CN212293607U - One-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit - Google Patents
One-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit Download PDFInfo
- Publication number
- CN212293607U CN212293607U CN202020441774.9U CN202020441774U CN212293607U CN 212293607 U CN212293607 U CN 212293607U CN 202020441774 U CN202020441774 U CN 202020441774U CN 212293607 U CN212293607 U CN 212293607U
- Authority
- CN
- China
- Prior art keywords
- chamber
- nucleic acid
- adsorption
- channel
- working port
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The patent discloses a one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit, which is an injection molding integrated structure and comprises a sample adding port (A), a cracking chamber (C1), an adsorption and elution chamber (C2), a waste liquid chamber (C3), an eluent chamber (C4), a fluorescence PCR chamber (C5) and a switching valve (VT 1); the switching valve (VT1) controls the communication of the adsorption and elution chamber (C2) with the lysis chamber (C1) and the waste liquid chamber (C3) or with the eluent chamber (C4) and the fluorescent PCR chamber (C5). The combined kit can ensure the whole nucleic acid extraction process and the real-time fluorescence PCR detection process, and can be completely completed in a totally-enclosed kit, so that aerosol pollution in the traditional automatic nucleic acid extraction process and the PCR test sample adding process is avoided, and the biological leakage and sample cross contamination risks are avoided.
Description
Technical Field
The patent relates to nucleic acid extraction, PCR amplification and real-time fluorescence detection technology in molecular biology, in particular to a one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit.
Background
The PCR nucleic acid detection technology is based on a molecular biological detection technology of a nucleic acid amplification technology invented by Kaly Moris in the United states in the last 80 th century, and the technology utilizes different specific sequences of DNA/RNA of organisms of different species to identify the types of biological cells, and the Nobel prize in 1993 is invented by Moris in the name of Moris. The technology is widely used for identifying the types of microbial pathogens in modern medicine, has the characteristics of high precision, high speed and high flux, and is increasingly paid more attention by people.
The existing PCR medical detection technology is combined with a fluorescence real-time detection technology, a micro-fluidic chip detection technology and a PCR technology, the existing PCR detection technology is transited from the traditional negative/positive qualitative detection to the level capable of accurately and quantitatively detecting the content of nucleic acid in a sample, the detection technology can provide one-hand quantitative analysis data for analysis such as medical diagnosis, treatment tracking, medical history development, epidemic disease tracking and the like, and is a powerful assistant for the current disease control, medical treatment and diagnosis.
At present, development of PCR detection instruments and kits is very important in developed countries in Europe and America and China. The mainstream nucleic acid detection technology is a real-time fluorescence PCR detection technology. The operation flow of the technology comprises three steps of sampling, nucleic acid extraction and real-time fluorescence PCR detection. The development technology of the PCR test instrument is also an application instrument mainly oriented to the 3 links.
The PCR technology is to realize the self-replication of nucleic acid in a sample through PCR circulation by acting a reagent on the sample. The concentration of nucleic acid in the sample doubles with self-replication every PCR cycle. This bio-amplification technique allows the nucleic acid in the sample to replicate 2^ n times after n cycles. Therefore, in principle, the PCR technology is adopted, and the detection precision can reach the resolution level of one cell, and such high precision is the advantage of the PCR detection technology and the defect of the technology, because if the sample in the sample testing environment and the aerosol exists, the pollution of one cell to the sample can also cause the detection result to be invalid.
In the technical scheme of the most advanced nucleic acid automatic extraction equipment which is popular internationally at present, in order to ensure that a magnetic rod for transferring and stirring magnetic beads can freely enter and exit each chamber, the spaces of all the chambers are open, and in the extraction process, high-temperature heating and high-frequency mechanical vibration stirring can generate a large amount of aerosol, so that the test environment is polluted, and even biological leakage accidents can be caused.
Therefore, the standard PCR laboratory has very high control requirements on the experimental environment, needs special equipment to form laminar flow and positive pressure of the experimental environment, and ensures that the experiment is not influenced by aerosol pollution through strict control of the laboratory environment. This has resulted in PCR nucleic acid tests that are only stable in high cost laboratories with stringent environmental controls.
