CN114015567A - Pneumatic integrated PCR detection kit based on rotary plunger and detection method thereof - Google Patents
Pneumatic integrated PCR detection kit based on rotary plunger and detection method thereof Download PDFInfo
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Abstract
The invention discloses a pneumatic integrated PCR detection kit based on a rotary plunger and a detection method thereof, wherein the kit comprises a kit body (1), and the kit body (1) is at least provided with a mixing bin (101) for cleaning and eluting a sample and a plurality of liquid storage bins (102) for respectively storing different reagents; the box body (1) is also provided with a plunger cavity, a rotary plunger (103) capable of rotating to different angles along the axial direction is arranged in the plunger cavity, a plurality of different liquid flow channels are arranged on the rotary plunger (103), and each liquid flow channel is communicated with the mixing bin (101) and the liquid storage bin (102) when the rotary plunger (103) rotates to different angles; the box body (1) is provided with an air pressure driving module (104) with an air inlet and an air outlet communicated to the mixing bin (101). The PCR detection kit and the detection method have the advantages of high space utilization rate, strong controllability, simple operation, difficult pollution introduction and higher detection precision.
Description
Technical Field
The invention relates to a pneumatic integrated PCR detection kit based on a rotary plunger and a detection method thereof, belonging to the technical field of medical detection.
Background
Polymerase Chain Reaction (PCR) is a method of using a DNA fragment as a template, and amplifying the DNA fragment to a sufficient amount for structural and functional analysis in the presence of DNA polymerase and nucleotide substrates. The PCR detection method has very important significance in clinically and rapidly diagnosing bacterial infectious diseases and the like.
When PCR detection is carried out, a collected sample is generally required to be subjected to cracking and cleaning and then to be subjected to reaction, a step-by-step operation mode is generally adopted in the early stage, and the sample is required to be continuously transferred to different containers and medicaments in the process, so that pollution is easily introduced, and the detection precision is seriously influenced. The microfluidic chip can transplant the steps of sample preparation, mixing, reaction, detection and the like to a very small chip in an operation unit mode, and material transfer is carried out through a microfluidic pipeline network on the chip, so that the chip combining the microfluidic chip and the PCR has the advantages of small reagent consumption, high analysis speed, high automation degree and the like, but the microfluidic pipeline is usually paved in a planar mode, and based on the requirement of small volume of the chip, the internal pipeline is thin, the requirement on size precision is high, and the control precision of flow is very high.
Disclosure of Invention
The invention aims to provide a pneumatic integrated PCR detection kit based on a rotary plunger and a detection method thereof. The PCR detection kit and the detection method have the advantages of high space utilization rate, strong controllability, simple operation, difficult pollution introduction and higher detection precision.
The technical scheme of the invention is as follows: the utility model provides a pneumatic integral type PCR detect reagent box based on rotatory plunger, its characteristics are: the device comprises a box body, wherein at least one mixing bin for cleaning and eluting a sample and a plurality of liquid storage bins for storing different reagents are arranged on the box body; the box body is also provided with a plunger cavity, a rotary plunger capable of rotating to different angles along the axial direction is arranged in the plunger cavity, a plurality of different liquid flow channels are arranged on the rotary plunger, and each liquid flow channel is communicated with the mixing bin and the liquid storage bin when the rotary plunger rotates to different angles; the box body is provided with an air pressure driving module communicated with an air inlet and an air outlet to the mixing bin;
among the aforesaid pneumatic integral type PCR detect reagent box based on rotatory plunger, its characterized in that: the liquid storage bins are provided with air holes. The mixing bin and the liquid storage bin are both provided with liquid holes for liquid to enter and exit and gas holes for gas to enter and exit. The box body is provided with an air valve, the rotary plunger is further provided with a plurality of air channels, and each air channel is communicated with the air valve and the air hole of the mixing bin when the rotary plunger rotates to different angles. When any one of the liquid storage bins is communicated with the mixing bin through the liquid flow channel, the liquid storage bin is communicated to the air valve through the air flow channel. The function of the air valve is to make the functional bin (liquid storage bin, etc.) freely enter and exit air, the pressure in the bin is constant, and when the air pressure in the air pressure driving module mixed bin changes, the liquid is transferred along the liquid path according to the pressure difference between the two bins.
In the pneumatic integrated PCR detection kit based on the rotary plunger, as an optimization, the box body is provided with a sample adding bin for sample cracking and conversion, the sample adding bin is provided with a sample adding hole, and the rotary plunger is provided with a liquid flow channel for communicating the sample adding bin with the mixing bin. If no sample adding bin is arranged, a sample adding hole is arranged in the mixing bin and is used for directly adding the converted sample (sample liquid after cracking treatment); the sample cracking and converting steps can be integrated by adding the sample loading bin.
