CN108660074B - Integrated solution for nucleic acid extraction PCR amplification detection - Google Patents
Integrated solution for nucleic acid extraction PCR amplification detection Download PDFInfo
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- CN108660074B CN108660074B CN201810525844.6A CN201810525844A CN108660074B CN 108660074 B CN108660074 B CN 108660074B CN 201810525844 A CN201810525844 A CN 201810525844A CN 108660074 B CN108660074 B CN 108660074B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention relates to a kit for realizing integration of nucleic acid extraction and PCR amplification detection and a test method thereof, wherein the kit comprises a nucleic acid extraction unit and a PCR amplification detection unit, and can be matched with a matched instrument to finish a full-automatic process of nucleic acid extraction and PCR amplification detection without plate transferring operation or manual intervention in the process.
Description
Technical Field
The invention relates to an experimental method for molecular biological analysis, in particular to an integrated solution for combining nucleic acid extraction and PCR amplification detection.
Background
Nucleic acid extraction and PCR amplification detection are important experimental means for research in molecular biology, early experiments are completed by pure hands, the time consumption is long, and experimental results are easily influenced by individual operation differences of experimenters. With the development of self-industrial automation, in the field of experimental science, traditional manual experiments are replaced by automation equipment more and more, the repeatability and the consistency of the automation equipment reduce errors of the traditional manual experiments, and the reliability and the experimental efficiency of the experiments are improved.
In terms of experimental procedures, a general nucleic acid analysis process basically comprises several parts of sample pretreatment, nucleic acid extraction and PCR amplification detection. The sample pretreatment is performed by different treatment modes such as centrifugation, filtration, grinding and the like according to different samples to be detected, and because the treatment modes are various, universal sample pretreatment equipment is difficult to design, so that the current situation is basically completed by means of semi-manual laboratory equipment such as a centrifuge, a temperature controller and the like in the link. The nucleic acid extraction process needs to complete the cracking, cleaning and purification of nucleic acid, common methods include a boiling method, a centrifugal column method, a magnetic bead method and the like, and the magnetic bead method is convenient for automatic integration, so the most adopted in the existing automatic nucleic acid extraction instrument is a magnetic bead method. Although the sample after nucleic acid extraction is eluted and concentrated, the concentration of the sample is very low, most samples cannot be directly used for hybridization for subsequent analysis, and the subsequent links are required to be carried out after PCR amplification, detection and quantification. The quantitative PCR instrument can complete PCR amplification and detection. It can be seen that the above three links are basically indispensable.
The demand is the development direction, and over the past decade, the automatic devices such as a sample preparation instrument, a nucleic acid extraction instrument and a quantitative PCR instrument which realize the three links are separated, and are continuously new and occupy the market. However, almost all the intermediate transfer operation steps of loading the prepared sample into a nucleic acid extractor and loading the sample after nuclear extraction into a quantitative PCR (polymerase chain reaction) extractor for amplification need to be completed manually by experimenters. How to get through this last link becomes a great burden for research. In recent years, manufacturers replace hands by means of manipulators, automatic arms and the like to complete sample transfer and transfer, but the system is huge and complex due to the fact that the mechanical arms are added to a plurality of discrete devices, manufacturing cost is extremely high, batch experiment operation is generally needed, and the speed of the whole experiment process is low.
In the nucleic acid extraction process, a magnetic frame is used in the traditional manual magnetic bead method, the required reagent amount is large, the extraction process is complex, and time and labor are consumed. Automated equipment uses roughly two methods: one method is to automate the traditional manual method, arrange a magnetic unit on the side wall or the bottom of the reaction cup, gradually add lysis solution, cleaning solution and eluent required by the nucleic acid extraction process into the reaction cup through an automatic liquid transfer device, suck, discharge and uniformly mix, and then remove waste liquid. Although this method saves labor and realizes automation, when the waste liquid is removed, a certain amount of waste liquid always remains at the bottom of the reaction cup, resulting in low purity of the sample after nucleic acid extraction. The other method is a method adopting a magnetic rod and a magnetic sleeve, wherein in the steps of cracking, cleaning and eluting for nucleic acid extraction, the magnetic rod and the magnetic sleeve wrapped outside the magnetic rod are used for moving and magnetically adsorbing at different reaction positions so as to achieve the purpose of transferring and extracting the nucleic acid. The method also realizes the automation of nucleic acid extraction, but the magnetic beads and liquid adsorbed on the magnetic sleeve are easy to drop into instruments and reaction cups in the transferring process, which can cause the pollution of the system. In addition, the method has large volume requirement of the eluent, is not beneficial to the concentration of the nuclear extraction sample, and is not easy to realize the requirement of high concentration of the nuclear extraction sample.
Conventionally, the amplification time of PCR is calculated in hours, which is the most offensive issue in the analysis process of molecular diagnosis, and the amplification time of PCR must be reduced to speed up the analysis process and speed up the analysis. The factor influencing the PCR amplification time is the temperature rise and fall rate of the PCR reaction system. In this regard, it has been instrumentally addressed to use conductive modules of high conductivity, such as silver; some have solved from the reagent, researched fast reaction reagent, reduced reaction system, etc.; some people solve the problem from consumable materials, and glass capillary tubes and the like are adopted.
