CN110862907B

CN110862907B - Nucleic acid extraction preliminary treatment amplification system

Info

Publication number: CN110862907B
Application number: CN201911240637.7A
Authority: CN
Inventors: 吴文明
Original assignee: Changchun Institute of Optics Fine Mechanics and Physics of CAS
Current assignee: Changchun Institute of Optics Fine Mechanics and Physics of CAS
Priority date: 2019-12-06
Filing date: 2019-12-06
Publication date: 2021-10-08
Anticipated expiration: 2039-12-06
Also published as: CN110862907A

Abstract

The invention discloses a nucleic acid extraction pretreatment amplification system, which comprises a filtering device, a DNA extraction device, a uniformly mixing device, a PCR amplification device and a driving device for driving liquid to flow; the method for processing the samples in sequence by different reagents saves labor time and improves the working efficiency of people. Through the exquisite pipeline design, functional units such as a filtering device, a DNA extraction device for extracting nucleic acid, a blending device, a PCR amplification device and the like are integrated in an embedded manner, so that the whole volume is obviously reduced; the device has the characteristics of simple structure, easy realization and operation, low cost and large-area popularization and use; in addition, the PCR amplification device is based on temperature cycle, so that the rapid amplification and signal amplification of nucleic acid molecules by polymerase are realized, and the detection time is greatly reduced; in addition, the detectable reagent can be widely used, and bacteria, viruses, fungi, chlamydia, rickettsia and the like can realize rapid detection.

Description

Nucleic acid extraction preliminary treatment amplification system

Technical Field

The invention relates to the technical field of nucleic acid extraction and amplification, in particular to a nucleic acid extraction pretreatment amplification system.

Background

Quantitative PCR is the most important molecular biology detection means at present, can quickly identify the types and drug resistance mutation of microorganisms such as bacteria, viruses and fungi, and has analysis sensitivity far exceeding that of other detection means; the method can accurately detect the number of different microbial populations in a short time of only three to forty minutes, so that the method has important application prospects in almost all life science fields of food detection, clinical inspection, disease control, inspection and quarantine, scientific research laboratories, food safety, cosmetic detection, environmental sanitation and the like.

The existing PCR amplification instrument mainly has the following defects:

1. the adopted temperature cycle needs a complex temperature control system to realize, and the technical cost is higher;

2. the whole machine has large volume and is not suitable for rapid detection in a field environment in due time;

3. the sample pretreatment and preparation process is complex and needs to be realized by a mechanical centrifugal motion system with high energy consumption and large volume.

Therefore, how to provide a nucleic acid extraction pretreatment amplification system with simple structure, small size and low cost is a problem to be solved by those skilled in the art.

Disclosure of Invention

In view of the above, the present invention provides a nucleic acid pretreatment amplification system with simple structure, small size and low cost.

In order to achieve the above purpose, the invention provides the following technical scheme:

a nucleic acid extraction pretreatment amplification system comprises a filtering device, a DNA extraction device, a uniformly mixing device, a PCR amplification device and a driving device for driving liquid to flow;

the filtering device includes: the buffer solution filter comprises a first pipeline for inputting a sample, a second pipeline for inputting a buffer solution and a third pipeline for communicating the middle part of the first pipeline with the middle part of the second pipeline, wherein the third pipeline is provided with a permeable membrane for filtering, a first valve for controlling opening and closing is arranged at an inlet and an outlet of the first pipeline, a second valve for controlling opening and closing is arranged at an inlet and an outlet of the second pipeline, and an outlet of the second pipeline is used for discharging filtered waste liquid;

the DNA extraction device comprises: the extraction pipeline and a heating device for heating the extraction pipeline are arranged, and the outlet of the first pipeline is connected to the inlet of the extraction pipeline;

the mixing device comprises a mixing pipeline for multiple shunting and confluence, one end of the mixing pipeline is provided with a buffer solution inlet connected with the outlet of the extraction pipeline and a reaction solution inlet for inputting a reaction solution, and the other end of the mixing pipeline is provided with a mixed solution outlet;

the PCR amplification apparatus includes: the device comprises a first heating area used for decomposing double-stranded DNA into single-stranded DNA, a second heating area used for copying the single-stranded DNA into the double-stranded DNA, and a circulating pipeline which is folded back and forth and arranged on the first heating area and the second heating area, wherein the inlet of the circulating pipeline is connected with the outlet of the mixed liquid.