In the pneumatic integrated PCR detection kit based on the rotary plunger, one of the reservoirs is preferably used as a PCR reaction reservoir. If the PCR reaction chamber is not arranged, the liquid for completing the sample treatment needs to be transferred to other kits or containers for PCR reaction, and the steps of amplification reaction can be integrated together by arranging the PCR reaction chamber. The PCR reaction chamber can be provided with certain transmittance, so that the fluorescence detection can be directly carried out from the outside, and the quantitative detection of nucleic acid can be realized.
In the pneumatic integrated PCR detection kit based on the rotary plunger, the box body is further provided with a second PCR reaction area, the second PCR reaction area comprises a one-to-many module, the one-to-many module comprises a plurality of one-to-many chambers which are transversely and sequentially arranged, and a main channel which is transversely communicated is arranged above the one-to-many chambers; a liquid flow channel for communicating the secondary mixing bin with one side of the main channel is arranged on the rotary plunger; each sub-multi-chamber is communicated with the second PCR reaction chamber through a bent liquid path at the lower side. The second PCR reaction area can be used for distributing products after PCR reaction and simultaneously carrying out multichannel PCR secondary reaction, so that a plurality of nested amplifications are realized, and the detection precision is further improved. The second PCR reaction chamber can be provided with certain transmittance, so that the fluorescence detection is conveniently carried out from the outside, and the quantitative detection of nucleic acid is realized.
In the pneumatic integrated PCR detection kit based on the rotary plunger, the other side of the main channel is provided with a waste liquid bin, and the waste liquid bin is provided with an air hole. The redundant reaction liquid is stored in a waste liquid bin. And the rotary plunger is provided with a gas channel for communicating the gas hole of the waste liquid bin with the gas valve.
In the pneumatic integrated PCR detection kit based on the rotary plunger, one of the liquid storage bins is internally used for storing sealing liquid and used for sealing the main channel after the diluted PCR reactant is distributed to the second PCR reaction bin, so that mutual influence among the channels is prevented.
In the pneumatic integrated PCR detection kit based on the rotary plunger, a secondary sample adding cavity is further arranged between the bent liquid path and the second PCR reaction bin, and a secondary sample adding hole is formed in the secondary sample adding cavity. The secondary sample adding holes are preset, so that the flexibility of the kit can be improved, and reaction liquid, enzyme and the like can be added according to actual detection requirements. And the reagent added into the secondary sample adding hole and the reagent in the multi-cavity chamber can enter the secondary PCR reaction chamber together only by once pressurization.
In the pneumatic integrated PCR detection kit based on the rotary plunger, the box body is provided with a secondary mixing bin connected with the PCR reaction bin; one of the liquid storage bins is internally stored with diluent and used as a dilution bin for PCR reaction products; the diluting bin is communicated with the secondary mixing bin through a liquid flow channel on the rotary plunger.
In the pneumatic integrated PCR detection kit based on the rotary plunger, the liquid flow channel on the rotary plunger consists of a groove arranged on the outer surface of the rotary plunger and the inner wall of the plunger cavity; the outer surface of the rotary plunger is made of flexible materials and forms interference fit with the inner wall of the plunger cavity, and sealing of a liquid flow channel is guaranteed. The rotary plunger is cylindrical, the surface area is large, and more flow passages can be arranged, so that different liquid flow passages can be used in each processing step, and the pollution caused by the repeated use of the flow passages is avoided.
In the aforesaid pneumatic integral type PCR detect reagent box based on rotatory plunger, the box body is the flat structure, including consumptive material main part and the closed membrane of compound in the consumptive material main part, mixed storehouse and stock solution storehouse in the box body enclose to close by the consumptive material main part and the closed membrane in both sides and form.
The detection method of the pneumatic integrated PCR detection kit based on the rotary plunger comprises the following steps:
cleaning: the magnetic beads absorbed with sample nucleic acid stay in the mixing bin by using an external magnet, then the mixing bin on the box body is sequentially communicated with one or more liquid storage bins containing cleaning liquid by using a liquid flow channel by rotating the rotary plunger to a specific angle, then the pressure intensity of the mixing bin is changed by using the air pressure driving module, so that the cleaning liquid enters the mixing bin, and the reagent can return to the mixing bin by reversely driving the air pressure driving module, so that one-time or multiple-time cleaning of the sample nucleic acid is realized;
elution: through rotating rotary plunger to specific angle, make the mixed storehouse on the box body utilize liquid runner and the stock solution storehouse that contains the eluant to realize the liquid circuit intercommunication, then utilize atmospheric pressure drive module to make the eluant inhale mixed storehouse, elute the nucleic acid on the magnetic bead to carry out subsequent PCR reaction.