How to solve the above three steps of real automation, reducing the cost of the instrument, compressing the time of the whole process, avoiding the pollution in the nucleic acid extraction process, improving the purity of the nucleic acid extraction, and accelerating the PCR amplification process is the main direction of our research and improvement.
Disclosure of Invention
Aiming at the problems, the invention provides a solution for realizing integration of nucleic acid extraction and PCR amplification detection, which comprises three experimental steps of molecular diagnosis and analysis, such as sample pretreatment, nucleic acid extraction, PCR amplification detection and the like.
In order to achieve the purpose, the invention provides the following technical scheme:
a kit for realizing integration of nucleic acid extraction and PCR amplification detection comprises a nucleic acid extraction unit and a PCR amplification detection unit.
Further, the kit can also comprise a sample pretreatment unit, a liquid transfer unit and an anti-pollution unit.
Wherein the nucleic acid extraction unit adopts a nucleic acid extraction mode of a magnetic bead method and at least comprises a lysis site, a cleaning site and an elution site; the PCR amplification detection unit at least comprises one PCR bit.
The invention can also be provided with a tube cover position, and the tube cover is tightly combined with the PCR position under the action of a tube cover folding unit of a matched instrument, thereby sealing the PCR system.
The liquid transfer unit comprises at least one pipetting head and one pipetting head storage location. The sample pretreatment unit comprises a sample position and also comprises a sample loading position, and when the kit comprises the sample loading position, the sample loading position is communicated with the sample position. The anti-pollution unit comprises a filter column inside the pipetting head, a filter membrane at the top end of each nucleic acid extraction position and paraffin oil of a PCR position internal closed system. And filter liquid membranes are sealed at the tops of the cracking position, the cleaning position and the elution position.
The invention also relates to a method for detecting by the kit for realizing integration of nucleic acid extraction and PCR amplification detection, which comprises the following steps:
(1) nucleic acid extraction step:
a. absorbing a sample and transferring the sample to a lysis site, uniformly mixing sample lysate, then carrying out cell lysis, and specifically combining free nucleic acid molecules after cell lysis with silicon hydroxyl on the surface of the magnetic beads;
b. after reacting for a certain time, carrying out imbibition operation by using the pipetting head, enabling a magnetic unit of a matched instrument to be close to the outer wall of the pipetting head and providing a corresponding magnetic field when carrying out imbibition operation, enabling magnetic beads coated with nucleic acid molecules in the pipetting head to be close to the inner wall of the pipetting head near the magnetic unit under the action of the magnetic field, completely absorbing lysate in a lysis position into the pipetting head by the imbibition operation, enabling the magnetic beads in the lysate in the pipetting head to be adsorbed to the vicinity of the inner wall of the pipetting head on one side of the magnetic unit after a certain magnetic adsorption time, inserting the pipetting head into the lysis position at the moment, carrying out drainage operation, and discharging the waste liquid after lysis into the lysis position;
c. inserting the liquid transfer head into the cleaning liquid at the cleaning position, enabling the magnetic unit to be far away from the liquid transfer head or removing the magnetic field, and carrying out uniform mixing operation, wherein impurities adsorbed on the surfaces of the magnetic beads after cracking are gradually dissociated in the cleaning liquid in the uniform mixing cleaning process;
d. moving the liquid transfer head with the magnetic bead system on the cleaned inner wall to an elution position, keeping the magnetic unit away from the liquid transfer head or removing the magnetic field, uniformly mixing the magnetic beads and the eluent, and performing the separation process of nucleic acid molecules and the magnetic beads;
e. the elution operation is finished, namely the nucleic acid extraction operation is finished, and a concentrated sample with higher purity is obtained;
(2) PCR amplification detection: the kit carries out PCR reaction, and a certain amount of eluted samples are absorbed from the elution position and transferred to the PCR position; and starting the temperature control device to provide the temperature of each stage required by the PCR reaction, and detecting the PCR reaction, wherein the detection device sequentially passes through the PCR bit of each kit to detect the real-time fluorescent signal in the detection stage.
Further, in the step (2), after the pipetting head leaves the PCR position, the tube cover folding unit of the matched instrument folds the tube cover position, and after folding, the top end bulge of the tube cover is sealed with the PCR position, so that the sealing of the inner body system of the PCR position is ensured to be non-volatile.
Specifically, the invention can complete the integrated solution of the steps, which comprises a liquid transfer unit, a sample pretreatment unit, a nucleic acid extraction unit, a PCR amplification detection unit and an anti-pollution unit. In order to simplify the system and reduce the cost, the units can be integrated on a specific kit, but the liquid transfer unit, the sample pretreatment unit and the anti-pollution unit are not necessary, and can be arranged outside the kit, and the system can also be provided with no units, but the inclusion of the units can obtain better user experience and more stable and reliable experimental results.
The kit is a consumable, one kit is required for each test, reagents and other related functional consumables can be pre-filled in the kit, and the top of the kit can be sealed to form a closed system.
