existing method is to integrate a testing instrument or connect a syringe pump or a peristaltic pump outside the instrument. During operation, only an excessive amount of sample solution needs to be added and there is no need to accurately control the volume, and then the accurate volume of the sample is transferred to the reaction mixture solution by controlling the syringe pump or the peristaltic pump.
- This method can reduce the requirements for the professional level of the operator, but if the instrument is integrated, it will increase the complexity of the instrument structure, resulting in an increase in the volume and cost of the instrument and a decrease in stability; if the external connection of the instrument is used
- the method that is, the equipment required for experiments is increased, and it is not suitable for scenarios such as mobile laboratories or on-site testing.
- microspheres For the detection of biological macromolecules, another existing method is to use a special kind of microsphere with a receptor that can specifically bind to the target biological macromolecule attached to the surface. Add the microspheres to the sample solution, separate the microspheres from the solution after the surface receptors are bound to the target molecules, and then add a fixed volume of elution solution to elute the target molecules from the surface of the microspheres. The reaction reagents are mixed for detection, or the microspheres connected to the target molecule are directly added to a fixed volume of the reaction solution for detection.
- the present invention provides a reaction tube, a reaction tube array, and a control participation
- the method of reacting sample volume and the above-mentioned reaction tube, reaction tube array, and method of controlling the volume of the sample involved in the reaction are in the fluorescence quantitative PCR reaction, the reaction of detecting whether there is a specific sequence of DNA molecules in the sample, and the reaction of determining the concentration of the specific sequence of DNA molecules In the application.
- a reaction tube which includes a liquid storage area 100, a sample transfer area 200, and a reaction area 300.
- the liquid storage area is open at one end and communicates with the sample transfer area at the other end;
- the sample transfer area includes At least one pipe, the portion of the first pipe 210 connected to the reaction zone in the at least one pipe has a first internal cross-sectional area, and the first pipe 210 has a continuous pipe length with a first internal cross-sectional area. Less than 0.5 mm, one end of the first pipe is connected to the liquid storage area, and the other end is connected to the reaction area; one end of the reaction area 300 is closed, and the other end is connected to the first pipe 210.
- the length of a region is not less than 2mm, and the first region is the region in the reaction zone that is connected to the first pipe and whose internal cross-sectional area is not less than 4 in the ratio of the first internal cross-sectional area.
- the volume is not more than 1ml.
- the reaction tube of the embodiment of the present invention is shown in FIG. 1.
- the shape of the liquid storage area is not specifically limited, for example, it may be funnel-shaped, cylindrical, or the like.
- the shape of the first pipe in the sample transfer area is not specifically limited, for example, it may be cylindrical.
- the internal cross-sectional area of the portion of the first pipe connected to the reaction zone refers to the internal cross-sectional area of the cross-section of the first pipe perpendicular to the length direction of the first pipe, and the first pipe has a continuous pipe with the first internal cross-sectional area.
- the length is not less than 0.5 mm, preferably not less than 2 mm.
- the shape of the reaction zone is not specifically limited, and may be cylindrical, conical, etc., for example.
- the internal cross-sectional area of the first zone in the reaction zone refers to the internal cross-sectional area of the cross section of the reaction zone perpendicular to the length direction of the first pipe.
- the internal cross-sectional area of the first zone is recorded as the second internal cross-sectional area. 2.
- the internal cross-sectional area is not greater than 500 mm 2 , preferably not greater than 200 mm 2 , and the ratio of the second internal cross-sectional area to the first internal cross-sectional area is not less than 4.
- the length of the first zone refers to the length of the first zone in a direction parallel to the length of the first pipe, the length of the first zone is not less than 2 mm, and the volume of the reaction zone is not more than 1 mL.
- the internal cross-sectional area of the reaction zone basically does not change in the entire reaction zone, and the first zone is basically equal to the reaction zone; when the shape of the reaction zone is conical, the upper half of the reaction zone It is the first region, and the internal cross-sectional area of the lower half of the reaction zone does not satisfy "the ratio of the internal cross-sectional area to the first internal cross-sectional area is not less than 4", and it does not belong to the first region.
- sample solution a liquid sample or sample solution
- the sample solution enters the liquid storage area from the open end of the liquid storage area, and then enters the first pipe connected with the liquid storage area. Subsequently, the sample solution flows in the first pipe and enters the reaction zone connected to the first pipe.
- the sample adding device can be extended into the liquid storage area and into the sample transfer area to directly add the sample solution to the first pipe. Then, the sample solution flows in the first pipe and enters the reaction zone connected to the first pipe. .
- the opening of the liquid storage area refers to a port that can be opened, and the opening may not be normally open.