Summarizing the empirical training since combating new coronary pneumonia epidemics, the following conclusions can be drawn:
1. the nucleic acid detection scheme is determined as a standard detection means for the diagnosis and detection of the virus infection epidemic disease source;
2. in the face of the situation that the epidemic situation of the people spreads suddenly and rapidly and a large number of suspected patients accumulate to be detected, the detection and diagnosis confirming means is an important link for controlling the development of the epidemic situation, ensures that a large number of suspected patients can be quickly and accurately detected and confirmed, and is a necessary condition for controlling the further diffusion of the epidemic situation;
3. although the nucleic acid detection technology is determined as the core detection means for resisting the epidemic situation by the characteristics of high sensitivity, high detection speed, large detection flux and the like, some defects are exposed in the process of resisting the epidemic situation:
(1) the detection speed is slow:
although the speed of nucleic acid detection is much higher than that of other classical pathogen detection methods, the detection still appears to be too slow in the case of detection of a large number of suspected cases, and doctors need a faster nucleic acid detection system in the case of outbreak of epidemic situations;
(2) the detection laboratory has high environmental requirements
The classical nucleic acid detection procedures, in the nucleic acid extraction stage, are open-ended operations, which, because of the high-frequency vibration stirring and heating of the liquid sample required in the nucleic acid extraction process, produce aerosols containing the detected pathogens, which, if dispersed in the test environment, would cause: a) risk of biological leakage; b) detecting cross contamination between samples to form a risk of interfering with detection results; in order to deal with the risks, a nucleic acid detection laboratory must be provided with laminar flow ventilation and biological safety equipment, and a strict biological safety control flow, so that high requirements are put on a testing laboratory, a management specification and operators, the number of laboratories with detection capability is limited, and a bottleneck for limiting the detection capability is formed;
pathogen residues still exist in the waste after detection is finished, so that the leakage prevention treatment is carried out on the waste generated by each step of operation in the detection process according to strict operation specifications;
for the above reasons, the nucleic acid detection laboratory is required to be equipped with expensive sample processing and testing equipment in the whole process, and also to have very high environmental conditions for preventing biological leakage and cross contamination, very strict operation and management specifications and very professional operators, so that the laboratory with the above conditions is few, a bottleneck of detection capability is formed, and the laboratory is not careful when facing a large number of suspected detection cases;
(3) there is an urgent need for a field nucleic acid detection technique that can be performed in the field:
in the epidemic situation development process, the house isolation is an effective isolation means compared with the centralized isolation before the suspected cases are diagnosed, so that the cross infection risk among the suspected cases can be effectively reduced, and meanwhile, the isolated suspected patients can be ensured to have a better living environment, and the recovery of the uninfected suspected patients is facilitated; along with the outbreak of epidemic situation, the case of isolating at home is constantly increased, for effectively managing and controlling isolation crowd to monitor the living area that patient pollutes, need urgently to develop an equipment that can carry out on-the-spot short-term test, have following requirement to this kind of equipment:
(a) the device can be powered by a battery or operated on a vehicle and is portable;
(b) the operation is carried out by one key, the operation is simple and convenient, and the requirement on personnel is low;
(c) can normally operate under the field/field test environment;
(d) the test is performed in a closed and automatic way, so that the biological leakage and the sample cross-contamination risk caused by aerosol and waste can be avoided;
(e) the operation is simple: after the sample is loaded, the whole process from nucleic acid extraction, PCR amplification and fluorescence real-time detection is automatically completed in a closed environment, one-key operation is carried out, the intermediate process does not need human intervention, and the sample does not need to be exposed;
(f) the operation cost is low, the structure is simple, the integration is realized, the whole process is finished by one device, and auxiliary devices are not needed;
(g) the biological leakage/cross contamination risk can be effectively controlled without a high-requirement laboratory environment, and aerosol pollution and waste pollution do not exist;
(h) without the need for highly skilled operators.
Disclosure of Invention
The purpose of this patent is overcome prior art's defect, provide a full-automatic closed nucleic acid of one-stop type and combine kit with real-time fluorescence PCR, can guarantee whole nucleic acid extraction process and real-time fluorescence PCR testing process, all accomplish in totally closed reagent box, in sample extraction process and PCR testing process, there is not any sample tested, or the aerosol that contains the sample can spill over outside the kit, in traditional automatic nucleic acid extraction flow and PCR test application of sample process have been avoided, the aerosol pollution of existence, biological leakage and the sample cross contamination risk that leads to.
This patent full-automatic closed nucleic acid of one-stop draws and real-time fluorescence PCR test joint kit, joint kit structure as an organic whole, including sample loading port A, schizolysis room C1, adsorb and elution room C2, waste liquid room C3, eluant room C4, fluorescence PCR room C5 and diverter valve VT1, the pipeline and the check valve that control fluid flow to of intercommunication each cavity, maintain pressure balance's breather valve, the piston PT that the drive liquid flows, the magnetic bead, wherein:
a lysate is prestored in the lysis chamber C1;
magnetic beads for adsorbing nucleic acid are pre-stored in the adsorption and elution chamber C2, and a piston PT is arranged in the adsorption and elution chamber; the piston PT is in sliding sealing contact with the inner wall of the adsorption and elution chamber C2;
eluent is pre-stored in the eluent chamber C4;
PCR reagents are prestored in the fluorescent PCR chamber C5;
a sample adding channel AH is arranged between the sample adding port A and the cracking chamber C1;
the first liquid inlet, the second liquid outlet, the third liquid inlet and the fourth liquid outlet of the switching valve VT1 are respectively communicated with the cracking chamber C1, the waste liquid chamber C3, the eluent chamber C4 and the fluorescent PCR chamber C5 through a first channel CH1, a second channel CH2, a third channel CH3 and a fourth channel CH 4;
the first working port and the second working port of the switching valve VT1 are both communicated with the adsorption and elution chamber C2; a pipeline of the second working port communicated with the adsorption and elution chamber C2 is provided with a filter F1 for preventing magnetic beads from passing through;
when the valve core of the switching valve VT1 is at different working positions, the first liquid inlet, the second liquid outlet or the third liquid inlet and the fourth liquid outlet are communicated with the first working port and the second working port;
one-way valves for preventing the reverse flow of the fluid are respectively arranged on the sample adding channel AH, the first channel CH1, the second channel CH2, the third channel CH3 and the fourth channel CH 4;
the top ends of the waste liquid chamber C3 and the eluent chamber C4 are respectively provided with vent valves for maintaining the pressure balance of the chambers;
and a communication pipeline is arranged between the eluent chamber C4 and the fluorescent PCR chamber C5, and a vent valve is arranged on the communication pipeline.