The kit can work together with a matched instrument, and the system completes the processes of sample pretreatment, nucleic acid extraction and PCR amplification detection. The matched instrument at least comprises a piston unit and can be matched with a liquid transfer unit to finish the transfer, liquid suction and discharge and uniform mixing operations of reaction liquid; the instrument may include a pressure control unit that cooperates with the sample pre-processing unit to perform a whole blood separation operation. The pressure control unit can be integrated with the piston unit in implementation, not only can be matched with the liquid transfer unit to carry out liquid transfer distribution, but also can be matched with the sample pretreatment unit to complete whole blood separation; the instrument is also provided with a magnetic unit which can be close to the liquid transfer unit and provides a magnetic field, and magnetic beads can be gathered when the magnetic unit is matched with the liquid absorption operation of reaction liquid; the instrument also needs to have an incubation module for nucleic acid extraction that can provide a lysis temperature and an elution temperature, respectively, and an incubation module for PCR reactions. The incubation module for PCR reaction can provide PCR temperature control for rapid temperature rise and fall and hot cover temperature control; the instrument also comprises a PCR detection unit, wherein the PCR detection unit at least comprises a detection channel with fluorescence of wavelength, the detection channel at least comprises an excitation light source and a detection device, and the detection channel can also comprise an optical filter, a lens and the like in implementation. The detection unit can move linearly to perform rapid scanning detection on a plurality of whole columns of reagent boxes. In the implementation, the instrument can also be provided with a bar code scanning unit, a liquid crystal display unit, a printer unit and the like, can finish automatic scanning identification of bar codes, liquid crystal display and result display of experiment processes, experiment result printing and the like, and can improve user experience by the aid of the units, but the units are not necessary.
The core of the invention is an integrated kit as a consumable, which comprises a nucleic acid extraction unit and a PCR amplification detection unit, and also comprises a sample pretreatment unit, a liquid transfer unit and an anti-pollution unit.
When the integrated kit comprises the liquid transfer unit, the liquid transfer unit at least comprises one pipetting head and one pipetting head storage position, and a plurality of pipetting heads and a plurality of pipetting head storage positions can be further included according to the requirements of the reagent system. The liquid transfer unit can also be arranged in an instrument matched with the kit, the matched instrument comprises a liquid transfer head storage position, and the liquid transfer head can be independently added by a user before an experiment. The pipetting head can be connected with a piston unit of a matched instrument for pipetting. During the connection, move liquid head top and the bottom tight fit of piston unit and guarantee the gas tightness, when the piston unit motion, move liquid head bottom point-end and get into and shift liquid, can move liquid operation and mixing operation. The liquid transferring head can also comprise a filter column inside, and the filter column can prevent liquid and volatile gas from entering the piston unit, so that pollution is avoided.
The sample preprocessing unit can comprise a sample position, and processed samples are added by experimenters before experiments. The kit can also comprise a sample loading position, when the kit comprises the sample loading position, the sample loading position is communicated with the sample position, a medium film capable of separating serum and plasma is arranged between the communicating channels, the size of the medium film can filter macromolecules such as red blood cells, when the kit is matched with an instrument for use, the pressure control unit of the instrument is tightly combined with the sample loading position or the sample position to ensure air tightness, whole blood at the sample loading position gradually permeates through the medium film under the action of positive pressure or negative pressure, and filtered serum enters the sample position through the communicating channels and can be used for subsequent nucleic acid extraction.
The nucleic acid extraction unit adopts a nucleic acid extraction mode of a magnetic bead method and at least comprises a cracking position, a cleaning position and an elution position. The nucleic acid extraction unit is matched with a piston unit and a magnetic unit of a matched instrument, so that the nucleic acid extraction process can be completed. The number of the cleaning positions can be one or more according to the requirements of a reagent system. The temperature control position can be set at the position where the matched instrument is connected with the cracking position, the cleaning position and the elution position, and the temperature required by cracking, cleaning and elution is provided according to the requirement of a reagent system.