- the end of the liquid storage area 100 with an opening may be provided with an upper cover 10, and the upper cover 10 may be provided when the sample is added. The cover is closed to avoid sample contamination.
- the reaction zone 300 is preloaded with a preloaded reagent 11
- the preloaded reagent 11 may be solid or liquid, and the volume of the preloaded reagent is smaller than the volume of the reaction zone 300.
- the pre-loaded reagent may contain all reaction components except the sample, and is added before the reaction tube is sold to the user. That is, the pre-loaded reagent is added by the manufacturer, and the manufacturer can use the sample manipulation equipment to ensure that the pre-loaded reagent is added to the reaction zone of the reaction tube in an accurate volume.
- the pre-installed reagents can be added before being sold to users, and the timing of addition is not specifically limited. For example, it can be added after the reaction tube is produced, or it can be added when the reaction zone is produced before being assembled with other parts of the reaction tube.
- the sample solution 12 After the sample solution 12 is added from the open end of the liquid storage area 100 (as shown in FIG. 2a), the sample solution 12 enters the first pipe connected with the liquid storage area, and then the sample solution 12 is in the first pipe 210 of the sample transfer area 200. It flows into the reaction zone 300 connected with the first pipe 210 (as shown in FIG. 2b) and is mixed with the pre-loaded reagent 11 in the reaction zone to obtain a mixed solution 21 (as shown in FIG. 2c). The sum of the volume of the sample 12 added to the opening of the liquid storage area and the volume of the pre-loaded reagent is greater than or equal to the volume of the reaction zone 300, so as to ensure that the sample 12 can fill the reaction zone 300 after entering the reaction zone with the pre-loaded reagent.
- the volume of the sample solution 12 entering the reaction zone 300 can be controlled by the volume of the reaction zone 300 and the volume of the pre-loaded reagent 11, without the user needing to control the volume of the sample solution added.
- the volume of the sample solution 12b entering the reaction zone is the volume of the reaction zone-the volume of the preloaded reagent, and the remaining The sample solution 12a stays in the first pipe or in the first pipe and the liquid storage area 100, and does not enter the reaction area.
- the sample solution in the mixed solution 21 and the preloaded reagents will react.
- the sample solution 12a and the mixed solution 21 that did not enter the reaction zone are connected but non-uniform solutions, so sample molecules will be generated in the solution.
- the diffusion of sample molecules in the solution can be analogous to the diffusion of the sample in a one-dimensional infinite space, and the diffusion perpendicular to the liquid flow direction is negligible.
- the concentration of the component M at the distance x in the reaction zone 300 from the lower boundary of the sample transfer zone 200 is C M.
- the ratio of the second internal cross-sectional area to the first internal cross-sectional area is S. Approximate calculations are made with the diffusion model of the sample in a one-dimensional infinite space. According to Fick’s second law, there is
- the concentration C M of component M at each x is shown in Table 1.
- the diffusion concentration within 1h is within an order of magnitude (ie, C M ⁇ 0.1C 0 ), and the distance is about 2 mm.
- the diffusion coefficient D will be further reduced.
- the actual reactive diffusion occurs within a limited length, and when the ratio of the second internal cross-sectional area to the first internal cross-sectional area is greater than 4, the final diffusion concentration will be lower than that in Table 1. Therefore, diffusion is negligible in the actual reaction system, especially in the reaction system with a short reaction time (less than 30min). It can be considered that only the sample 12b entering the reaction zone 300 participated in the reaction, and the sample 12a located in the transfer zone 200 did not participate in the reaction. .
- the reaction tube of the embodiment of the present application uses the first pipe of the sample transfer area and the reaction zone of a specific size, so that the sample solution 12b that enters the reaction zone is mixed and reacted with the preloaded reagents, and the sample solution 12a that has not entered the reaction zone hardly reacts with the preloaded reagents.
- the reagents are mixed to react, so that the amount of sample involved in the reaction can be precisely controlled by controlling the size of the reaction zone and the size of the pre-loaded reagents.
- the reaction tube of the embodiment of the present application controls the volume of the sample that can enter the reaction zone by pre-installing a set volume of reagent in the reaction zone; on the other hand, because of the first inner part of the liquid circulation pipe in the sample transfer zone of the reaction tube
- the cross-sectional area is much smaller than the second inner cross-sectional area of the first zone in the reaction zone.
- the first inner cross-sectional area is not greater than 100 mm 2 , preferably not greater than 25 mm 2 .