In the one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit, the switching valve VT1 further comprises a third working port and a fourth working port, and the third working port and the fourth working port are both communicated with the adsorption and elution chamber C2; a second filter F2 for preventing magnetic beads from passing through is arranged on a pipeline of the fourth working port communicated with the adsorption and elution chamber C2;
when the valve core of the switching valve VT1 is in different working positions, the first liquid inlet and the second liquid outlet are respectively communicated with the first working port and the second working port, or the third liquid inlet and the fourth liquid outlet are respectively communicated with the third working port and the fourth working port.
In the one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit, the piston PT is connected with the piston rod BA, the top end of the adsorption and elution chamber is provided with the mounting port, and the piston rod is arranged in the mounting port in a vertically moving manner.
In the above one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit, the switching valve VT1 is located below the adsorption and elution chamber C2.
The one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit adopts a plurality of connected combined kits for testing a plurality of independent samples in parallel.
In the one-stop full-automatic closed nucleic acid extraction and real-time fluorescent PCR test combined kit, the fluorescent PCR chamber C5 adopts a constant-temperature hot-plate PCR reaction technology.
In the one-stop full-automatic closed nucleic acid extraction and real-time fluorescent PCR test combined kit, the fluorescent PCR chamber C5 is a flat PCR reaction tube.
When the combined kit is unsealed for use, a valve core of a switching valve VT1 in the kit is located at an initial working position in an initial state, at the moment, the first channel CH1 and the second channel CH2 are respectively communicated with the adsorption and elution chamber C2 through a first liquid inlet and a first working port, and the second liquid outlet and the second working port, and the third channel CH3 and the fourth channel CH4 are both blocked from the adsorption and elution chamber C2;
when the nucleic acid extraction flow advances to the elution stage, the valve core of the switching valve VT1 moves to another working position, at this time, the first channel CH1 and the second channel CH2 are both blocked from the adsorption and elution chamber C2, and the third channel CH3 and the fourth channel CH4 are respectively communicated with the adsorption and elution chamber C2 through the third liquid inlet and the first working port, the fourth liquid outlet and the second working port.
When the combined kit is unsealed for use, a valve core of a switching valve VT1 in the kit is located at an initial working position in an initial state, at the moment, the first channel CH1 and the second channel CH2 are respectively communicated with the adsorption and elution chamber C2 through a first liquid inlet and a first working port, and the second liquid outlet and the second working port, and the third channel CH3 and the fourth channel CH4 are both blocked from the adsorption and elution chamber C2;
when the nucleic acid extraction process advances to the elution stage, the valve core of the switching valve VT1 moves to another working position, at this time, the first channel CH1 and the second channel CH2 are both blocked from the adsorption and elution chamber C2, and the third channel CH3 and the fourth channel CH4 are respectively communicated with the adsorption and elution chamber C2 through the third liquid inlet and the third working port, the fourth liquid outlet and the fourth working port.
The utility model discloses a full-automatic closed nucleic acid of one-stop type draws and real-time fluorescence PCR test joint kit, adopt disposable joint kit that can injection moulding, wherein embedded the pipeline of each cavity of intercommunication, switch and prevent the check valve of adverse current, and the lysate has been prestored, adsorb magnetic bead and eluant, nucleic acid extraction operation and PCR test completion back, the discarded object and the final reaction product of all intermediate processes, all seal up and preserve in sealed joint kit, the risk that does not have the biological leakage, the abandonment process flow is convenient, and is simple and low-cost. Compared with the prior art, the one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit has the following advantages:
(1) one-stop operation, a nucleic acid detection group process and one-stop full-automatic completion;
(2) one-key test operation is realized, the operation is simple, and the requirement on personnel is low;
(3) the test device runs in a totally-enclosed manner, the test process is totally enclosed, and the risks of biological leakage and sample cross contamination caused by aerosol are avoided; the waste of the middle procedure of the test is also stored in a used kit, and the risk of environmental pollution and biological leakage is avoided;
(4) the method is rapid, the constant temperature hot plate PCR reaction technology with the patent application number of CN202010105445.1 is adopted, the time for raising and lowering the temperature of the temperature changing platform is saved, and the test speed is more than one time faster than that of the classical PCR method;
(5) the high sensitivity, adopt the flat PCR reaction test tube of patent application No. CN202010106989.X, expand the clear aperture of the fluorescence test by one time, the reaction area expands by 4 times, greatly increase the sensitivity of the fluorescence detection;
(6) the kit is portable, can be used for field test, and can meet the requirements of rapid detection on quarantine fields, which cannot be realized by conventional nucleic acid detection;
(7) the low running cost, an instrument is a nucleic acid detection laboratory, does not need special high-cost nucleic acid detection laboratory, also does not need professional training's operating personnel, greatly reduced test running cost improves detection speed and efficiency.