Preferably, the filtering device comprises a first substrate, the upper end face of the first substrate is provided with a first pipe groove and a second pipe groove which penetrate through the first substrate, and a third pipe groove which communicates the middle part of the first pipe groove with the middle part of the second pipe groove, a first cover plate which is hermetically attached to the first substrate is covered on the first substrate, the first cover plate and the first pipe groove, the second pipe groove and the third pipe groove respectively form a first pipeline, the second pipeline and the third pipeline, and the permeable membrane is arranged at the joint of the second pipe groove and the first pipe groove.

Preferably, the first valve comprises a first one-way valve arranged at an inlet of the first pipe groove and a first electromagnetic valve arranged at an outlet of the first pipe groove, and the two valves comprise a second one-way valve arranged at an inlet of the second pipe groove and a second electromagnetic valve arranged at an outlet of the second pipe groove.

Preferably, the DNA extraction device comprises a second substrate, an extraction pipe groove which is folded back and forth is processed at the upper end of the second substrate, a second cover plate which is hermetically attached to the second substrate is covered on the upper end of the second substrate, the second cover plate and the extraction pipe groove form the extraction pipeline, and the heating device is a heating plate which is attached to the lower end of the second substrate.

Preferably, a first heat preservation layer is further packaged on the outer sides of the second substrate and the second cover plate, and a second isolation layer for preventing liquid from entering is further packaged on the outer side of the first heat preservation layer.

Preferably, the mixing device includes the third base plate, the up end processing of third base plate has the mixed tube groove of reposition of redundant personnel and confluence many times, is used for the buffer solution pipe socket of input buffer solution, is used for the reaction liquid pipe socket of input reaction liquid and is used for exporting the mixed liquid pipe socket of mixed liquid, the buffer solution pipe socket with reaction liquid pipe socket converge and communicate in the one end of mixed tube groove, the other end of mixed tube groove communicate in mixed liquid pipe socket, the sealed laminating of up end of third base plate has the third apron, the third apron with mixed tube groove buffer solution pipe socket, reaction liquid pipe socket and mixed liquid pipe socket forms respectively the hybrid tube the buffer solution pipeline, reaction liquid pipeline and mixed liquid pipeline.

Preferably, the PCR amplification device includes a fourth substrate, a circulation tube slot is processed on the upper end surface of the fourth substrate, a fourth cover plate is hermetically attached to the upper end surface of the fourth substrate, the circulation tube slot and the fourth cover plate form the circulation pipeline, a first heating sheet is attached to one side of the lower end surface of the fourth cover plate below the circulation tube slot to form the first heating zone, and a second heating sheet is attached to the other side of the lower end surface of the fourth cover plate below the circulation tube slot to form the second heating zone; and a second heat-insulating layer is packaged on the outer side of the PCR amplification device, and a fourth isolating layer is packaged on the outer side of the second heat-insulating layer.

Preferably, the PCR amplification device includes a first heat preservation box, a second heat preservation box and a third heat preservation box are arranged in the first heat preservation box, the second heat preservation box and the third heat preservation box are mutually sealed and are filled with oil, the circulating pipeline is reciprocally folded to penetrate through the second heat preservation box and the third heat preservation box, a first heating device is arranged in the first heat preservation box, a second heating device is arranged in the second heat preservation box, so that the oil in the second heat preservation box forms the first heating area, and a third heating device is arranged in the third heat preservation box, so that the oil in the third heat preservation box forms the second heating area.

Preferably, the inlet of the second pipeline is connected with a buffer solution storage bag for storing a buffer solution, the reaction solution inlet is connected with a reaction solution storage bag for storing a reaction solution, the driving device is a peristaltic pump arranged at the outlet of the circulating pipeline, the buffer solution inlet is provided with a third valve, the reaction solution inlet is provided with a fourth valve, and the controller further comprises a signal connected to the peristaltic pump, the first valve, the second valve, the third valve and the fourth valve.