- the first internal cross-sectional area of the part of the first pipe connected to the reaction zone is relatively small. Due to the surface tension, the sample solution will not enter the first pipe from the liquid storage area and enter the reaction from the first pipe under the action of gravity. In order to enter the first pipe and enter the reaction area from the first pipe, external forces such as centrifugation and hand shaking are required. In this way, the user can control the mixing time by himself, thereby controlling the reaction start time, which is beneficial to sample preservation and avoid side reactions. This method of controlling the reaction start time is particularly suitable for reactions that start as soon as the sample is mixed and cannot be started by controlling the reaction conditions.
- the specific time of centrifugation or hand shaking is not limited, so that the sample solution enters and fills the reaction zone. Generally, centrifugation in a palm centrifuge is sufficient for a few seconds.
- the at least one pipe includes an outer pipe 220 and at least one inner pipe 210, the inner pipe is located inside the outer pipe, and the inner pipe is a first pipe for sample flow.
- the sample transfer area 200 can be realized by pipes of different structures.
- the at least one pipe in the sample transfer area only includes the first pipe for sample flow, and the sample transfer area has a single-layer pipe structure.
- the sample transfer area 200, the liquid storage area 100, and the reaction area 300 can be integrally formed, or the three can be formed separately and then assembled.
- the at least one pipe in the sample transfer area includes an outer pipe 220 and at least one inner pipe 210, the inner pipe is located inside the outer pipe, and the inner pipe is used for samples.
- the sample transfer area has a double-layer pipe structure.
- the cross-sectional view of the reaction tube of this structure is shown in FIG. 4, where the cross-section of the reaction area is 310.
- the outer wall of the inner tube is attached to the inner wall of the outer tube.
- the inner tube and the outer tube are detachable, and the outer wall of the inner tube 210 can be closely attached to the inner wall of the outer tube 220 so that the inner tube is fixed in the outer tube.
- the outer tube 220 in the sample transfer area 200, the liquid storage area 100, and the reaction area 300 can be integrally formed, or the three can be formed separately and then assembled.
- the inner tube can be formed separately, and then the separately formed inner tube is inserted into the outer tube.
- the outer tube includes a partition, and the partition divides the outer tube into at least one inner tube.
- the inner area of the outer tube is divided into at least one inner tube by a partition.
- the separator can be processed separately and inserted into the outer tube.
- the specific pipe structure used in the sample transfer area can be selected according to the difficulty of processing.
- the liquid storage zone, the sample transfer zone and the reaction zone are integrally formed, or at least one of the liquid storage zone, the sample transfer zone and the reaction zone is formed separately, and the separately formed zone is formed with Other areas are directly connected or connected through accessories.
- the connection is threaded, glued, welded, welded or fitted.
- the liquid storage area, the sample transfer area, and the reaction area may be made of the same material or different materials, and the types of materials include, but are not limited to, plastic, glass, and ceramic.
- the reaction tube can be formed at one time by injection molding or 3D printing, or it can be formed by a combination of three areas. The three areas can be directly connected in the order of the liquid storage area, the sample transfer area, and the reaction area by threading, gluing, welding, welding or fitting, or by other connecting accessories.
- the inner wall of the first pipe is made of a material with strong hydrophobicity.
- a material with strong hydrophobicity refers to a material with a contact angle greater than 90 degrees.
- the sample solution is generally hydrophilic.
- the inner wall of the first pipe uses a highly hydrophobic material to further ensure that the sample will enter the first pipe from the liquid storage area and enter the reaction area from the first pipe through external forces such as centrifugation or hand shaking. , Users can control the mixing time by themselves, and then control the reaction start time, which is conducive to sample preservation and avoid side reactions.
- reaction areas and sample transfer areas there are multiple sample transfer areas in communication with the liquid storage area, and the reaction areas and sample transfer areas have a one-to-one correspondence.
- one liquid storage area 100 can be connected to more than one sample transfer area 200, wherein each sample transfer area 200 is respectively connected to a reaction zone 300, and each reaction zone 300 can contain the same volume and the same component of the sample transfer zone.
- the reagent 11 is used for multiple parallel reactions.
- the pre-loaded reagent 11 with different volumes of the same components can also be used for concentration gradient detection.
- the pre-loaded reagent 11 with different components can also be used for different detections on the same sample.
- the volume of each reaction zone 300 can also be different.
- the liquid storage area 100 is connected to three sample transfer areas 200c, 200d, and 200e, respectively.
- the three sample transfer areas 200c, 200d, and 200e correspond to the reaction areas 300c, 300d, and 300e, respectively.
- the zone contains the respective pre-loaded reagents 11c, 11d and 11e.
- the volume of the sample solution 12 added to the liquid storage area 100 should be greater than or equal to the total volume of all reaction areas and the preloaded reagents contained in all reaction areas The difference between the total volume.
- the volume of the sample solution 12 in the storage area 100 is added.
- the volume should be greater than or equal to V1+V2+V3-Y1-Y2-Y3.