The further specific operating scheme of the invention is as follows:
when the kit is used for extracting nucleic acid, a piston unit of a matched instrument is connected with a liquid transferring head to absorb a certain amount of pretreated sample from a sample position and transfer the sample to a lysis position, the piston unit moves up and down to suck, discharge and uniformly mix sample lysate, the lysis position system is used for cell lysis at a lysis temperature provided by the matched instrument, and free nucleic acid molecules after cell lysis are specifically combined with silicon hydroxyl on the surface of magnetic beads. In the process of combining the cracking magnetic beads, the liquid-moving head can be matched with the piston to continuously suck and discharge, so that the reaction process is accelerated, and the cracking time is shortened. After reacting the uniform time, the piston connection moves the liquid head and carries out the imbibition operation, the magnetic unit of supporting instrument is close to moving liquid head outer wall and providing corresponding magnetic field when carrying out the imbibition operation, move the inside magnetic bead of coating nucleic acid molecule of liquid head and be close to near the first inner wall that moves of magnetic unit under the effect in magnetic field, the lysate in the position of will cracking is absorbed completely to moving liquid in the head to the imbibition operation, through certain magnetism adsorption time, move the magnetic bead in the inside lysate of liquid head and all adsorbed near the first inner wall that moves liquid of magnetic unit one side, move the liquid head and insert the position of cracking this moment, the liquid discharge operation is carried out in the slow decline of piston unit, waste liquid after the cracking is arranged to the position of cracking. At the moment, the piston moves to a cleaning position under the control of a two-dimensional or three-dimensional motion system connected with the piston, the liquid transfer head connected with the piston is inserted into cleaning liquid of the cleaning position, the magnetic unit is far away from the liquid transfer head or the magnetic field is removed, the piston moves up and down to carry out uniform mixing operation, and impurities adsorbed on the surfaces of the magnetic beads are gradually dissociated in the cleaning liquid in the uniform mixing and cleaning process. After the cleaning operation is completed, the cleaning mixed liquid is completely absorbed into the liquid moving head to perform the magnetic adsorption operation according to the magnetic adsorption process, and the waste liquid is discharged to the cleaning position after the magnetic adsorption. If the reagent system needs, the cleaning position can be multiple, and the cleaning operation is repeatedly executed. The inner wall after washing has the liquid transfer head of magnetic bead system, moves to the elution position under the drive of piston, and magnetic unit is far away from the liquid transfer head or removes the magnetic field, and the piston up-and-down motion is repeatedly absorbed and discharged and is mixed magnetic bead and eluant, and the elution system carries out the process of breaking away from of nucleic acid molecule and magnetic bead under the elution temperature that supporting instrument provided. After a certain time, the elution operation is finished, namely the nucleic acid extraction operation is finished, and a concentrated sample with higher purity is obtained. At the moment, the elution position also comprises a magnetic bead after reaction elution, so that the magnetic bead can be prevented from influencing the subsequent PCR amplification detection, the magnetic bead can be subjected to magnetic adsorption again, and the liquid transfer head with the magnetic bead adsorbed on the inner wall is transferred to the liquid transfer head position for recycling.
The PCR amplification detection unit at least comprises a PCR position and also comprises a tube cover position, and the tube cover can be tightly combined with the PCR position under the action of a tube cover folding unit of a matched instrument to seal a PCR system. The PCR can be in various shapes such as conical shape, cylindrical shape and the like, and can also be in a flat cuboid structure in implementation, the structure can increase the contact area with a PCR temperature control module of a matched instrument, improve the lifting speed of a PCR system and further compress the PCR amplification time. The tube cover position can be turned over under the action of a matched instrument, and the top end bulge of the turned tube cover position can be sealed with the PCR position, so that the sealing and non-volatilization of an internal system of the PCR position are ensured.
When the kit is used for PCR reaction, a piston unit of a matched instrument is connected with a liquid transferring head to absorb a certain amount of eluted samples from an elution position, the eluted samples pass through a filter liquid film at the top end of the kit and a paraffin oil sealing layer of a PCR position and are transferred to the PCR position, and the piston unit moves up and down to repeatedly suck, discharge and mix a PCR system uniformly. After the pipetting head leaves the PCR position, the tube cover turning-over unit of the matched instrument can turn over the tube cover position, and the top end bulge of the turned-over tube cover is sealed with the PCR position, so that the sealing and non-volatilization of the inner system of the PCR position are ensured. After the preparation of the PCR system is completed, the PCR temperature control device of the matched instrument is started to provide the temperature of each stage required by the PCR reaction, the PCR reaction to be detected is carried out, and in the detection stage, the detection device of the matched instrument sequentially passes through the PCR position of each kit to detect the real-time fluorescent signal. The detection device of the matched instrument is generally provided with a light source and a detection device, a lens can be added when the optical collection efficiency is required to be improved, the detection device can also comprise an optical filter and the like for an instrument with a plurality of optical detection channels, the devices can be integrated on a moving device, and the detection of the PCR position of each kit can be completed through the movement of the moving device.
The anti-pollution unit can comprise a filter column inside the pipetting head, a filter membrane at the top end of each nucleic acid extraction position and paraffin oil of a PCR in-situ closed system. The contamination prevention unit is not an essential component of the present invention, but with the various arrangements of the contamination prevention unit, the possibility of contamination is reduced.
The liquid transferring head can be preset at the liquid transferring head position, a filter medium film can be preset near the connection position of the sample separating position and the sample position, lysis solution, cleaning solution and eluent for nucleic acid extraction can be preset at the lysis position, the cleaning position and the elution position, a system for carrying out PCR reaction or quantitative PCR reaction can be preset at the PCR position, and a medium closed system such as paraffin oil can be pre-sealed on the system.
The schizolysis position, wash the position, elution position top can also seal the filter membrane, this membrane can be rubber, soft material that silica gel etc. do not react with the system, the opening of shape such as cross can have at its membrane surface center, move the liquid head and can get into corresponding position through this opening smoothly and move the liquid operation, when moving the liquid head and break away from the filter membrane, move the liquid that liquid head bottom outer wall carried and can hang by this filter membrane and drop and remain in corresponding position, can not carry out the kit and fall in instrument or kit, the possibility of pollution has been reduced.