And a piston rod BA is connected to the piston PT, the piston rod BA is connected with a piston driving mechanism of the testing equipment in the testing process, and the mechanism drives the piston to move up and down to provide power for moving the sample liquid among the chambers in the testing process.
The valve core deflector rod of the switching valve VT1 embedded in the kit is connected with a driving device of the testing equipment, and in the testing process, the testing equipment can select the connection between the first channel CH1 and the second channel CH2 and the adsorption and elution chamber C2, or the connection between the third channel CH3 and the fourth channel CH4 and the adsorption and elution chamber C2, or vice versa, by driving the valve core of the switching valve to move.
The nucleic acid detection equipment (PCR workstation) adopting the combined kit can realize the full-automatic detection of nucleic acid extraction and real-time fluorescence PCR detection, the whole test operation process is completely carried out in the totally-enclosed integrated combined kit, and the possibility of cross contamination of equipment and laboratories polluted by generated aerosol and parallel test samples in the traditional detection process does not exist. The realization of one detection device is the target of a nucleic acid detection laboratory.
Drawings
FIG. 1 is a schematic diagram showing the external structure of a one-stop fully-automatic closed nucleic acid extraction and real-time fluorescence PCR test kit according to the present invention;
FIG. 2 is a schematic view of the working principle of the one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit.
FIG. 3 is a schematic diagram of the operation principle of another one-stop fully-automatic closed nucleic acid extraction and real-time fluorescence PCR test kit.
Detailed Description
In order to make the technical solution of the present patent better understood by those skilled in the art, the following detailed description is given with reference to the accompanying drawings:
referring to fig. 1 and 2, in the best embodiment of the present invention, a one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit is an injection-molded integrated structure, and includes a sample port a, a lysis chamber C1, an adsorption and elution chamber C2, a waste liquid chamber C3, an eluent chamber C4, a fluorescence PCR chamber C5, and a switching valve VT 1.
The sample adding port A, the cracking chamber C1, the adsorption and elution chamber C2, the waste liquid chamber C3, the eluent chamber C4 and the fluorescent PCR chamber C5 are sequentially arranged from left to right; a lysate is prestored in the lysis chamber C1; the adsorption and elution chamber C2 is pre-stored with magnetic beads for adsorbing nucleic acid, a piston PT is arranged in the adsorption and elution chamber C2, a piston rod BA is connected to the piston PT, in the testing process, the piston rod BA is connected with a piston driving mechanism of testing equipment (such as testing equipment of a rapid PCR workstation) and can drive the piston PT to move up and down in the adsorption and elution chamber C2 through the piston rod BA, and power is provided for transferring liquid of corresponding steps between corresponding chambers in the testing process.
Eluent is pre-stored in an eluent chamber C4; PCR reagents are pre-stored in the fluorescent PCR chamber C5; the fluorescent PCR chamber C5 adopts the constant temperature hot plate PCR reaction technology with the patent application number of CN202010105445.1, so that the time for raising and lowering the temperature of the temperature changing platform is saved, and the testing speed is more than one time faster than that of the classical PCR method; the fluorescent PCR chamber C5 can be a flat PCR reaction tube with the patent application number of CN202010106989.X, the clear aperture of the fluorescent test is enlarged by one time, the reaction area is enlarged by 4 times, and the sensitivity of the fluorescent detection is greatly increased.
A sample addition channel AH is arranged between the sample addition port A and the lysis chamber C1, and a one-way valve V6 which only allows liquid to flow into the lysis chamber C1 is arranged on the sample addition channel AH.
The switching valve VT1 is embedded in the kit for controlling the switching of the first channel CH1, the second channel CH2, the third channel CH3 and the fourth channel CH4 connected to the corresponding chambers during the corresponding experimental steps.
The upper part of the switching valve VT1 is provided with a first working port, a second working port, a fourth working port and a third working port from left to right which are communicated with the adsorption and elution chamber C2, a pipeline of the second working port communicated with the adsorption and elution chamber C2 is provided with a filter F1 for preventing magnetic beads from passing through, and a pipeline of the fourth working port communicated with the adsorption and elution chamber C2 is provided with a second filter F2 for preventing magnetic beads from passing through.
The lower part of the switching valve VT1 is provided with a first liquid inlet, a second liquid outlet, a fourth liquid outlet and a third liquid inlet from left to right, wherein the first liquid inlet, the second liquid outlet, the fourth liquid outlet and the third liquid inlet are respectively communicated with a cracking chamber C1, a waste liquid chamber C3, a fluorescent PCR chamber C5 and an eluent chamber C4 through a first channel CH1, a second channel CH2, a fourth channel CH4 and a third channel CH 3.