Preferably, an oil inlet for inputting oil is further formed at the inlet of the circulation pipeline, so that the oil and the mixed liquid are converged and then flow into the circulation pipeline.

The nucleic acid extraction pretreatment amplification system provided by the invention has the advantages that the method for sequentially processing the samples by different reagents, such as reaction liquid and buffer liquid, is adopted, the labor time is saved, and the working efficiency of people is improved. Through the exquisite pipeline design, the functional units such as a filtering device for purifying a sample reagent, a DNA extraction device for extracting nucleic acid, a blending device, a PCR amplification device and the like are integrated in an embedded manner, so that the whole volume is obviously reduced; the device has the characteristics of simple structure, easy realization and operation, low cost and large-area popularization and use; in addition, the PCR amplification device is based on temperature cycle, so that the rapid amplification and signal amplification of nucleic acid molecules by polymerase are realized, and the detection time is greatly reduced; in addition, the detectable reagent can be widely used, and bacteria, viruses, fungi, chlamydia, rickettsia and the like can realize rapid detection.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a schematic diagram of an embodiment of a nucleic acid extraction pretreatment amplification system provided in the present invention;

FIG. 2 is a schematic view of a filtration apparatus;

FIG. 3 is a schematic view of a DNA extraction apparatus;

FIG. 4 is a schematic view of a homomixer;

FIG. 5 is a schematic view of a PCR amplification apparatus;

FIG. 6 is a schematic view of the heat-insulating layer of the PCR amplification apparatus.

Wherein, 1-a waste liquid outlet, 2-a sample outlet, 3-a DNA extraction device, 4-a filtration device, 5-a first electromagnetic valve, 6-a second electromagnetic valve, 7-a buffer solution storage bag, 8-a peristaltic pump, 9-a sample inlet, 10-a blending device, 11-a PCR amplification device, 12-a first one-way valve, 13-a second one-way valve, 14-a reaction solution storage bag, 15-a partition plate, 16-a storage battery, 17-a first pipeline, 18-a second pipeline, 19-a permeable membrane, 20-a second isolation layer, 21-a first heat preservation layer, 22-a second cover plate, 23-an extraction pipeline inlet, 24-a second substrate, 25-a heating plate, 26-an extraction pipeline outlet, 27-a third isolation layer, 28-a third cover plate, 29-a buffer solution inlet, 30-a third base plate, 31-a reaction solution inlet, 32-a mixed solution outlet, 33-an oil liquid inlet, 34-a circulating pipeline inlet, 35-a circulating pipeline, 36-a circulating pipeline outlet, 37-a first heat preservation box, 38-a second heat preservation box, 39-a third heat preservation box, 40-a fourth heat preservation box and 41-a fifth heat preservation box.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The core of the invention is to provide a nucleic acid extraction pretreatment amplification system which has simple structure, small size and low cost.

Referring to FIGS. 1 to 5, FIG. 1 is a schematic diagram of an embodiment of a nucleic acid extraction pretreatment amplification system according to the present invention; FIG. 2 is a schematic view of a filtration apparatus; FIG. 3 is a schematic view of a DNA extraction apparatus; FIG. 4 is a schematic view of a homomixer; FIG. 5 is a schematic view of a PCR amplification apparatus; FIG. 6 is a schematic view of the heat-insulating layer of the PCR amplification apparatus.