In the detection method, after the mixing bin is communicated with the liquid path of any one of the liquid storage bins, the gas channel on the rotary plunger also communicates the gas hole of the liquid storage bin with the air valve, so that the reagent in the liquid storage bin can be smoothly transferred when the air pressure driving module operates.
In the above-described detection method, preferably, before the step (i), a sample preprocessing step is further included: add into sample and the required medicament of schizolysis/conversion in the application of sample storehouse, form the sample mixed liquid, and sample schizolysis/conversion is accomplished in the heating, rotation through rotatory plunger makes application of sample storehouse and mixed storehouse switch on, utilize the atmospheric pressure drive module to make the sample mixed liquid shift to mixed storehouse, rotate rotatory plunger to another angle, make the stock solution storehouse of storing the bonding liquid switch on with mixed storehouse, and operation atmospheric pressure drive module sneaks into mixed storehouse with the bonding liquid, then use outside magnet to produce magnetic force and adsorb the magnetic bead in mixing the storehouse, after the standing, treat that the sample mixed liquid becomes clear, send the supernatant fluid out and mix the storehouse, then kept sample nucleic acid on the magnetic bead. The magnetic beads are added from the sample adding bin or directly added from the mixing bin.
In the above-mentioned detection method, preferably, after the step (ii), a PCR reaction step is further included: the rotary plunger is rotated to a specific angle, so that the mixing bin on the box body is communicated with the PCR reaction bin through the liquid flow channel, and then the eluent with eluted nucleic acid is sent to the PCR reaction bin through the air pressure driving module, so that the nucleic acid is subjected to PCR reaction in reaction liquid in the PCR reaction bin.
In the above-mentioned detection method, preferably, the PCR reaction step further comprises a second PCR reaction step (nested PCR):
the rotary plunger is rotated to a specific angle, so that the dilution bin on the box body is communicated with the mixing bin through a liquid path, then the diluent is sucked into the mixing bin through the air pressure driving module, the rotary plunger is rotated again, so that the mixing bin is communicated with the PCR reaction bin through the liquid path, the diluent is sequentially pressed into the PCR reaction bin and the secondary mixing bin through the air pressure driving module, so that the PCR reactant is diluted, and then the diluted PCR reactant is sucked back to the mixing bin;
rotating the plunger to enable the mixing bin to be communicated with a main channel liquid path of the one-to-many module, pressurizing the mixing bin by the air pressure driving module, enabling diluted PCR reactants to sequentially fill one-to-many chambers in the one-to-many module, and enabling redundant PCR reactants to enter a waste liquid bin; this step can open the vent of the waste bin;
the air pressure driving module continuously pressurizes, so that diluted PCR reactants in one-component multi-cavity are pressed into a second PCR reaction bin corresponding to the lower part of the air pressure driving module, and a second PCR reaction is carried out on the reaction liquid in the second PCR reaction bin; this step closes the vent of the waste bin.
After the diluted PCR reactant is pressed into a second PCR reaction bin corresponding to the lower part of the diluted PCR reactant, the rotary plunger is rotated to enable the mixing bin to be communicated with the liquid storage bin filled with the sealing liquid, then the sealing liquid is sucked into the mixing bin by the air pressure driving module, then the rotary plunger is rotated to enable the mixing bin to be communicated with the main channel, the sealing liquid is fed into the main channel, the main channel and the one-component multi-cavity are filled and sealed, and then the second PCR reaction is continuously carried out.
Compared with the prior art, the invention integrates the functional bins such as the mixing bin and the liquid storage bin on the kit, uses the rotary plunger as a carrier of liquid and gas flow passages, communicates different functional bins by rotating the angle of the rotary plunger to realize function switching, and simultaneously realizes the transfer of liquid in the functional bins by changing the air pressure of the functional bins by using the air pressure driving module, thereby realizing processing functions such as mixing, cleaning, elution and the like. The invention fully exerts the bridge function of the rotary plunger, changes the flow channel for switching the function into a three-dimensional type and fully utilizes the space of the kit. The function switching can be controlled by rotating the rotary plunger, the liquid transfer can be quantitatively carried out by the air pressure driving module, the forward and reverse repeated driving can also play a role in uniformly mixing, the function realization of the invention is easier to operate, and the function can be realized by simply matching with an external motor, so the requirement on matched detection equipment is lower. The invention has high integral integration level, realizes all functions without repeatedly using the same liquid flow channel, avoids the influence of residual medicament on detection, and can completely isolate the whole treatment process from the outside, thereby having higher detection precision.