- a reaction tube array comprising a plurality of the above-mentioned reaction tubes and a connecting member 400 connecting the plurality of the above-mentioned reaction tubes, and a plurality of the reaction tubes share a liquid storage area or a plurality of the above-mentioned reaction tubes.
- Each reaction tube in the reaction tube has its own independent liquid storage area.
- Fig. 6 shows a situation where each reaction tube in a plurality of reaction tubes has its own independent liquid storage area.
- a plurality of reaction tubes can be connected and used through the connecting member 400.
- the structure and volume of the connected reaction tubes may be the same or different, and the pre-loaded reagent 11 contained in the reaction zone 300 of each reaction tube may be the same or different.
- the connecting piece 400 is respectively connected with the liquid storage areas 100f, 100g and 100h of the three reaction tubes, and the three reaction tube groups are connected for use.
- the three liquid storage areas are respectively connected to the respective sample transfer areas 200f, 200g and 200h, and each sample transfer area is respectively connected to the respective reaction areas 300f, 300g and 300h, and each reaction area contains its own pre-loaded reagents 11f, 11g and 11h .
- the reaction tube array of the embodiment of the present invention can perform multiple parallel reactions as required.
- a method for controlling the volume of a sample involved in a reaction comprising: adding a liquid sample or a sample solution to the liquid storage area of the above-mentioned reaction tube or reaction tube array, and reacting by centrifugation or hand shaking.
- the reaction tube makes the reaction zone of the reaction tube or the reaction tube array filled with the liquid sample or sample solution, wherein the reaction zone of the reaction tube contains the pre-loaded reagent, and the pre-loaded reagent is in the liquid
- the sample or sample solution is added to the reaction zone before adding the sample solution to the liquid storage zone.
- volume of the liquid sample or sample solution added to the liquid storage area needs to be greater than or equal to the total volume of the reaction zone connected to the liquid storage area-the total volume of the pre-filled liquid in the reaction zone connected to the liquid storage area, In order to ensure that the liquid sample or sample solution can fill the reaction area after centrifugation or hand shaking.
- the volume of the sample that can enter the reaction zone is controlled by pre-installing a set volume of reagent in the reaction zone; on the other hand, due to the first internal transverse of the liquid circulation pipe in the sample transfer zone of the reaction tube
- the cross-sectional area is much smaller than the second inner cross-sectional area of the first zone in the reaction zone.
- a reaction tube a reaction tube array, and a method for controlling the volume of a sample involved in a reaction in a fluorescent quantitative PCR reaction to detect whether there is a specific sequence of DNA molecules in the sample, or to determine the specific sequence of DNA molecules.
- a method for controlling the volume of a sample involved in a reaction in a fluorescent quantitative PCR reaction to detect whether there is a specific sequence of DNA molecules in the sample, or to determine the specific sequence of DNA molecules.
- reaction tube an application of the reaction tube, the reaction tube array, and the method for controlling the volume of the sample involved in the reaction in the color reaction for qualitatively detecting the pH of the sample solution.
- the application of the reaction tube, the reaction tube array, the method for controlling the volume of the sample involved in the reaction, and the application of the above-mentioned reaction tube, the reaction tube array, and the method for controlling the volume of the sample involved in the reaction is pre-installed in the reaction zone
- a fixed volume of reagent is used to control the volume of sample that can enter the reaction zone;
- the first internal cross-sectional area of the liquid circulation pipe in the sample transfer zone of the reaction tube is much smaller than the second internal cross-sectional area of the first zone in the reaction zone Area, the sample that did not enter the reaction zone due to the filling of the reaction zone only slowly diffuses into the reaction zone at a negligible diffusion rate, so the amount of sample involved in the reaction is approximately equal to the amount of sample entering the reaction zone, thus ensuring that only simple instruments and simplicity are required
- the operation can precisely control the amount of sample involved in the reaction in the small-volume liquid phase reaction.
- Figure 1 is a front view of a reaction tube provided by an embodiment of the present invention.
- FIG. 2 is a schematic diagram of a process of a liquid sample or a sample solution entering the reaction zone of the reaction tube in the embodiment of the present invention
- Figure 3 is a front view of a reaction tube provided by an embodiment of the present invention, showing two different sample transfer area structures;
- FIG. 4 is a cross-sectional view of a reaction tube having a transfer zone structure provided by an embodiment of the present invention
- Figure 5 is a front view of a reaction tube with multiple transfer zones and multiple reaction zones provided by an embodiment of the present invention
- Fig. 6 is a front view of a reaction tube array provided by an embodiment of the present invention.
- Fig. 7 shows the fluorescent quantitative PCR results obtained when using reaction tube A in Example 2 of the present invention when different volumes of sample solutions are added.