The nucleic acid extraction pretreatment amplification system comprises a filtering device 4, a DNA extraction device 3, a uniformly mixing device 10, a PCR amplification device 11 and a driving device for driving liquid to flow;

the filter device 4 includes: the buffer solution filter comprises a first pipeline 17 for inputting a sample, a second pipeline 18 for inputting a buffer solution and a third pipeline for communicating the middle part of the first pipeline 17 with the middle part of the second pipeline 18, wherein the third pipeline is provided with a permeable membrane 19 for filtering, the inlet and the outlet of the first pipeline 17 are both provided with a first valve for controlling opening and closing, the inlet and the outlet of the second pipeline 18 are both provided with a second valve for controlling opening and closing, and the outlet of the second pipeline 18 is used for discharging filtered waste liquid;

the DNA extraction apparatus 3 includes: an extraction duct and heating means for heating the extraction duct, the outlet of the first duct 17 being connected to the inlet 23 of the extraction duct;

the kneading apparatus 10 includes: a mixing pipeline for mixing liquid, wherein one end of the mixing pipeline is provided with a buffer liquid inlet 29 connected with the outlet 26 of the extraction pipeline and a reaction liquid inlet 31 for inputting reaction liquid, and the other end of the mixing pipeline is provided with a mixed liquid outlet 32;

the PCR amplification apparatus 11 includes: a first heating area for decomposing the double-stranded DNA into single-stranded DNA, a second heating area for duplicating the single-stranded DNA into double-stranded DNA, and a circulating pipeline 35 which is folded back and forth and arranged on the first heating area and the second heating area, wherein the inlet 34 of the circulating pipeline is connected with the mixed liquid outlet 32.

Among them, since some organic or inorganic molecules inhibiting the PCR reaction, such as humic acid or mineral salt elements, often exist in the field in-situ sample, and have a large influence on the amplification efficiency of the PCR reaction, the filtering device 4 is mainly used for separating pathogens from the field sample before performing the PCR amplification, storing the pathogens in a weakly alkaline buffer solution, and preparing for breaking the walls of bacteria and collecting DNA. The filter means 4 is therefore required to separate bacteria from the aerosol or liquid sample and to put the pathogens in the buffer.

When filtering, a field sample containing biological warfare agents can be input from the inlet of the first pipeline 17, the opening of the inlet of the first pipeline 17 and the closing of the outlet of the first pipeline 17 are controlled by the first valve, the opening of the outlet and the closing of the inlet of the first pipeline 17 are controlled by the second valve, so that the sample flows into the third pipeline and the osmotic membrane 19 on the third pipeline from the inlet of the first pipeline 17, is filtered and flows out from the outlet of the second pipeline 18, microorganisms are attached to the osmotic membrane 19 at the moment, and the outlet of the second pipeline 18 is connected to a liquid discharge pipe and serves as a waste liquid outlet 1 for discharging filtered waste liquid; then, the inlet of the first pipe 17 is controlled to be closed and the outlet is controlled to be opened by the first valve, the inlet of the second pipe 18 is controlled to be opened and the outlet is controlled to be closed by the second valve, the buffer solution is injected from the inlet of the second pipe 18 so that the buffer solution flows through the third pipe and the permeable membrane 19 thereon, the microorganism is contained in the buffer solution, and the buffer solution containing the bacteria flows out from the outlet of the first pipe 17 so that the buffer solution containing the microorganism is transferred to the DNA extracting apparatus 3. Wherein, the aperture of the permeable membrane 19 can be freely selected according to the species and size of the microorganism to be detected, and the replacement is convenient. The inlet of the first pipe 17 is used for connecting a pipeline for inputting a sample to be used as the sample inlet 9.

Before amplification, nucleic acid molecules in microorganisms need to be extracted, and since nucleic acid is mainly present in cell nuclei, the microorganisms need to be treated to rupture cell walls or cell membranes, release genetic materials in the microorganisms, and finish collection of DNA. Since most microorganisms have their cell walls broken at high temperature and thus release their genetic materials, the DNA extraction apparatus 3 will lyse bacteria or viruses by pyrolysis and release nucleic acid molecules inside.

The DNA extraction device 3 comprises an extraction pipeline and a heating device, wherein the heating device is used for heating the extraction pipeline, so that when the buffer solution containing the microorganisms entering the extraction pipeline flows in the extraction pipeline, under the action of high temperature, the microorganisms break the wall and release genetic materials, so that the buffer solution containing nucleic acid molecules flows out of the outlet 26 of the extraction pipeline.