The detection method of the present invention is derived from the use of a pneumatic integrated PCR detection kit based on a rotary plunger. The magnetic bead is used as a tool for intercepting nucleic acid, and the sample can be conveniently cracked, converted, cleaned, eluted and subjected to PCR reaction by operating the rotation of the rotary plunger and different movement modes of the pneumatic driving module.
Furthermore, the detection method can also utilize the air pressure driving module to evenly divide the PCR reaction products in sequence, the air pressure driving module does not need to be finely operated, the use is very convenient, and further, the second PCR reaction of the second equivalent raw materials can be carried out.
Drawings
FIG. 1 is a schematic structural view of a kit of the present invention;
FIG. 2 is a schematic view of the back side structure of the reagent cartridge shown in FIG. 1
Fig. 3 is a schematic view of the structure of the rotary plunger of the present invention.
Detailed Description
The invention is further illustrated by the following figures and examples, which are not to be construed as limiting the invention.
Example 1: a pneumatic integrated PCR detection kit (nested PCR detection kit) based on a rotary plunger comprises a kit body 1, as shown in figure 1-2, wherein the kit body 1 is at least provided with a mixing bin 101 for cleaning and eluting a sample and a plurality of liquid storage bins 102 for respectively storing different reagents, and respectively stores one or more cleaning liquids, binding liquids, eluents, diluents and the like; the box body 1 is also provided with a plunger cavity, a rotary plunger 103 which can rotate to different angles along the axial direction is arranged in the plunger cavity, a plurality of different liquid flow channels are arranged on the rotary plunger 103, and each liquid flow channel is respectively communicated with the mixing bin 101 and the liquid storage bin 102 when the rotary plunger 103 rotates to different angles; the box body 1 is provided with an air pressure driving module 104 with an air inlet and an air outlet communicated to the mixing bin 101; the liquid storage bins 102 are all provided with air holes. The mixing bin 101 and the liquid storage bin 102 are both provided with liquid holes for liquid to enter and exit and air holes for gas to enter and exit. The pneumatic driving module 104 may be a piston (in this embodiment, a piston is used), or an air nozzle connected to an external air pump may be directly used. The rotary plunger 103 and the pneumatic drive module 104 may be provided in plural for sharing different functions.
The box body 1 is provided with an air valve 105, the rotary plunger 103 is further provided with a plurality of air channels, and each air channel is respectively communicated with the air valve 105 and the air holes of the mixing bin 101 when the rotary plunger 103 rotates to different angles. When any one of the liquid storage chambers 102 communicates with the mixing chamber 101 through a liquid flow path, the liquid storage chamber 102 communicates with the damper 105 through a gas flow path.
The box body 1 is provided with a sample adding bin 106 for sample cracking and conversion, the sample adding bin 106 is provided with a sample adding hole 106a, and the rotary plunger 103 is provided with a liquid flow channel for communicating the sample adding bin 106 with the mixing bin 101.
One of the reservoirs 102 is used as a PCR reaction chamber 102a, the front end of the PCR reaction chamber 102a can be connected with a secondary sample adding cavity with a secondary sample adding hole for adding reaction liquid, enzyme and the like, and when the eluent with nucleic acid enters the PCR reaction chamber 102a, the reagent in the secondary sample adding hole can be brought in together.
The box body 1 is also provided with a second PCR reaction area, the second PCR reaction area comprises a one-to-many module, the one-to-many module comprises a plurality of one-to-many chambers 1101 which are transversely arranged in sequence, and a main channel 1102 which is transversely communicated is arranged above the one-to-many chambers 1101; a secondary mixing bin 107 is arranged on the box body 1, and a gas flow channel for communicating the secondary mixing bin 107 with the pneumatic driving module 104 and a liquid flow channel for communicating one side of the secondary mixing bin 107 with the main channel 1102 are arranged on the rotary plunger 103; each of the divided chambers 1101 is connected to the second PCR reaction chamber 1104 via a bent liquid path 1103 on the lower side. This embodiment adopts an eight-in-one method. The one minute multi-chamber 1101 bottom inclines, and the bottom surface that is close to liquid feeding direction one side is higher, the liquid way 5 that buckles communicates to the lower one side of bottom surface, and the liquid way entry angle and the position design of second time PCR reaction storehouse 6 are more reasonable for two kinds of solutions can directly reach the reaction chamber bottom after the liquid way flows in, and can not produce the bubble.
The other side of main channel 1102 is equipped with waste liquid storehouse 1105, and waste liquid storehouse 1105 is equipped with the gas pocket. The rotary plunger 103 is provided with a gas channel for communicating the gas hole of the waste liquid bin 1105 with the gas gate 105.