- Fig. 8 shows the fluorescent quantitative PCR results obtained when different volumes of sample solutions are added using the reaction tube B in Example 2 of the present invention.
- Icon 100-liquid storage area; 200-sample transfer area; 300-reaction area; 10-reaction tube upper cover; 11-preloaded reagents; 220-outer tube of sample transfer area; 210-tube in sample transfer area; 12- Liquid sample or sample solution; 12a-liquid sample or sample solution that does not enter the reaction zone; 12b-liquid sample or sample solution that enters the reaction zone; 21-liquid sample or sample solution that enters the reaction zone or a mixed solution of sample solution and pre-loaded reagents; 310-Reaction zone inner wall; 400-Connector.
- This example shows a specific reaction tube and its preparation method.
- Hollow hose made of material, in which the outer diameter of the glass capillary tube is 2.00 ⁇ 0.05mm, the inner diameter is 1.50 ⁇ 0.05mm, and the length is 36mm; the outer diameter of the PFA hose is 1.50 ⁇ 0.05mm, the inner diameter is 0.50 ⁇ 0.02mm, and the length is 24mm.
- the part of the PFA hose in the glass capillary that is not inserted is used as the reaction zone 300 with a capacity of about 20 ⁇ l.
- the area where the cross-sectional area meets the ratio requirement is the first area; the part of the glass capillary tube with the PFA hose inside, that is, the cavity part in the PFA hose, serves as the first pipe of the sample transfer area 200, with a capacity of about 5 ⁇ l; glass capillary tube
- the opening is connected with the opening at the lower end of the funnel-shaped liquid storage tank.
- the liquid storage tank serves as the liquid storage area 100 of the reaction tube, with a capacity of about 60 ⁇ l.
- a fluorescent quantitative PCR reaction was used to verify the influence of the ratio of cross-sectional area in the sample transfer area of the reaction tube and the reaction area.
- the reaction tube prepared in Example 1 was used as the reaction tube A.
- another reaction tube B is made in this example, which does not contain a PFA hose inside, and the rest of the structure, size and material are the same as the reaction tube A.
- the difference between the two reaction tubes is: the inner diameter of the sample transfer area 200 of the reaction tube A is 0.50 ⁇ 0.02 mm, and the inner diameter of the sample transfer area 200 of the reaction tube B is 1.50 ⁇ 0.05 mm.
- the reaction zone 300 of the reaction tube A and the reaction tube B are both pre-loaded with 10 ⁇ l of the fluorescence quantitative PCR reaction premix, that is, the above-mentioned pre-loaded reagent 11.
- This master mix contains: 0.5U/ ⁇ l KAPA2G Fast DNA Polymerase (KAPA BIOSYSTEMS), 2x KAPA2G Buffer (KAPA BIOSYSTEMS), 4mM MgCl 2 , 2 ⁇ M forward primer, 2 ⁇ M reverse primer, 400 ⁇ M each dNTP, 2x SYBR Green dye.
- the sequence of the forward primer is: 5'-GGGCCAATGTTGTATCCTTCTC-3'
- the sequence of the reverse primer is: 5'-GCCCATCGGTCACTTACACTTC-3'.
- Step 1 Add sample solution to the liquid storage area 100 of the two reaction tubes.
- Step 2 The sample solution 12 enters the reaction zone 300 of the reaction tube by centrifugation, and the volume exceeds the volume of the reaction zone 300-the sample solution with the pre-loaded reagent volume is stored in the sample transfer area 200, or stored in the transfer area 200 and the liquid storage area 100.
- Step 3 Put the reaction tube after adding the sample solution 12 and centrifuged into the fluorescence quantitative PCR device, so that the temperature near the reaction zone 300 can be controllably switched between different temperatures, so that the liquid in the reaction zone 300 can perform PCR reaction.
- the fluorescence quantitative PCR device contains a blue LED light source and corresponding imaging equipment, which can record the fluorescence intensity of the liquid in the reaction zone 300 at the end of the low temperature of each cycle.
- the specific reaction temperature program is as follows: 95°C for 5s melting, 60°C for 15s extension, 40 cycles in this way, recording the fluorescence intensity of the reaction zone 300 at the end of each cycle at 60°C for 15s.
- FIG. 7 shows the fluorescence intensity results of 4 sets of experiments using reaction tube A. It can be seen that the excess sample solution (the sample solution that cannot enter the reaction zone because the reaction zone is filled) will not enter the reaction zone due to diffusion. The sample volume has no effect on the fluorescence quantitative PCR reaction.
- Figure 8 shows the fluorescence intensity results of 4 sets of experiments using reaction tube B.