The buffer solution flowing out of the DNA extraction device 3 needs to be fully mixed with the reaction solution before entering the PCR amplification device 11 for amplification, otherwise, the amplification efficiency of the reaction reagent is affected. In order to realize rapid mixing of reagents in a small volume, the mixing device 10 provided by the invention carries out rapid mixing by arranging a mixing pipeline for multiple shunting and confluence, buffer solution and reaction solution are respectively introduced into the mixing device 10 from a buffer solution inlet 29 and a buffer solution inlet 29, the reaction solution and the buffer solution containing genetic materials can be converged at the inlets of the mixing pipeline, the two liquids can be fully mixed by multiple shunting and confluence, and the mixed liquid is transmitted to the PCR amplification device 11 through the outlet of the mixing pipeline.

The PCR amplification device 11 is the core part of the whole system, and the performance of the PCR amplification device 11 determines whether the whole biological warfare agent in-situ PCR instrument can complete the preset design task and index. Amplification of bacterial nucleic acid molecules requires temperature cycling, which is determined by the nature of polymerase chain reaction PCR. Generally, a double-stranded DNA molecule is denatured at a high temperature of about 95 ℃ and the double-stranded DNA is decomposed into two single-stranded DNAs. Under the condition of low temperature of about 60 ℃, single-stranded DNA can react according to the base complementary pairing principle under the action of a primer and polymerase, and two single-stranded DNAs can be converted into two double-stranded DNAs. After a high-temperature denaturation and low-temperature annealing cycle, the original DNA molecular weight is changed to 2 times of the original DNA molecular weight, and after a plurality of temperature cycles, the number of the DNA molecules is exponentially increased, so that a high-concentration DNA fragment is obtained in a short time, and the high-concentration DNA fragment is output from a sample outlet 2 of the system, thereby realizing high-specificity in-situ accurate identification and absolute quantification.

The mixed solution output from the outlet of the mixing pipe enters the inlet 34 of the circulating pipe to enter the PCR amplification device 11, wherein the first heating area is about 95 degrees at the high temperature area, the second heating area is about 60 degrees at the low temperature area, and the circulating pipe 35 is arranged on the first heating area and the second heating area in a reciprocating and turning-back manner, so that the mixed solution can sequentially and repeatedly pass through the high temperature area and the low temperature area when flowing along the circulating pipe 35, nucleic acid molecules in the reaction solution can be amplified through multiple cycles, and DNA fragments with high concentration are output at the outlet.

It should be noted that the accuracy of the reaction temperature and the reaction time of the PCR are key factors for ensuring the reaction efficiency, and thus the reaction temperature and the reaction time need to be accurately controlled. Considering the specific way of communication between the devices, it is preferable that silicone tubing with a wall thickness of 0.5mm and a tube diameter of 2mm be used to connect the parts.

In summary, the nucleic acid extraction pretreatment amplification system provided by the invention uses different reagents, such as reaction solution and buffer solution, to sequentially process the sample, thereby saving labor time and improving working efficiency of people. Through the exquisite pipeline design, the functional units such as the filtering device 4 for purifying the sample reagent, the DNA extraction device 3 for extracting nucleic acid, the blending device 10, the PCR amplification device 11 and the like are integrated in an embedded manner, so that the whole volume is obviously reduced; the device has the characteristics of simple structure, easy realization and operation, low cost and large-area popularization and use; in addition, the PCR amplification device 11 is based on temperature cycle, so that the rapid amplification and signal amplification of nucleic acid molecules by polymerase are realized, and the detection time is greatly reduced; in addition, the detectable reagent can be widely used, and bacteria, viruses, fungi, chlamydia, rickettsia and the like can realize rapid detection.

On the basis of the above embodiment, in consideration of the specific structural arrangement of the filtering device 4, preferably, the filtering device 4 includes a first substrate, the upper end surface of the first substrate is provided with a first pipe groove and a second pipe groove penetrating through the first substrate, and a third pipe groove communicating the middle part of the first pipe groove with the middle part of the second pipe groove, the first pipe groove is covered with a first cover plate hermetically attached to the first substrate, the first cover plate and the first pipe groove, the second pipe groove and the third pipe groove form a first pipeline 17, a second pipeline 18 and a third pipeline, respectively, and the permeable membrane 19 is disposed at the joint of the second pipe groove and the first pipe groove.