The top of the sample separation position, the sample position, the liquid transfer head position, the cracking position, the cleaning position, the elution position and the PCR position can be sealed with a protective film, and the film can be made of an aluminum-plastic film and other materials which are easy to puncture or tear and do not react with the system. The surface of the protective film can also be marked with bar codes, and the bar codes can carry relevant information of reagents, such as detection items, reaction processes and the like. The protective film can keep the sealing of the solid and liquid preset at each position and can also ensure the cleanness of each position.
In practice, the kit may be pre-loaded and pre-filled with the pipetting head and the related reaction solution as described above, followed by sealing the filtrate membrane and finally sealing the protective membrane. In the experimental operation process, some instruments that set up need the user to use external scanning rifle to sweep the sign indicating number and tear off the protection film and load the kit to supporting instrument in and operate, and also some instrument users that set up only need load the kit in supporting instrument, by supporting instrument automatic execution scan bar code and puncture the operation of protection film.
Due to the adoption of the technical scheme, the invention has the following advantages: 1. the real automation of sample pretreatment, nucleic acid extraction and PCR amplification detection is realized; 2. the structure is simple, the system integration level is high, the material consumption cost is low, the experimental process time is short, and the method is suitable for rapid detection with low flux; 3. the possibility of pollution is reduced by various means, and the validity of an experimental result is ensured.
Drawings
FIG. 1 is a schematic diagram of an integrated solution of the present invention;
FIG. 2 is a schematic structural view of a liquid transfer unit of the present invention;
FIG. 3 is a schematic view of the structure of the contamination-preventive unit of the present invention;
FIG. 4 is a schematic diagram of a sample pre-processing unit according to the present invention;
FIG. 5 is a schematic diagram showing the structure of a nucleic acid extraction unit according to the present invention;
FIG. 6 is a schematic diagram of the construction of the PCR amplification detecting unit system according to the present invention;
FIG. 7 is a schematic diagram of the ready construction of the PCR amplification detecting unit system of the present invention;
FIG. 8 is a schematic view showing the structure of the PCR amplification detecting unit of the present invention at the time of detection.
Detailed Description
The invention is further illustrated with reference to the following figures and specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Further, it should be understood that various changes and modifications can be made by those skilled in the art after reading the teaching of the present invention, and those equivalents also fall within the scope of the appended claims of the present application.
As shown in fig. 1 and 3, the embodiment of the present invention is composed of a sample pretreatment unit 1, a liquid transfer unit 2, a nucleic acid extraction unit 3, a PCR amplification detection unit 4, and an anti-contamination unit. The above units are integrated on one reagent kit, which is the integrated detection reagent kit of this embodiment.
The integrated kit of the embodiment is a consumable, and each kit can be matched with an instrument to perform a complete sample pretreatment, nucleic acid extraction and PCR amplification detection process. The apparatus in the embodiment has a piston unit 61 (fig. 2) which can be connected to the liquid transfer head 221 of the liquid transfer unit 2 in an airtight manner to perform liquid transfer, pipetting and mixing operations. The associated instrument also has a magnet unit 62, which is comprised of a magnetic material, in the embodiment a permanent magnet. The permanent magnet can provide a magnetic field, and magnetic bead adsorption and transfer can be performed in cooperation with the action of the liquid transfer head 221 in the nucleic acid extraction process. The complete apparatus further has temperature control units 631, 632 (FIG. 5) and 633 (FIG. 6 and FIG. 7), which can provide the required temperatures for the lysis, elution and PCR reactions of nucleic acid extraction, respectively. In the embodiment, the matching instrument is provided with a tube cover 42 folding device, so that the tube cover 42 can be folded after a sample is filled, and the top end protrusion of the tube cover 42 is tightly connected with the top end of the PCR position 41. Meanwhile, the turnover device also provides the temperature of the hot cover required by the PCR reaction. The complete instrument also has a detection unit 64 (fig. 6-8) for real-time excitation and detection of the fluorescence signal of the PCR bit 41. In one embodiment, the detection unit 64 has 5 optical paths, each with a respective excitation light source 641 and detection device 642, and different optical paths may also have lenses and color filters. The excitation light source 641 and the detection device 642 are integrated on a moving device, which can be driven by a motor 644 to perform a rapid scanning motion along a linear guide 643, so as to perform optical detection on the PCR sites 41 of the respective reagent boxes in turn.
As shown in FIGS. 2 to 8, the integrated kit according to the embodiment of the present invention includes a sample loading position 11, a sample position 12, pipette tip positions 211 and 212, pipette tips 221 and 222 preset in the pipette tip positions 211 and 212, a lysis position 31, a washing position 32, an elution position 33, a PCR position 41, and a cap 42. The pipetting heads 221 and 222 are provided with a filter column 51 to prevent liquid or volatile gas from entering the piston unit 61 of the associated instrument, thereby preventing contamination. Wherein, a sample loading position 11 is preset with a medium film 111 which can filter macromolecules such as red blood cells, a lysis position 31 is filled with lysis solution in advance, a cleaning position 32 is filled with cleaning solution in advance, an elution position 33 is filled with eluent in advance, and a PCR reaction system and paraffin oil 52 are filled in advance in a PCR position 41. After the solid and liquid are preset, the filtrate membrane 53 is pressed in the lysis position 31, the cleaning position 32 and the elution position 33, and finally the whole body from the sample loading position 11 to the PCR position 41 is subjected to membrane sealing by a protective membrane, so that the testable integrated kit is formed.