When the valve core of the switching valve VT1 is in the initial left working position P1, the first liquid inlet and the second liquid outlet are respectively communicated with the first working port and the second working port; when the spool of the switching valve VT1 moves to the right operating position P2, the third and fourth liquid inlets communicate with the third and fourth operating ports.
The first channel CH1 is provided with a one-way valve V1 for preventing the liquid from flowing back from the adsorption and elution chamber C2 to the cracking chamber C1; the second channel CH2 is provided with a filter F1 for preventing magnetic beads from passing through and a one-way valve V2 for preventing the fluid from flowing back to the adsorption and elution chamber C2 from the waste liquid chamber C3, and the filter F1 is positioned at the connecting end of the second channel CH2 and the adsorption and elution chamber C2; the third channel CH3 is provided with a one-way valve V3 which prevents the liquid from flowing back from the adsorption and elution chamber C2 to the eluent chamber C4; the fourth channel CH4 is provided with a filter F2 for preventing magnetic beads from passing through and a one-way valve V4 for preventing liquid from flowing back from the fluorescent PCR chamber C5 to the adsorption and elution chamber C2, and the second filter F2 is positioned at the connecting end of the fourth channel CH4 and the adsorption and elution chamber C2.
The top end of the waste liquid chamber C3 is provided with a one-way vent valve V7 for maintaining the pressure balance in the cavity, and the top end of the eluent chamber C4 is provided with a one-way vent valve V8 for maintaining the pressure balance in the cavity; a communication channel is arranged between the eluent chamber C4 and the fluorescent PCR chamber C5, and a one-way vent valve V5 is arranged on the communication channel for maintaining the pressure balance in the fluorescent PCR chamber.
The one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit has the advantages that when the kit is unsealed (in an initial state), the valve core of the switching valve VT1 is located at the left initial position P1, at the moment, the first channel CH1 and the second channel CH2 are both communicated with the adsorption and elution chamber C2, and the third channel CH3 and the fourth channel CH4 are both cut off from the adsorption and elution chamber C2; before entering an elution step, the equipment shifts the valve core of the switching valve VT1 to another working position P2 on the right side, at this time, the first channel CH1 and the second channel CH2 are both cut off from the adsorption and elution chamber C2, and the third channel CH3 and the fourth channel CH4 are both communicated with the adsorption and elution chamber C2.
The one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit has the working principle as follows:
(1) when the combination kit is unsealed, the initial state is as follows: the valve body of the switching valve VT1 embedded in the combination kit is at position P1, where the first channel CH1 and the second channel CH2 embedded in the combination kit are open (in communication with the adsorption and elution chamber C2), and the third channel CH3 and the fourth channel CH4 are closed (not in communication with the adsorption and elution chamber C2). The piston PT in the adsorption and elution chamber C2 is in the low position. The lysis chamber C1 has a pre-stored lysate. The adsorption and elution chamber C2 is pre-filled with magnetic beads to which nucleic acids are adsorbed. The waste liquid chamber C3 is empty. Eluent is prestored in the eluent chamber C4. PCR reagents were prestored in the fluorescent PCR chamber C5.
(2) Sample addition: the sample is added into a lysis chamber C1 from a sample adding port A through a sample adding channel AH embedded in the combined reagent box, and the sample can be prevented from flowing backwards through a one-way valve V6 on the sample adding channel AH;
(3) after sample adding is completed, the combined kit after sample adding can be inserted into testing equipment such as a rapid PCR workstation and the like to carry out one-stop nucleic acid extraction and real-time fluorescence PCR test. When the combined reagent kit is inserted into the workstation, the positioning device in the workstation automatically positions and clamps the combined reagent kit, and the accurate positioning of each working position is ensured. The piston driving rod of the workstation is pressed down and is connected with a piston rod BA in the adsorption and elution chamber C2. A reversing deflector rod of the switching valve in the workstation clamps a reversing switch of the switching valve VT1 in the joint kit;
(4) after entering the cracking chamber C1, the sample and the lysis solution in the cracking chamber C1 carry out biochemical reaction, the ultrasonic module of the workstation stirs the liquid in the cracking chamber C1, and the heating module heats the cracking chamber C1 so as to accelerate the reaction speed and ensure the uniformity of the reaction, the cracking reaction promotes the cell nucleus in the biological cells in the sample to crack, and the nucleic acid substances encapsulated in the cell nucleus are released into the lysis solution;