Wherein, the first base plate can be acrylic plate or organic glass, first chase, second chase and third chase can be the chase of processing at acrylic plate or organic glass upper surface, first apron can be the same material with first base plate, first apron covers the laminating at first base plate upper surface, in order to cover first chase, second chase and third chase, and form first pipeline 17, second pipeline 18 and third pipeline with first chase, second chase and third chase, and set up osmotic membrane 19 in the junction of third chase and first chase, thereby form and be used for filterable passageway. The first base plate and the first cover plate can be welded into a whole through an ultrasonic welding machine, and interfaces required by connection can be machined at two ends of the first pipe groove and the second pipe groove.

In the basis of the above embodiment, in consideration of the specific arrangement of the first valve, it is preferable that the first valve includes a first check valve 12 provided at the inlet of the first pipe chase and a first solenoid valve 5 provided at the outlet of the first pipe chase, and the two valves include a second check valve 13 provided at the inlet of the second pipe chase and a second solenoid valve 6 provided at the outlet of the second pipe chase. Due to the effect of the one-way conduction of the first one-way valve 12 and the second one-way valve 13, a sample can be input from the inlet of the first pipeline 17, and after the filtering is finished, a buffer solution can be input from the inlet of the second pipeline 18, and in the process, the opening and closing of the first electromagnetic valve 5 and the second electromagnetic valve 6 only need to be controlled.

In addition, to prevent aerosol contaminants in the sample from entering the device during long field experiments, the tubing interface to the device needs to be sealed. And a first isolation layer is encapsulated at the outer sides of the first substrate and the first cover plate to enhance the sealing effect.

In addition to the above embodiments, in consideration of the specific arrangement of the DNA extraction device 3, the DNA extraction device 3 includes a second substrate 24, an extraction tube slot folded back and forth is formed at the upper end of the second substrate 24, a second cover plate 22 hermetically attached to the second substrate 24 is capped at the upper end of the second substrate 24, the second cover plate 22 and the extraction tube slot form an extraction duct, and the heating device is a heating plate 25 attached to the lower end of the second substrate 24.

The second substrate 24 and the second cover plate 22 may be acrylic plates or organic glass, etc., the first substrate is provided with a reciprocating extraction pipe groove, the first substrate and the first cover plate may be welded together by an ultrasonic welding machine, and then the two ends of the extraction pipe grooves on the two sides are provided with interfaces required for connection. The lower end surface of the first substrate is further provided with a heating plate 25 for heating to heat the extraction pipeline, so that the wall of the microorganism flowing through the extraction pipeline is broken to release genetic materials.

In addition to the above-described embodiments, in order to prevent the salt and the mineral element permeating the silicone oil from entering the DNA extraction device 3, the first heat insulating layer 21 is further enclosed on the outer sides of the second base plate 24 and the second cover plate 22, and the second isolation layer 20 for preventing the liquid from entering is further enclosed on the outer side of the first heat insulating layer 21. Preferably, the second isolation layer 20 may be made of glass, the outlet of the first tube 17 may be connected to the inlet of the extraction tube through a quartz tube, the quartz tube is connected to the sealed second cover plate 22 and the sealed second cover plate 22, and then the whole device is encapsulated with an insulating material to form a first insulating layer 21, and the second isolation layer 20 is encapsulated outside the first insulating layer 21 to prevent the salt and mineral elements penetrating into the silicone oil from entering the DNA extraction device 3.

In addition to the above embodiments, in consideration of the specific installation manner of the mixing device 10, preferably, the mixing device 10 includes a third substrate 30, a mixing pipe groove for multiple diversion and confluence, a buffer pipe groove for inputting buffer solution, a reaction liquid pipe groove for inputting reaction liquid, and a mixed liquid pipe groove for outputting mixed liquid are processed on the upper end surface of the third substrate 30, the buffer pipe groove and the reaction liquid pipe groove are converged and communicated with one end of the mixing pipe groove, the other end of the mixing pipe groove is communicated with the mixed liquid pipe groove, a third cover plate 28 is hermetically attached to the upper end surface of the third substrate 30, and the third cover plate 28 and the mixing pipe groove, the buffer pipe groove, the reaction liquid pipe groove, and the mixed liquid pipe groove form a mixing pipeline, a buffer pipe, a reaction liquid pipe, and a mixed liquid pipe, respectively.