In this example, separation of a whole blood sample can be performed, and as shown in FIG. 2, if the sample from which nucleic acid is extracted is plasma, pretreatment of the sample can be performed using the kit of the present invention. The user now adds the whole blood 131 into the sample loading position 11, and the piston head 611 of the matching instrument is tightly connected with the top end 121 of the sample position 12, so that the airtightness is ensured. The piston rod 612 moves upwards in the piston cylinder to form negative pressure, the negative pressure enables the whole blood 131 in the sample loading position 11 to gradually pass through the medium film 111, macromolecules 112 such as red blood cells are retained in the medium film, and the filtered plasma 132 enters the sample position 12 through a communication channel between the sample loading position 11 and the sample position 12. Thus, the pretreatment of the whole blood sample is completed. If the sample for nucleic acid extraction is plant tissue, the user needs to complete the sample pretreatment in advance outside the instrument and the kit, and the treated sample is directly added into the sample position 12.
The present invention can extract nucleic acid, as shown in fig. 5, the pipetting head 221 is connected to the piston unit of the matching instrument, first a certain amount of pre-treated sample is aspirated from the sample position, and transferred to the lysis position 31 (a), the piston unit moves up and down to aspirate and discharge the mixing system, and at the same time, the incubation device 631 of the matching instrument works to provide the lysis temperature to the lysis position 31. In the cracking process, the liquid transferring head 221 can be used for sucking, discharging and uniformly mixing at random, so that the cracking reaction is accelerated. After the time of the lysis reaction is up, the pipetting head 221 enters the lysis site 31, the magnet 62 of the matched instrument is close to the pipetting head 221, the piston unit is connected with the pipetting head 221 to start the pipetting operation, and the magnetic beads in the lysate entering the pipetting head 221 in the pipetting process are gradually adsorbed on the side wall (B) of the pipetting head 221 close to the magnet 62 under the action of the magnetic field of the magnet 62. After the magnetic adsorption for a certain period of time (C), the piston unit is connected to the pipetting head 221 to begin the pipetting operation, and the lysed waste liquid is pipetted into the lysis site 31 (D). At this time, the piston unit and the reagent kit platform move in a combined manner, so that the liquid transfer head 221 carries the magnetic bead system to move to the cleaning position 32, the magnet 62 is far away from the liquid transfer head 221, the piston unit is connected with the liquid transfer head 221 to perform liquid suction and discharge mixing operation, and impurities such as protein and the like adsorbed on the surfaces of the magnetic beads after cracking are separated from the magnetic beads and are dissociated in the cleaning liquid (E). The magnetic adsorption operation is performed again, and the washing waste liquid is discharged to the washing level 32. In the examples, 3 washing sites were provided, and the purity of nucleic acid extraction was ensured by repeating the washing in the same manner as in the above washing procedures. The washed pipetting head 221 moves to the elution position 33, the magnet 62 is far away from the pipetting head 221, and the piston is connected with the pipetting head 221 to perform repeated suction, discharge and uniform mixing operation, so that the magnetic beads connected with the nucleic acid are uniformly distributed in the eluent (F). The incubation device 632 of the kit operates to provide an elution temperature to the elution site 33. After reacting for a certain time, separating the nucleic acid from the magnetic beads to complete the extraction of the nucleic acid. The magnet 62 is close to the pipetting head 221, and a pipetting operation is performed, and magnetic beads having detached nucleic acids in the eluate are adsorbed to the side wall of the pipetting head 221 close to the magnet 62. The pipette tip 221 having the magnetic beads adsorbed thereon is recovered at the pipette tip position 211.
In this embodiment, as shown in fig. 6 to 8, the piston unit is connected to the pipetting head 222 to aspirate a certain amount of nucleic acid from the elution site, and then the sample is moved to the PCR site 41, the pipetting head 222 penetrates the paraffin oil 52 sealing the PCR system and is inserted into the PCR system to perform the pipetting operation, and then the nucleic acid sample is discharged into the PCR system, and the PCR reaction system is aspirated and mixed repeatedly. After the pipetting head 222 is removed, the tube cover 42 is folded by a folding device in the matching instrument, the bulge on the top end of the tube cover 42 enters the top end of the PCR position 41, and the two are tightly matched to ensure the air tightness, so that the system is not volatilized in the PCR process. In the embodiment, the PCR bit 41 is a flat cuboid structure, and has a larger contact surface with a PCR temperature control bit of a matched instrument, so that the temperature rise and fall speed is higher, and the PCR reaction time length can be shortened. The PCR experiment can be carried out by adding the prepared PCR position of the sample flip cover, the flip cover device of the matched instrument can be a hot cover at the same time, the temperature of the hot cover can be provided, and the PCR temperature control device of the matched instrument works to provide target temperatures of all stages required by the PCR. In the detection section of amplification, the detection unit 64 works, moves along the plane vertical direction of the placing direction of the reagent kit, sequentially passes through the PCR position 41 of each reagent kit, and performs excitation and fluorescence collection on the system in the PCR position 41.