A secondary sample adding cavity is also arranged between the bent liquid path 1103 and the second PCR reaction bin 1104, and a secondary sample adding hole 1106 is arranged on the secondary sample adding cavity. The secondary sample adding holes on the PCR reaction bin 102a may be the same as the secondary sample adding holes 1106 therein, a rubber plug may be disposed on the secondary sample adding holes 1106, and when the rubber plug is extruded into the secondary sample adding holes 1106, the sample added into the secondary sample adding holes 1106 may be actually extruded into the secondary sample adding cavity due to air pressure.
One of the liquid storage bins 102 is internally stored with diluent and used as a dilution bin for PCR reaction products; the dilution bin is in communication with the secondary mixing bin 107 via a liquid flow channel in the rotating plunger 103.
The liquid flow channel on the rotary plunger 103 consists of a groove arranged on the outer surface of the rotary plunger 103 and the inner wall of the plunger cavity; the outer surface of the rotary plunger 103 is made of flexible materials and forms interference fit with the inner wall of the plunger cavity, and sealing of a liquid flow channel is ensured. The structure of the rotary plunger 103 is shown in fig. 3.
The box body 1 is of a flat structure and comprises a consumable main body and a sealing film compounded on the consumable main body (the sealing film can be used on one side or both sides), and the mixing bin 101 and the liquid storage bin 102 in the box body 1 are formed by enclosing the consumable main body and the sealing film on both sides.
The detection method using the kit of example 1 was as follows:
cracking and converting: adding reagents (lysis solution or methylation reagent and the like) required by the cracking conversion and a sample into the sample adding bin, and performing circulating heating to perform the cracking conversion of the sample;
magnetic bead binding: add the magnetic bead liquid in mixing storehouse 101, rotation through rotatory plunger 103 makes application of sample storehouse 106 and mix storehouse 101 and switch on, utilize air pressure drive module 104 to make the sample mix liquid and shift to mixing storehouse 101, rotate rotatory plunger 103 to another angle, make the stock solution storehouse 102 of storing the bonding liquid and mix the storehouse and switch on, and operation air pressure drive module will combine liquid to mix storehouse 101, then use outside magnet to produce magnetic force and adsorb the magnetic bead in mixing storehouse 101, after the standing, treat that the sample mixes the liquid and become clear, send out mixing storehouse 101 with the supernatant. In practical use, the magnetic beads can be replaced by filter membranes which are arranged on the liquid holes of the mixing bin 101, nucleic acid can be retained on the filter membranes when all cleaning liquid leaves the mixing bin, and finally the nucleic acid is discharged from the filter membranes through the elution liquid. This eliminates the need for external magnets.
Thirdly, cleaning: the rotary plunger 103 is rotated to a specific angle, so that the mixing bin 101 on the box body 1 is sequentially communicated with one or more liquid storage bins 102 containing cleaning liquid by using a liquid flow channel, then the pressure of the mixing bin 101 is changed by using the movement of the air pressure driving module 104, so that the cleaning liquid enters the mixing bin 101, and the reagent can return to the mixing bin 101 by reversely moving the air pressure driving module 104, so that the cleaning of the sample nucleic acid for one time or more times is realized;
and fourthly, elution: the rotary plunger 103 is rotated to a specific angle, so that the mixing bin 101 on the box body 1 is communicated with the liquid storage bin 102 containing eluent by using a liquid flow channel, then the eluent is sucked into the mixing bin 101 by using the movement of the air pressure driving module 104, and the nucleic acid on the magnetic beads is eluted, so that the subsequent PCR reaction is carried out;
PCR reaction: the rotary plunger 103 is rotated to a specific angle, so that the mixing bin 101 on the box body 1 is communicated with the PCR reaction bin 102a through a liquid flow channel, and then the air pressure driving module 104 is used for moving to send the eluted nucleic acid eluent into the PCR reaction bin 102a, so that the nucleic acid is subjected to PCR reaction in the reaction liquid (which can be arranged in a secondary sample adding cavity) in the PCR reaction bin 102 a;
sixthly, performing the second PCR reaction: the rotary plunger 103 is rotated to a specific angle, so that the dilution bin on the box body 1 is communicated with the mixing bin 101 through a liquid path, then the diluent is sucked into the mixing bin 101 through the air pressure driving module 104, then the rotary plunger 103 is rotated, so that the mixing bin 101 is communicated with a liquid path of the PCR reaction bin 102a, the diluent is sequentially pressed into the PCR reaction bin 102a and the secondary mixing bin 107 through the air pressure driving module to dilute the PCR reactant, and then the diluted PCR reactant is sucked back to the mixing bin 101;
the plunger 103 is rotated to make the mixing bin 101 and the main channel 1102 of the one-to-many module in fluid communication, the pneumatic driving module pressurizes the mixing bin 101, the diluted PCR reactant sequentially fills the one-to-many chambers 1101 of the one-to-many module by the pneumatic pressure, and redundant solution remains in the waste fluid bin 1105; here, the bent liquid path 1103 should be as small as possible to prevent the solution from directly entering the second PCR reaction chamber 1104.