The third substrate 30 and the third cover plate 28 may be acrylic plates or organic glass, a mixing channel for multiple diversion and confluence is processed on the third substrate 30, and the third cover plate 28 may seal the third cover plate 28 and the third substrate 30 together by an ultrasonic welding machine to form a microfluidic chip with a mixing channel. And the inlet of the buffer solution pipeline, the inlet of the reaction solution pipeline and the outlet of the mixed solution pipeline are all processed and connected with required interfaces, a quartz pipeline is installed, and a third isolating layer 27 made of glass can be used for packaging, so that the blending device 10 can be manufactured. The buffer solution and the reaction solution are respectively introduced into the mixing device 10 from the two inlets, the reaction solution and the buffer solution containing genetic materials can be converged at the inlet of the mixing pipeline, the two solutions can be fully mixed through multiple times of flow division and confluence, and the mixed solution is transmitted to the PCR amplification device 11 through the quartz tube.

In view of the specific arrangement of the PCR amplification apparatus 11, it is preferable that the PCR amplification apparatus 11 includes a fourth substrate, a circulation pipe groove is formed on an upper end surface of the fourth substrate, a fourth cover plate is further hermetically attached to the upper end surface of the fourth substrate, the circulation pipe groove and the fourth cover plate form a circulation pipe 35, a first heating plate is attached to a lower end surface of the fourth cover plate on one side below the circulation pipe groove to form a first heating area, and a second heating plate is attached to a lower end surface of the fourth cover plate on the other side below the circulation pipe groove to form a second heating area.

The fourth substrate and the fourth cover plate may also be acrylic plates or organic glass, the upper surface of the first substrate is processed with a reciprocating and returning circulation tube slot, the fourth cover plate can be sealed on the fourth substrate by an ultrasonic sealing technology, the lower end surface of the fourth substrate is provided with a first heating sheet and a second heating sheet, the fourth substrate is a high temperature region in the region provided with the first heating sheet, the temperature is about 95 ℃, the fourth substrate is a low temperature region in the region provided with the second heating sheet, the temperature is about 60 ℃, so that the circulating tube slot can pass through the high temperature region and the low temperature region in a reciprocating and returning manner, in order to ensure the amplification effect, preferably, the number of cycles of the circulation tube 35 passing through the low temperature region of the high temperature region can be 40 cycles, and after 40 cycles of the circulation at a temperature of 95 ℃ to 60 ℃, the DNA fragment to be amplified successfully.

On the basis of the above embodiment, in order to prevent the heat loss from being too fast, it is preferable that a second insulating layer is packaged outside the PCR amplification device 11, and a fourth insulating layer is packaged outside the second insulating layer in order to prevent the salt and mineral elements penetrating into the silicone oil from entering the PCR reaction device. That is, the whole reaction device is wrapped by the fourth isolating layer made of glass.

Therefore, the nucleic acid extraction pretreatment amplification system provided by the invention can prevent external liquid from permeating by arranging the isolation layers on all parts, can be used on land, is also suitable for water bodies, such as directly used in the sea, and obviously improves the use convenience.

In another embodiment of the PCR amplification apparatus, the PCR amplification apparatus 11 includes a first heat-preservation box 37, a second heat-preservation box 38 and a third heat-preservation box 39 are disposed in the first heat-preservation box 37, the second heat-preservation box 38 and the third heat-preservation box 39 are sealed with each other and are filled with oil, a circulation pipeline is reciprocally folded and inserted into the second heat-preservation box 38 and the third heat-preservation box 39, a first heating device is disposed in the first heat-preservation box 37, a second heating device is disposed in the second heat-preservation box 38, so that the oil in the second heat-preservation box 38 forms a first heating zone, and a third heating device is disposed in the third heat-preservation box 39, so that the oil in the third heat-preservation box 39 forms a second heating zone.