- the reaction tube A since the reaction tube A includes a transfer zone with a smaller inner diameter and a longer length, when the volume of the reaction zone 300 and the volume of the pre-installed fluorescent quantitative PCR reaction solution 11 and the concentration of the components are constant, It is not necessary to accurately control the volume of the sample solution 12 added to the reaction tube, and the volume of the sample solution participating in the reaction remains unchanged.
- the results of this example show that the reaction tube A can accurately control the volume of the sample involved in the fluorescence quantitative PCR reaction.
- an indigo carmine acid-base indicator was used to perform qualitative pH measurement to verify the influence of the ratio of cross-sectional area in the sample transfer zone of the reaction tube and the reaction zone.
- reaction zone 300 of the reaction tube A and the reaction tube B is pre-loaded with 10 ⁇ l of indigo carmine aqueous solution with a mass fraction of 0.04% as the pre-loaded reagent 11.
- Step 1 Prepare NaOH aqueous solutions with concentrations of 200 nM, 100 nM, 50 nM, and 10 nM as sample solution 12.
- Control group 1 Use 4 reaction tubes A to which 10 ⁇ l of indigo carmine aqueous solution has been added, respectively add 10 ⁇ l of NaOH aqueous solutions of four concentrations to the storage area 100 of the 4 reaction tubes, and centrifuge to make the sample solution 12 completely enter the reaction area 300. There is no solution remaining in the sample transfer area 200 or the liquid storage area 100.
- Control group 2 Use 4 reaction tubes B to which 10 ⁇ l of indigo carmine aqueous solution has been added, respectively add 40 ⁇ l of four concentrations of NaOH aqueous solution to the storage area 100 of the 4 reaction tubes, and centrifuge to make the sample solution 12 enter and fill the reaction area 300 , The part whose volume exceeds the reaction zone 300 remains in the sample transfer zone 200 or the liquid storage zone 100.
- Experimental group Use 4 reaction tubes A to which 10 ⁇ l of indigo carmine aqueous solution has been added, respectively add 40 ⁇ l of four concentrations of NaOH aqueous solution to the storage area 100 of the 4 reaction tubes, and centrifuge to make the sample solution 12 enter and fill the reaction area 300.
- the part whose volume exceeds the reaction zone 300 remains in the sample transfer zone 200 or the liquid storage zone 100.
- the color development results of the solution in the reaction zone 300 of the 12 reaction tubes in the three groups are as follows:
- the experimental results of the 4 reaction tubes in the experimental group are the same as those in the control group 1, and the NaOH concentration is corresponding to the solution from high to low
- the colors are yellow, yellow-green, blue-green and blue, indicating that only the 10 ⁇ l sample solution 12 that enters the reaction zone 300 participates in the mixing with the pre-installed acid-base indicator, while the 30 ⁇ l sample solution that does not enter the reaction zone 300 does not participate in and preliminarily Load the mix of acid-base indicator.
- the results of the three experiments with NaOH concentration of 200nM-50nM in control group 2 are all yellow, and the solution is more alkaline, indicating that the volume of sample solution 12 involved in mixing with the pre-installed acid-base indicator is greater than 10 ⁇ l, and some of them did not enter the reaction.
- the sample solution in zone 300 enters the reaction zone through diffusion and participates in the mixing with the pre-installed acid-base indicator.
- reaction tube A contains a transfer zone with a smaller inner diameter and a longer length, it is not necessary if the capacity of the reaction zone 300 and the volume and component concentration of the pre-installed color solution 11 are constant.
- the volume of the sample solution 12 added to the reaction tube is accurately controlled, and the volume of the sample solution participating in the color reaction remains unchanged.
- the results of this example show that the reaction tube A can accurately control the volume of the sample involved in the color reaction.
- reaction tubes B, C, and D three reaction tubes are used, namely reaction tubes B, C, and D.
- the reaction tube B is the same as the reaction tube B in Example 2
- the reaction tube C is the PFA hose in the reaction tube A in Example 2 replaced with a hose with an outer diameter of 1.50 ⁇ 0.05mm and an inner diameter of 0.75 ⁇ 0.02mm
- the reaction tube D is the PFA hose in the reaction tube A in Example 2 is replaced by a hose with an outer diameter of 1.50 ⁇ 0.05 mm and an inner diameter of 0.56 ⁇ 0.02 mm.
- Sample solution No. 1 contains mixed-source human male genomic DNA at a concentration of about 2 ng/ ⁇ l, and contains NaOH at a concentration of about 100 mM.
- the No. 2 sample solution contains the same mixed-source human male genomic DNA at a concentration of about 2ng/ ⁇ l, and does not contain NaOH.
- the pre-loaded reagent 11 pre-added in the reaction zone 300 of each reaction tube is 18 ⁇ l of a fluorescent quantitative PCR reaction premix.