The air hole in the waste liquid bin 1105 is closed, the air pressure driving module continues to pressurize, so that the diluted PCR reactant in the one-component multi-chamber 1101 is pressed into the second PCR reaction bin 1104 corresponding to the lower part, and the second PCR reaction is carried out with the reaction liquid in the second PCR reaction bin 1104.
According to the embodiment, nested PCR can be performed in one reagent kit, so that external pollution during secondary PCR can be avoided, and the detection precision is improved.
Experimental example: in order to compare LOD (minimum detection limit) of the detection method of this example and the direct qPCR (real-time fluorescent quantitative nucleic acid amplification detection) method, the inventors detected 13 target markers using both the method of this example and the direct qPCR method. The reagents and steps of the direct qPCR method were the same as the method of this example, except that there was no pre-amplification step. In each method, 13 target markers are amplified/pre-amplified simultaneously, but each target marker is quantified separately. Comparison of LODs for the pre-amplification method and the direct qPCR method for the target marker 1 is shown below. Comparison of LOD for the pre-amplification method and the direct qPCR method for the other 12 target markers used a similar procedure and is not shown here.
Briefly, specifically, CRC tissue DNA was incorporated into blood cell DNA at a rate of 0.5% and 0.2%. 40ng of DNA was treated with bisulfite (Methycode Bisulfit Conversion Kit), where half of the converted DNA was used in the method of this example and the remaining half of the converted DNA was used directly in qPCR (i.e., direct qPCR method). The final concentration of the primers in the pre-amplification step (first PCR) of this example was 50 nM. 25 μ L of LPCR mixture containing 10 μ L of the transformed DNA, 2.5 μ L of a premix containing the above primers, and 12.5 μ L of LPCR reagent: (
Universal Probe qPCR Master Mix (NEB)). The PCR program was set to 95 ℃ for 3 minutes; 95 ℃ for 30 seconds, 56 ℃ for 60 seconds, 8 cycles. The product obtained after the pre-amplification step was diluted 10 times for further qPCR (second PCR). The qPCR mix consisted of 10 μ L template DNA, 2.5 μ L primer/probe pool and 12.5 μ L LUNA master mix. qPCR was run on ABI 7500Real-Time PCR System, programmed at 95 ℃ for 5 minutes; 95 ℃ for 15 seconds, 56 ℃ for 40 seconds (fluorescence acquisition), 50 cycles. In parallel 4 timesAnd (6) repeating. The results are shown in table 5 below.
Comparison of the results of the method of this example with those of the direct qPCR method
As shown in table 5, the method of this example showed improved LOD (0.50% vs.0.20% CRC DNA percentage), better stability and higher detection sensitivity compared to the direct qPCR method. The results of the pre-amplification method for the other 12 target markers were better or not worse than the direct qPCR method, and the results are not shown here.
Example 2: on the basis of the kit in example 1, a sample adding bin, a PCR reaction bin and a second PCR reaction zone are omitted, and during detection, a sample (containing magnetic beads) subjected to lysis conversion is directly added into a mixing bin, and then magnetic bead combination, cleaning and elution are sequentially carried out by using the same steps as those in example 1. And after the elution is finished, sending the reagent out of the kit, and carrying out external PCR reaction and detection. This embodiment places the heating step outside the kit, and only the sample is washed and eluted, and the external equipment can be more compact.
Example 3: on the basis of the kit in the embodiment 1, only a second PCR reaction area is omitted, the lysis conversion, the cleaning, the elution, the PCR reaction and the detection of the sample can be realized, and the functions are more complete compared with those in the embodiment 2.
Implementation 4: on the basis of the kit of embodiment 1, the second PCR reaction bin 1104 in the second PCR reaction area is omitted, and a liquid outlet is arranged at the end of the bent liquid path 1103 and is directly connected with the test tube, that is, the kit performs sample pretreatment, the first PCR and the dilution of PCR products and divides the products into multiple parts, and the multiple parts of PCR products are subjected to the second PCR reaction in other test tubes or kits. The kit is more flexible than the embodiment, and the primer and the probe of the second PCR reaction can be added into other kits according to the requirement.