Waste recovery can be carried out in the kit after the experiment according to the experiment requirements, and the matched instrument of the embodiment does not contain a pipeline and an air circuit, so that the maintenance is convenient, and the non-liquefaction operation of the instrument is realized.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also technical features in the above embodiments or in different embodiments may be combined and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (15)
1. A device for realizing integration of nucleic acid extraction and PCR amplification detection is characterized in that: the kit comprises a matched instrument and a plurality of whole-column kits, wherein each kit comprises a nucleic acid extraction unit and a PCR amplification detection unit; the matched instrument is provided with a PCR detection unit, the PCR detection unit at least comprises a detection channel with wavelength fluorescence, the detection channel at least comprises an excitation light source and a detection device, the excitation light source and the detection device are integrated on a moving device and are driven by a motor to rapidly scan and move along a linear guide rail, and the PCR positions of a plurality of aligned kits are sequentially subjected to optical detection; the kit also comprises a sample pretreatment unit, a liquid transfer unit and an anti-pollution unit; the nucleic acid extraction unit of the kit adopts a nucleic acid extraction mode of a magnetic bead method and at least comprises a lysis site, a cleaning site and an elution site; the kit also comprises a tube cover position, and the matched instrument also comprises a tube cover folding device, wherein the tube cover of the kit is folded after a sample is filled, so that the bulge at the top end of the tube cover is tightly connected with the top end of the PCR position; the matching instrument also comprises a piston unit which is matched with the liquid transfer unit to complete the transfer, liquid suction and discharge and uniform mixing operations of the reaction liquid.
2. The apparatus of claim 1, wherein: the PCR amplification detection unit of the kit at least comprises one PCR bit.
3. The apparatus of claim 1, wherein: the anti-pollution unit of the kit comprises a filter column inside the pipetting head, a filter membrane at the top end of each nucleic acid extraction position and paraffin oil of a PCR position internal closed system.
4. The apparatus of claim 1, wherein: the top of the cracking position, the cleaning position and the elution position of the kit is also sealed with a filter liquid membrane.
5. The apparatus of claim 1, wherein: the liquid transfer unit of the kit at least comprises a pipetting head and a pipetting head storage position.
6. The apparatus of claim 1, wherein: the sample pretreatment unit of the kit comprises a sample position.
7. The apparatus of claim 1, wherein: the sample pretreatment unit of the kit comprises a sample position and a sample loading position, wherein the sample loading position is communicated with the sample position.
8. The apparatus of claim 7, wherein: the part of the kit, which is communicated with the sample loading position, is provided with a medium film capable of separating serum and plasma.
9. The apparatus according to any one of claims 1-8, wherein: the corollary instrument also comprises a magnetic unit made of magnetic materials.
10. The apparatus according to any one of claims 1-8, wherein: the corollary instrument also comprises a temperature control unit.
11. The apparatus of claim 1, wherein: the complete set of instrument still includes the accuse pressure unit, accuse pressure unit and sample pretreatment unit cooperation are accomplished whole blood separation operation.
12. A method for detecting by using the device for realizing integration of nucleic acid extraction and PCR amplification detection of any one of claims 1-11, wherein the device comprises a matched instrument and a plurality of aligned kits, and the kits comprise a nucleic acid extraction unit and a PCR amplification detection unit; the matched instrument is provided with a PCR detection unit, the PCR detection unit at least comprises a detection channel with wavelength fluorescence, the detection channel at least comprises an excitation light source and a detection device, the excitation light source and the detection device are integrated on a moving device and are driven by a motor to rapidly scan and move along a linear guide rail, and the PCR positions of a plurality of aligned kits are sequentially subjected to optical detection; the method comprises the following steps:
(1) nucleic acid extraction step:
a. absorbing a sample and transferring the sample to a lysis site, uniformly mixing sample lysate, then carrying out cell lysis, and specifically combining free nucleic acid molecules after cell lysis with silicon hydroxyl on the surface of the magnetic beads;
b. after reacting for a certain time, carrying out imbibition operation by using the pipetting head, enabling a magnetic unit of a matched instrument to be close to the outer wall of the pipetting head and providing a corresponding magnetic field when carrying out imbibition operation, enabling magnetic beads coated with nucleic acid molecules in the pipetting head to be close to the inner wall of the pipetting head near the magnetic unit under the action of the magnetic field, completely absorbing lysate in a lysis position into the pipetting head by the imbibition operation, enabling the magnetic beads in the lysate in the pipetting head to be adsorbed to the vicinity of the inner wall of the pipetting head on one side of the magnetic unit after a certain magnetic adsorption time, inserting the pipetting head into the lysis position at the moment, carrying out drainage operation, and discharging the waste liquid after lysis into the lysis position;
c. inserting the liquid transfer head into the cleaning liquid at the cleaning position, enabling the magnetic unit to be far away from the liquid transfer head or removing the magnetic field, and carrying out uniform mixing operation, wherein impurities adsorbed on the surfaces of the magnetic beads after cracking are gradually dissociated in the cleaning liquid in the uniform mixing cleaning process;
d. moving the liquid transfer head with the magnetic bead system on the cleaned inner wall to an elution position, keeping the magnetic unit away from the liquid transfer head or removing the magnetic field, uniformly mixing the magnetic beads and the eluent, and performing the separation process of nucleic acid molecules and the magnetic beads;
e. the elution operation is finished, namely the nucleic acid extraction operation is finished, and a concentrated sample with higher purity is obtained;
(2) PCR amplification detection: the kit carries out PCR reaction, and a certain amount of eluted samples are absorbed from the elution position and transferred to the PCR position; and (3) starting the PCR temperature control device, providing the temperature of each stage required by the PCR reaction, and detecting the PCR reaction, wherein in the detection stage, the PCR detection unit sequentially passes through the PCR bit of each kit to detect the real-time fluorescent signal.