- This master mix contains: 0.25U/ ⁇ l KAPA2G Fast DNA Polymerase (KAPA BIOSYSTEMS), 1x KAPA2G Buffer (KAPA BIOSYSTEMS), 2mM MgCl 2 , 1 ⁇ M forward primer, 1 ⁇ M reverse primer, 200 ⁇ M each dNTP, 1x SYBR Green dye.
- the sequence of the forward primer is: 5'-GGGCCAATGTTGTATCCTTCTC-3'
- the sequence of the reverse primer is: 5'-GCCCATCGGTCACTTACACTTC-3'.
- Control group Use a reaction tube B with 18 ⁇ l of pre-loaded reagents, add 18 ⁇ l of No. 2 sample solution to the liquid storage area 100 of the reaction tube, centrifuge to make the sample solution enter and fill the reaction zone 300, and the volume exceeds the reaction zone 300 Part of it remains in the sample transfer area 200 or the liquid storage area 100.
- Experimental group 1 Use a reaction tube B with 18 ⁇ l of pre-filled reagents, add 18 ⁇ l of No. 1 sample solution to the liquid storage area 100 of the reaction tube, centrifuge to make the sample solution enter and fill the reaction area 300, and the volume exceeds the reaction area 300 The part remains in the sample transfer area 200 or the liquid storage area 100.
- Experimental group 2 Use 3 reaction tubes C that have been added with 18 ⁇ l of pre-loaded reagents, add 18 ⁇ l of sample solution No. 1 to the liquid storage area 100 of each reaction tube, centrifuge to make the sample solution enter and fill the reaction area 300, and the volume exceeds the reaction Part of the area 300 remains in the sample transfer area 200 or the liquid storage area 100.
- Experimental group 3 Use 3 reaction tubes D with 18 ⁇ l of pre-loaded reagents, add 18 ⁇ l of No. 1 sample solution to the liquid storage area 100 of each reaction tube, centrifuge to make the sample solution enter and fill the reaction area 300, and the volume exceeds the reaction Part of the area 300 remains in the sample transfer area 200 or the liquid storage area 100.
- the target sequence of double-stranded DNA of about 10 5 molecules / ⁇ l corresponding to the amplified product was: 5 '-GGGCCAATGT TGTATCCTTC TCAGTGTTTC TTCGGCCTTT CTAGTGGAGA GGTGCTCTCG GGGAAGTGTA AGTGACCGAT GGGC-3'.
- the centrifugation allows the sample solution to enter the reaction zone 300, and the part whose volume exceeds the reaction zone 300 remains in the sample transfer zone 200 or the liquid storage zone 100 and the sample transfer zone 200.
- the reaction tube after adding the sample solution 12 and centrifuging is placed in a fluorescent quantitative PCR device, so that the temperature near the reaction zone 300 can be controllably switched between different temperatures, so that the liquid in the reaction zone 300 can undergo PCR reaction.
- the device contains a blue LED light source and corresponding imaging equipment, which can record the fluorescence intensity of the liquid in the reaction zone 300 at the end of the low temperature of each cycle.
- the specific reaction temperature program is as follows: 95°C for 5s melting, 60°C for 15s extension, 40 cycles in this way, recording the fluorescence intensity of the reaction zone 300 at the end of each cycle at 60°C for 15s.
- the Quantagne q225 fluorescent quantitative PCR instrument (Beijing Kubo Technology Co., Ltd.) was used for verification experiments.
- the reaction solutions in different reaction wells contain mixed-source human male genomic DNA at a concentration of about 2ng/ ⁇ l and NaOH at a concentration of 40mM, 20mM, 10mM or no NaOH, each with three parallel reaction wells for each NaOH concentration.
- the volume of the reaction zone of the reaction tube is 20 ⁇ l, and the volume of the preloaded reagent is 18 ⁇ l, so the volume of the sample solution that can enter the reaction zone is 2 ⁇ l. If the NaOH in the sample solution that has not entered the reaction zone does not diffuse into the reaction zone, when the NaOH concentration in the sample solution added to the storage zone is 100 mM, the NaOH concentration in the mixed solution in the reaction zone is about 10 mM. If the NaOH in the sample solution that has not entered the reaction zone diffuses into the reaction zone, the concentration of NaOH in the mixed solution in the reaction zone is greater than 10 mM.