Example 5: on the basis of the kit of example 1, the PCR reaction chamber 102a is omitted, and after the elution step, the eluate is divided into multiple parts by the second PCR reaction region directly, and then a multi-channel PCR reaction is performed, which is actually a first PCR reaction performed multiple times simultaneously by using the kit. The kit can be used for multi-channel PCR of samples, and the detection efficiency is improved.
Claims (17)
1. The utility model provides a pneumatic integral type PCR detect reagent box based on rotatory plunger which characterized in that: the kit comprises a kit body (1), wherein the kit body (1) is at least provided with a mixing bin (101) for cleaning and eluting a sample and a plurality of liquid storage bins (102) for storing different reagents respectively; the box body (1) is also provided with a plunger cavity, a rotary plunger (103) capable of rotating to different angles along the axial direction is arranged in the plunger cavity, a plurality of different liquid flow channels are arranged on the rotary plunger (103), and each liquid flow channel is communicated with the mixing bin (101) and the liquid storage bin (102) when the rotary plunger (103) rotates to different angles; the box body (1) is provided with an air pressure driving module (104) with an air inlet and an air outlet communicated to the mixing bin (101).
2. The pneumatic integrated PCR detection kit based on the rotary plunger according to claim 1, wherein: the air holes are formed in the liquid storage bin (102), the air valves (105) are arranged on the box body (1), the rotating plunger (103) is further provided with a plurality of air flow channels, and each air flow channel is respectively communicated with the air holes of the air valves (105) and the liquid storage bin (102) when the rotating plunger (103) rotates to different angles.
3. The pneumatic integrated PCR detection kit based on the rotary plunger according to claim 1, wherein: the sample adding device is characterized in that a sample adding bin (106) for sample cracking or conversion is arranged on the box body (1), a sample adding hole (106a) is formed in the sample adding bin (106), and a liquid flow channel for communicating the sample adding bin (106) with the mixing bin (101) is formed in the rotary plunger (103).
4. The pneumatic integrated PCR detection kit based on the rotary plunger according to claim 1, wherein: one of the reservoirs (102) serves as a PCR reaction reservoir (102 a).
5. The pneumatic integrated PCR detection kit based on the rotary plunger according to claim 4, wherein: the box body (1) is also provided with a second-time PCR reaction area, the second-time PCR reaction area comprises a one-to-many module, the one-to-many module comprises a plurality of one-to-many chambers (1101) which are transversely and sequentially arranged, and a main channel (1102) which is transversely communicated is arranged above the one-to-many chambers (1101); a liquid flow channel for communicating the mixing bin (101) and one side of the main channel (1102) is arranged on the rotary plunger (103); each of the one-divided chambers (1101) is connected to the second PCR reaction chamber (1104) through a bent liquid path (1103) on the lower side.
6. The pneumatic integrated PCR detection kit based on the rotary plunger according to claim 5, wherein: and a waste liquid bin (1105) is arranged on the other side of the main channel (1102), and an air hole is formed in the waste liquid bin (1105).
7. The pneumatic integrated PCR detection kit based on the rotary plunger according to claim 5, wherein: one of the liquid storage bins (102) is internally used for storing sealing liquid.
8. The pneumatic integrated PCR detection kit based on the rotary plunger according to claim 5, wherein: and a secondary sample adding cavity is also arranged between the bent liquid path (1103) and the second PCR reaction bin (1104), and a secondary sample adding hole (1106) is formed in the secondary sample adding cavity.
9. The pneumatic integrated PCR detection kit based on the rotary plunger according to claim 5, wherein: a secondary mixing bin (107) connected with the PCR reaction bin (102a) is arranged on the box body (1); one of the liquid storage bins (102) is internally used for storing diluent and used as a dilution bin for PCR reaction products; the diluting bin is communicated with the secondary mixing bin (107) through a liquid flow channel on the rotary plunger (103).
10. The pneumatic integrated PCR detection kit based on the rotary plunger according to claim 1, wherein: the liquid flow channel on the rotary plunger (103) consists of a groove arranged on the outer surface of the rotary plunger (103) and the inner wall of the plunger cavity; the outer surface of the rotary plunger (103) is made of flexible materials and forms interference fit with the inner wall of the plunger cavity, so that the sealing of a liquid flow passage is ensured.
11. The pneumatic integrated PCR detection kit based on the rotary plunger according to claim 1, wherein: the box body (1) is of a flat structure and comprises a consumable main body and a sealing film compounded on the consumable main body, and the mixing bin (101) and the liquid storage bin (102) in the box body (1) are formed by enclosing the consumable main body and the sealing film on two sides.