13. The method of claim 12, wherein: in the step (2), after the pipetting head leaves the PCR position, the tube cover folding unit of the matched instrument folds the tube cover position, and the top end bulge of the folded tube cover is sealed with the PCR position, so that the system in the PCR position is sealed and is not volatilized.
14. The method of claim 12, wherein: the kit comprises a sample pretreatment unit, wherein the sample pretreatment unit at least comprises one sample position, and a treated sample is directly added.
15. The method of claim 14, wherein: the sample pretreatment unit further comprises a sample loading position, a communicating channel is arranged between the sample loading position and the sample position, a medium film capable of separating serum and plasma is arranged between the communicating channels, the size of the medium film can filter erythrocyte macromolecules, when the medium film is used in cooperation with a matched instrument, a pressure control unit of the matched instrument is tightly combined with the sample loading position or the sample position to ensure air tightness, whole blood at the sample loading position gradually permeates through the medium film under the action of positive pressure or negative pressure, and filtered serum enters the sample position through the communicating channel to be used for subsequent nucleic acid extraction.
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| CN109337793B (en) * | 2018-10-23 | 2022-01-25 | 宁波艾捷康宁生物科技有限公司 | Full-automatic nucleic acid extraction detecting system |
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| CN110331084A (en) * | 2019-08-06 | 2019-10-15 | 中国人民解放军军事科学院军事医学研究院 | Nucleic acid extraction instrument and detection system |
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| CN113462556B (en) * | 2021-09-02 | 2021-12-28 | 德诺杰亿（北京）生物科技有限公司 | Heating assembly of nucleic acid extractor |
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| CN114634853B (en) * | 2022-03-13 | 2023-05-02 | 首玺(广州)医疗科技有限责任公司 | Nucleic acid detection reagent device |
| CN115747025B (en) * | 2022-10-14 | 2023-09-22 | 北京卓诚惠生生物科技股份有限公司 | Full-automatic nucleic acid extraction and PCR detection all-in-one |
| CN116875425B (en) * | 2023-07-24 | 2024-03-12 | 北京万泰生物药业股份有限公司 | Nucleic acid extraction and purification device, reagent combination, nucleic acid extraction and purification system and method |
| CN117070314B (en) * | 2023-10-11 | 2024-01-30 | 苏州大学 | Nucleic acid extraction device, method of using the same, and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1281462A (en) * | 1997-12-06 | 2001-01-24 | Dna研究仪器有限公司 | Isolation of nucleic acids |
| WO2009049268A1 (en) * | 2007-10-12 | 2009-04-16 | Rheonix, Inc. | Integrated microfluidic device and methods |
| CN206556968U (en) * | 2016-11-23 | 2017-10-13 | 杭州杰毅麦特医疗器械有限公司 | A kind of detection of nucleic acids pre-treatment automation equipment |
-
2018
- 2018-05-28 CN CN201810525844.6A patent/CN108660074B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1281462A (en) * | 1997-12-06 | 2001-01-24 | Dna研究仪器有限公司 | Isolation of nucleic acids |
| WO2009049268A1 (en) * | 2007-10-12 | 2009-04-16 | Rheonix, Inc. | Integrated microfluidic device and methods |
| CN206556968U (en) * | 2016-11-23 | 2017-10-13 | 杭州杰毅麦特医疗器械有限公司 | A kind of detection of nucleic acids pre-treatment automation equipment |
Non-Patent Citations (2)
| Title |
|---|
| Comparison of Three Magnetic Bead Surface Functionalities for RNA Extraction and Detection;Nicholas M Adams,et al.;《ACS Appl. Mater. Interfaces》;20150325;第7卷(第11期);第6062-6069页 * |
| 核酸提取方法的研究进展;张丽等;《中国动物检疫》;20111231;第28卷(第12期);第75-78页 * |
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