(5) after the cracking step is finished, a nucleic acid adsorption step is started, a piston driving rod in the workstation drives a piston PT in an adsorption and elution chamber C2 to lift through a piston rod BA, so that a cracking solution in which nucleic acid in sample cells is dissolved in a cracking chamber C1 is sucked into an adsorption and elution chamber C2 in which adsorption magnetic beads are prestored through a first channel CH1, and in the process, a check valve V2 pre-embedded in a second channel CH2 of the kit can prevent liquid/gas from being sucked into a waste liquid chamber C3. After the lysate enters the adsorption and elution chamber C2, the magnetic beads in the adsorption and elution chamber C2 are fully combined with the nucleic acid in the lysate with the assistance of the magnetic stirring module and the heating module in the workstation, so that the nucleic acid is fully adsorbed on the surfaces of the magnetic beads;
(6) after the nucleic acid is fully adsorbed, the workstation drives a piston PT in an adsorption and elution chamber C2 to press down through a piston driving rod, residual liquid in the adsorption and elution chamber C2 is discharged into a waste liquid chamber C3 through a second channel CH2, at the moment, a check valve V1 pre-embedded in a first channel CH1 can prevent the liquid from flowing back to a cracking chamber C1, and meanwhile, a filter F1 can block magnetic beads adsorbed with the nucleic acid, retain the magnetic beads in the adsorption and elution chamber C2 and wait for elution;
(7) the workstation automatically shifts a reversing switch of a switching valve VT1 pre-buried in the kit, shifts a valve core of the switching valve VT1 from a left starting position P1 to another right working position P2, cuts off a communication channel between an adsorption and elution chamber C2 and a cracking chamber C1 and a communication channel between an adsorption and elution chamber C2 and a waste liquid chamber C3 (namely, cuts off a first channel CH1 and a second channel CH2 from the adsorption and elution chamber C2), and simultaneously communicates a communication channel between the adsorption and elution chamber C2 and an eluent chamber C4 and a communication channel between the adsorption and elution chamber C2 and a fluorescence PCR chamber C5 (namely, a third channel CH3 and a fourth channel CH4 are communicated with the adsorption and elution chamber C2);
(8) the workstation drives the piston PT in the adsorption and elution chamber C2 to lift again, so that the eluent prestored in the eluent chamber C4 is sucked into the adsorption and elution chamber C2 through the third channel CH3, the eluent is mixed with the magnetic beads which are retained in the adsorption and elution chamber C2 and adsorb nucleic acid, and at the moment, the one-way valve V4 pre-embedded in the fourth channel CH4 can prevent liquid from being sucked from the fluorescent PCR chamber C5. After the eluent enters the adsorption and elution chamber C2, the eluent is fully mixed with the magnetic beads under the action of a magnetic stirring module and a heating module of the workstation, and the nucleic acid adsorbed on the magnetic beads is eluted by the eluent and dissolved in the eluent;
(9) the workstation drives the piston PT in the adsorption and elution chamber C2 to press down, the eluent with the nucleic acid dissolved in the adsorption and elution chamber C2 is pressed into the fluorescence PCR chamber C5, the check valve V3 pre-embedded in the third channel CH3 prevents the liquid from flowing back to the eluent chamber C4, and meanwhile, the nucleic acid solution pressed into the fluorescence PCR chamber C5 (hot plate PCR reaction tube) is mixed with the PCR reagent pre-stored in the fluorescence PCR chamber C5 to wait for PCR reaction and real-time fluorescence detection. In this process, the second filter F2 prevents the magnetic beads from entering the fluorescent PCR chamber C5, and the magnetic beads are retained in the closed adsorption and elution chamber C2 and discarded with the kit;
(10) a constant-temperature hot plate type rapid real-time fluorescence PCR module in the workstation starts to work, so that the sample nucleic acid in the fluorescence PCR chamber C5 is mixed with a PCR reagent to carry out PCR reaction, and meanwhile, a fluorescence detection system carries out real-time monitoring on the fluorescence change in the PCR reaction process;
(11) after the real-time fluorescence PCR detection is finished, a piston driving rod of the workstation is separated from the connection with a piston rod penetrating through the adsorption and elution chamber C2, a reversing deflector rod of the workstation is separated from the joint kit, the positioning and clamping device releases the joint kit, the joint kit can be drawn out of testing equipment such as a PCR workstation, and all reaction process products and final products are sealed in the used joint kit and discarded together with the joint kit.
See fig. 3 for another one-stop fully automatic closed nucleic acid extraction and real-time fluorescence PCR assay kit. In fig. 3, the upper part of the switching valve VT1 is provided with a first working port and a second working port from left to right, and the first working port and the second working port are both communicated with the adsorption and elution chamber C2; a filter F1 for preventing magnetic beads from passing through is arranged on a pipeline of the second working port communicated with the adsorption and elution chamber C2. When the valve core of the switching valve VT1 is in the initial left working position P1, the first liquid inlet and the second liquid outlet are respectively communicated with the first working port and the second working port; when the spool of the switching valve VT1 moves to the right working position P2, the third liquid inlet and the fourth liquid outlet communicate with the first working port and the second working port.