- Table 2 shows the reaction results in reaction tubes B, C, and D. Comparing the reaction results of the verification experiment with the experimental results of the three reaction tubes shown in Table 2, it can be seen that when 18 ⁇ l of sample solution No. 1 containing 100 mM NaOH was added to reaction tube B, no amplification was seen, because the NaOH concentration in the reaction solution was too high. The PCR reaction cannot be performed normally due to the large size; when 18 ⁇ l of sample solution No. 1 containing 100mM NaOH is added to the reaction tube C, the amplification can be carried out, but the standard deviation of the Ct value of three parallel experiments is very large, and the average value is very different from the control group. ; When 18 ⁇ l of sample solution No.
- reaction tube D 1 containing 100mM NaOH was added to reaction tube D, the Ct value of the three parallel experiments was very close, and was close to the result of the control group. It can be seen that when the inner diameter of the reaction zone 300 of the reaction tube is 1.5 mm, and the inner diameter of the first pipe of the sample transfer zone 200 is 0.56 mm, the ratio of the second internal cross-sectional area to the first internal cross-sectional area is approximately
- the application of controlling the volume of samples involved in the reaction can be carried out at 7 o'clock, which can be used for quantitative detection; the inner diameter of the sample transfer area 200 is 0.75mm, that is, the ratio of the second inner cross-sectional area to the first inner cross-sectional area is about 4.
- the application of controlling the volume of the sample involved in the reaction cannot be performed stably, so it cannot be used for quantitative detection, but can only be used for qualitative detection;
- the inner diameter of the sample transfer zone 200 is the same as the reaction zone, that is, the second inner cross-sectional area is When the ratio of the internal cross-sectional area is 1, the application of controlling the volume of the sample involved in the reaction cannot be performed.
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Abstract
Description
Claims (13)
- 一种反应管,其特征在于,包括储液区、样品转移区和反应区,所述储液区一端开口,另一端与样品转移区连通;所述样品转移区包含至少一个管道,所述至少一个管道中的第一管道中与所述反应区连接的部分具有第一内横截面积,所述第一管道具有第一内横截面积的连续管道长度不小于0.5mm,所述第一管道的一端与储液区连通,另一端与反应区连通;所述反应区一端封闭,另一端与所述第一管道连通,所述反应区中第一区域的长度不小于2mm,所述第一区域为反应区中与所述第一管道连接的且其内横截面积与第一内横截面积比值不小于4的区域,所述反应区的容积不大于1ml。
- 根据权利要求1所述的反应管,其特征在于,所述反应区中预装有预装试剂,所述预装试剂为固体或液体,所述预装试剂的体积小于所述反应区的容积。
- 根据权利要求1或2所述的反应管,其特征在于,所述至少一个管道包括一个外管和至少一个内管,所述内管位于所述外管内部,所述内管为第一管道。
- 根据权利要求3所述的反应管,其特征在于,所述内管的外壁与所述外管的内壁贴合。
- 根据权利要求3所述的反应管,其特征在于,所述外管中包含分隔物,所述分隔物将所述外管分隔成至少一个所述内管。
- 根据权利要求1-5任一项所述的反应管,其特征在于,所述储液区、样品转移区和反应区为一体成型,或所述储液区、样品转移区和反应区中的至少一个区单独成型,且单独成型的区在成型后与其他区直接连接或通过附件连接。
- 根据权利要求6所述的反应管,其特征在于,所述连接为螺纹、胶粘、焊接、熔接或嵌合连接。
- 根据权利要求1-7任一项所述的反应管,其特征在于,所述第一管道的内壁为疏水性较强的材料。
- 根据权利要求1-8任一项所述的反应管,其特征在于,与所述储液区连通的所述样品转移区有多个,所述反应区与样品转移区一一对应。
- 一种反应管阵列,包含多个权利要求1-9任一项所述的反应管和将多个权利要求1-9任一项所述的反应管连接起来的连接件,多个所述反应管共用一个储液区或多个所述反应管中的每个反应管有各自独立的储液区。
- 一种控制参与反应的样品体积的方法,所述方法包括:向权利要求1-9任一项所述的反应管或权利要求10所述的反应管阵列的储液区加入液体样品或样品溶液;通过离心或手甩所述反应管或所述反应管阵列使所述反应管或所述反应管阵列的反应区被所述液体样品或样品溶液填满;其中,所述反应管或所述反应管阵列的反应区内包含所述预装试剂,所述预装试剂在所述液体样品或样品溶液加入所述储液区前加入所述反应区。
- 一种权利要求1-9任一项所述的反应管、权利要求10所述的反应管阵列或权利要求11所述的控制参与反应的样品体积的方法在荧光定量PCR反应,检测样品中是否存在特定序列DNA分子的反应或测定特定序列DNA分子浓度的反应中的应用。
- 一种权利要求1-9任一项所述的反应管、权利要求10所述的反应管阵列或权利要求11所述的控制参与反应的样品体积的方法在定性检测溶液酸碱度的显色反应中的应用。
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