12. The detection method using the pneumatic integrated PCR detection kit based on the rotary plunger according to any one of claims 1 to 11, characterized by comprising the steps of:
cleaning: the method comprises the steps that magnetic beads adsorbed with sample nucleic acids stay in a mixing bin (101) by using an external magnet, then a rotating plunger (103) is rotated to a specific angle, the mixing bin (101) on a box body (1) is sequentially communicated with one or more liquid storage bins (102) containing cleaning liquid by using a liquid flow channel, then the pressure of the mixing bin (101) is changed by using an air pressure driving module (104), so that the cleaning liquid enters the mixing bin (101), and reagents can return to the mixing bin (101) by reversely driving the air pressure driving module (104), so that one or more times of cleaning of the sample nucleic acids are realized;
elution: the rotary plunger (103) is rotated to a specific angle, so that the mixing bin (101) on the box body (1) is communicated with the liquid storage bin (102) containing eluent by a liquid flow channel, then the eluent is sucked into the mixing bin (101) by the air pressure driving module (104), and the nucleic acid on the magnetic beads is eluted, so that the subsequent PCR reaction is carried out.
13. The detection method according to claim 12, characterized in that: when the mixing bin (101) is communicated with the liquid path of any one of the liquid storage bins (102), the gas flow channel on the rotary plunger (103) also communicates the air hole of the liquid storage bin (102) with the air valve (105), so that the reagent in the liquid storage bin (102) can be smoothly transferred when the air pressure driving module operates.
14. The detection method according to claim 12, further comprising, before the step (r), a sample preprocessing step of: adding a sample and a reagent required for cracking/conversion into a sample adding bin (106) to form a sample mixed solution, heating to finish sample cracking or conversion, conducting the sample adding bin (106) and a mixing bin (101) by rotating a rotary plunger (103), moving an air pressure driving module (104) to transfer the sample mixed solution to the mixing bin (101), rotating the rotary plunger (103) to another angle to conduct a liquid storage bin (102) for storing a binding solution and the mixing bin, operating the air pressure driving module to mix the binding solution into the mixing bin (101), then using an external magnet to generate magnetic force to adsorb magnetic beads into the mixing bin (101), standing, sending supernatant out of the mixing bin (101) after the sample mixed solution becomes clear, and retaining sample nucleic acid on the magnetic beads; the magnetic beads are added from the sample adding bin (106) or directly added from the mixing bin (101).
15. The detection method according to claim 12, further comprising a PCR reaction step after the step (II): the rotary plunger (103) is rotated to a specific angle, so that the mixing bin (101) on the box body (1) is communicated with the PCR reaction bin (102a) through a liquid flow channel, and then the eluent with the eluted nucleic acid is sent to the PCR reaction bin (102a) through the air pressure driving module (104), so that the nucleic acid is subjected to PCR reaction in the reaction liquid in the PCR reaction bin (102 a).
16. The assay of claim 15, wherein the PCR reaction step is followed by a second PCR reaction step:
the method comprises the steps that a rotary plunger (103) is rotated to a specific angle, a dilution bin on a box body (1) is communicated with a mixing bin (101) through a liquid path, then diluent is sucked into the mixing bin (101) through an air pressure driving module (104), then the rotary plunger (103) is rotated, the mixing bin (101) is communicated with a PCR reaction bin (102a) through the liquid path, the diluent is sequentially pressed into the PCR reaction bin (102a) and a secondary mixing bin (107) through the air pressure driving module to enable PCR reactants to be diluted, and then the diluted PCR reactants are sucked back into the mixing bin (101);
rotating a plunger (103) to enable a mixing bin (101) to be communicated with a main channel (1102) of the one-to-many module through a liquid path, and pressurizing the mixing bin (101) through an air pressure driving module (104) to enable diluted PCR reactants to sequentially fill one-to-many chambers (1101) in the one-to-many module;
the air pressure driving module continuously pressurizes, so that the diluted PCR reactant in the one-component multi-chamber (1101) is pressed into a second PCR reaction chamber (1104) corresponding to the lower part, and a second PCR reaction is carried out with the reaction liquid in the second PCR reaction chamber (1104).
17. The detection method according to claim 15, characterized in that: after the diluted PCR reactant is pressed into a second PCR reaction bin (1104) corresponding to the lower part, the rotary plunger (103) is rotated to enable the mixing bin (101) to be communicated with the liquid storage bin (102) filled with the sealing liquid, then the sealing liquid is sucked into the mixing bin (101) by the air pressure driving module (104), then the rotary plunger (103) is rotated to enable the mixing bin (101) to be communicated with the main channel (1102), the sealing liquid is fed into the main channel (1102), the main channel (1102) and the one-to-many chamber (1101) are filled and sealed, and then the second PCR reaction is continued.
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