The main differences between fig. 3 and fig. 2 are: the switching valve VT1 in fig. 3 has only two working ports, namely the first working port and the second working port, while the switching valve VT1 in fig. 2 has four working ports, namely the first working port, the second working port, the third working port and the fourth working port; in fig. 3, only the pipeline connecting the second working port and the adsorption and elution chamber C2 is provided with a filter F1 for preventing magnetic beads from passing through, in fig. 2, the pipeline connecting the second working port and the adsorption and elution chamber C2 is provided with a filter F1 for preventing magnetic beads from passing through, and the pipeline connecting the fourth working port and the adsorption and elution chamber C2 is provided with a second filter F2 for preventing magnetic beads from passing through.
Referring to fig. 1, the one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit can adopt a quadruple kit structure, and can analyze four groups of samples at one time, thereby greatly improving the working efficiency.
The one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR combined kit has wide application markets, such as:
(1) quarantine of crisis epidemic situation;
(2) detecting conventional pathogens;
(3) quarantine by veterinarians;
(4) customs site quarantine;
(5) species detection for endangered animal and plant protection;
(6) performing field scientific investigation;
(7) detecting environmental protection;
(8) forensic identification application.
To sum up, the one-stop full-automatic closed nucleic acid of this patent draws and real-time fluorescence PCR test joint kit, can guarantee whole nucleic acid extraction process and real-time fluorescence PCR testing process, all accomplish in totally closed joint kit, in the sample extraction in-process and PCR test process, there is not any sample to be tested, or the aerosol that contains the sample can spill over outside joint kit, avoided traditional automatic nucleic acid extraction flow in with PCR test application of sample in-process, the aerosol pollution of existence, the biological leakage that leads to and sample cross contamination risk.
It should be understood by those skilled in the art that the above embodiments are only for illustrating the patent and are not used as limitations of the patent, and that changes and modifications to the above described embodiments are within the scope of the claims of the patent as long as they are within the spirit and scope of the patent.
Claims (7)
1. A station-type full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit is characterized in that the combined kit is of an integrated structure and comprises a sample adding port (A), a cracking chamber (C1), an adsorption and elution chamber (C2), a waste liquid chamber (C3), an eluent chamber (C4), a fluorescence PCR chamber (C5) and a switching valve (VT1), pipelines for communicating all chambers and one-way valves for controlling the flow of fluid, vent valves for maintaining pressure balance, Pistons (PT) for driving liquid to flow, and magnetic beads, wherein:
a lysate is prestored in the lysis chamber (C1);
magnetic beads for adsorbing nucleic acid are pre-stored in the adsorption and elution chamber (C2), and a Piston (PT) is arranged in the adsorption and elution chamber; the Piston (PT) is in sliding sealing contact with the inner wall of the adsorption and elution chamber (C2);
an eluent is pre-stored in the eluent chamber (C4);
the fluorescent PCR chamber (C5) is pre-stored with PCR reagents;
a sample adding channel (AH) is arranged between the sample adding port (A) and the cracking chamber (C1);
the first liquid inlet, the second liquid outlet, the third liquid inlet and the fourth liquid outlet of the switching valve (VT1) are respectively communicated with the cracking chamber (C1), the waste liquid chamber (C3), the eluent chamber (C4) and the fluorescent PCR chamber (C5) through a first channel (CH1), a second channel (CH2), a third channel (CH3) and a fourth channel (CH 4);
the first working port and the second working port of the switching valve (VT1) are both communicated with an adsorption and elution chamber (C2); a filter (F1) for preventing magnetic beads from passing through is arranged on a pipeline of the second working port, which is communicated with the adsorption and elution chamber (C2);
when the valve core of the switching valve (VT1) is at different working positions, the first liquid inlet, the second liquid outlet or the third liquid inlet and the fourth liquid outlet are communicated with the first working port and the second working port;
the sample adding channel (AH), the first channel (CH1), the second channel (CH2), the third channel (CH3) and the fourth channel (CH4) are respectively provided with a one-way valve for preventing the reverse flow of the fluid;
the top ends of the waste liquid chamber (C3) and the eluent chamber (C4) are respectively provided with a vent valve for maintaining the pressure balance of the chambers;
and a communication pipeline is arranged between the eluent chamber (C4) and the fluorescent PCR chamber (C5), and a vent valve is arranged on the communication pipeline.
2. The one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit according to claim 1, wherein the switching valve (VT1) further comprises a third working port and a fourth working port, and the third working port and the fourth working port are both communicated with the adsorption and elution chamber (C2); a second filter (F2) for preventing magnetic beads from passing through is arranged on a pipeline of the fourth working port, which is communicated with the adsorption and elution chamber (C2);
when the valve core of the switching valve (VT1) is at different working positions, the first liquid inlet and the second liquid outlet are respectively communicated with the first working port and the second working port, or the third liquid inlet and the fourth liquid outlet are respectively communicated with the third working port and the fourth working port.
3. The one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit according to claim 1 or 2, wherein the Piston (PT) is connected with a piston rod (BA), the top end of the adsorption and elution chamber is provided with a mounting port, and the piston rod is arranged in the mounting port in a vertically moving manner.
4. The one-stop fully automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit according to claim 1 or 2, wherein the switching valve (VT1) is located below the adsorption and elution chamber (C2).
5. The one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit according to claim 1 or 2, wherein a plurality of connected combined kits are adopted for testing a plurality of independent samples in parallel.
6. The one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit according to claim 1 or 2, wherein the fluorescence PCR chamber (C5) adopts a constant temperature hot plate PCR reaction technology.
7. The one-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit according to claim 1 or 2, wherein the fluorescence PCR chamber (C5) is a flat PCR reaction tube.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202020441774.9U CN212293607U (en) | 2020-03-30 | 2020-03-30 | One-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202020441774.9U CN212293607U (en) | 2020-03-30 | 2020-03-30 | One-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN212293607U true CN212293607U (en) | 2021-01-05 |
Family
ID=73961250
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202020441774.9U Active CN212293607U (en) | 2020-03-30 | 2020-03-30 | One-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN212293607U (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112831399A (en) * | 2021-02-24 | 2021-05-25 | 梅里医疗科技(洋浦)有限责任公司 | Automatic detection reagent bottle group, kit, reagent bin and detection method for intelligent hospital |
| CN113913289A (en) * | 2021-12-07 | 2022-01-11 | 北京芯源视界生物科技有限公司 | Detection kit and detection method for pathogenic nucleic acid under airtight condition |
-
2020
- 2020-03-30 CN CN202020441774.9U patent/CN212293607U/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112831399A (en) * | 2021-02-24 | 2021-05-25 | 梅里医疗科技(洋浦)有限责任公司 | Automatic detection reagent bottle group, kit, reagent bin and detection method for intelligent hospital |
| CN112831399B (en) * | 2021-02-24 | 2024-01-05 | 通用技术集团健康管理科技有限公司 | Nucleic acid detection kit, kit bin and detection method for full-automatic chemiluminescence immunoassay analyzer in intelligent hospital |
| CN113913289A (en) * | 2021-12-07 | 2022-01-11 | 北京芯源视界生物科技有限公司 | Detection kit and detection method for pathogenic nucleic acid under airtight condition |
| CN113913289B (en) * | 2021-12-07 | 2022-03-22 | 北京芯源视界生物科技有限公司 | Detection kit and detection method for pathogenic nucleic acid under airtight condition |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111500675A (en) | One-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit | |
| JP6838127B2 (en) | Test cartridge with integrated transfer module | |
| JP5049274B2 (en) | Cartridge for automated medical diagnosis | |
| CN112226361B (en) | Nucleic acid detection card box based on magnetic bead transfer and valve control liquid transfer and detection method | |
| CN110075935B (en) | Multi-index detection microfluidic card box and application method | |
| CN107199061B (en) | Application method of multi-task full-automatic biochemical detection chip | |
| CN207933420U (en) | A kind of micro-fluidic chip of genetic test | |
| US10906043B2 (en) | Microfluidic based integrated sample analysis system | |
| CN212293607U (en) | One-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit | |
| US20110244466A1 (en) | Nucleic acid testing device and method | |
| CN111909835A (en) | Closed micro-fluidic nucleic acid detection card box | |
| KR20090084823A (en) | Cartridge system | |
| EP3304031A1 (en) | A component of a device, a device, and a method for purifying and testing biomolecules from biological samples | |
| KR20170024827A (en) | The Quantitative PCR Cartridge with Microchannel-Film Reactor, Nucleic Acid Extraction Module and qPCR Reagents Module, and The Rapid qPCR System Using the Same | |
| CN108160125A (en) | Biochemistry detection micro-fluidic chip and biochemistry detection micro-fluidic chip system and their application | |
| WO2022174471A1 (en) | Fully-integrated pathogen nucleic acid test microfluidic chip | |
| Li et al. | A self-contained and fully integrated fluidic cassette system for multiplex nucleic acid detection of bacteriuria | |
| CN215757272U (en) | Nucleic acid amplifier | |
| CN111534575A (en) | Microfluidic nucleic acid detection card box and kit | |
| KR20210065460A (en) | Micro-chip for analyzing fluids and method for amplification of genes using the same | |
| US8808643B1 (en) | Fluidics platform and method for sample preparation and analysis | |
| CN211035866U (en) | Plunger type nucleic acid detection integrated card box | |
| CN207533259U (en) | ELISA detects micro-fluidic chip and ELISA detection micro-fluidic chip systems | |
| TWI390201B (en) | Fluid manipulation gene array analysis wafers | |
| EP4000729A1 (en) | Method for determining the presence of a pathogen |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220325 Address after: 519000 floor 2, phase II plant, No. 2, Chuangxin Fourth Road, high tech Zone, Zhuhai, Guangdong Patentee after: ZHUHAI BLACK HORSE BIOTECHNOLOGY Co.,Ltd. Patentee after: Zhuhai Heima Medical Instrument Co., Ltd Address before: 519080 2nd floor, phase II plant, No.2, Chuangxin 4th Road, high tech Zone, Zhuhai City, Guangdong Province Patentee before: ZHUHAI BLACK HORSE BIOTECHNOLOGY Co.,Ltd. |
|
| TR01 | Transfer of patent right |