The temperature of the first heat preservation tank 37 may be 30 degrees to reduce the speed of heat dissipation from the second heat preservation tank 38 and the third heat preservation tank 39 to the outside, and the oil in the second heat preservation tank 38 needs to form a first heating area of the heating circulation pipeline 35, so the temperature of the oil in the second heat preservation tank 38 should be about 95 degrees, and similarly, the oil in the third heat preservation tank 38 should be about 60 degrees, the second heat preservation tank 38 and the third heat preservation tank 39 may be arranged in parallel in the third heat preservation tank 39, or the third heat preservation tank 39 may be arranged in the second heat preservation tank 38 to further reduce energy consumption loss, and in addition, other heat preservation layers may be arranged outside the first heat preservation tank 37 as needed.

In consideration of the specific arrangement mode of the circulation pipeline, specifically, a plurality of small holes for penetrating the circulation pipeline may be formed in the side walls of the second heat insulation box 38 and the third heat insulation box 39, and the circulation pipeline should be penetrated and arranged in the small holes in a sealing manner to ensure the sealing property between the second heat insulation box 38 and the third heat insulation box 39, so that the circulation pipeline can be inserted between the second heat insulation box 38 and the third heat insulation box 39 in a reciprocating and returning manner.

In this embodiment, the PCR amplification apparatus can achieve lower energy consumption compared to the previous thermal insulation system, and can also meet the use requirement in the deep sea high pressure environment, because the first thermal insulation tank 37, the second thermal insulation tank 38, and the third thermal insulation tank 39 are all filled with oil, the formation of oil bath heating is formed by heating the oil, the uniformity of heating is improved, and in addition, because the thermal insulation tanks are filled with oil, even if the apparatus is placed in the deep sea or other high pressure application environments, the first thermal insulation tank 37, the second thermal insulation tank 38, and the third thermal insulation tank 39 filled with oil can bear the high pressure in the deep sea, and can be used in the deep sea. Compared with the previous embodiment, the PCR amplification device of the present embodiment is used in environments including shallow sea, air, land, etc., and can be applied to deep sea. The oil in the compensation heat-preservation box of the oil bag can realize the external high-pressure resistance bearing capacity.

In addition, in order to further reduce energy consumption, a fourth heat preservation box 40 may be disposed between the first heat preservation box 37 and the second heat preservation box 38, specifically, the temperature of the fourth heat preservation box 40 may also be 95 degrees, a fifth heat preservation box 41 may be disposed between the first heat preservation box 37 and the third heat preservation box 39, specifically, the temperature of the fifth heat preservation box 41 may be 60 degrees, and the fifth heat preservation box 41 may be further sleeved outside the fourth heat preservation box 40, so as to further reduce energy consumption. The core thought of the structure is that the low energy consumption of the whole system is realized through multilayer embedded heat preservation.

In order to further improve the convenience of the nucleic acid extraction pretreatment amplification system provided by the present invention in use, it is preferable that the inlet of the second tube 18 is connected to a buffer storage bag 7 for storing a buffer, the reaction solution inlet 31 is connected to a reaction solution storage bag 14 for storing a reaction solution, the driving device is a peristaltic pump 8 disposed at the outlet 36 of the circulation tube, the buffer inlet 29 is provided with a third valve, the reaction solution inlet 31 is provided with a fourth valve, and the system further includes a controller in signal connection with the peristaltic pump 8, the first valve, the second valve, the third valve and the fourth valve.

In the embodiment, the driving device is a peristaltic pump 8, the peristaltic pump 8 provides driving power for the liquid flowing in the pipeline, a buffer storage bag 7 for storing buffer is connected to the inlet of the second tube 18, a reaction solution storage bag 14 for storing reaction solution is connected to the reaction solution inlet 31, and a third valve is provided at the buffer inlet 29, a fourth valve is arranged at the reaction liquid inlet 31, and the third valve and the fourth valve can be both electromagnetic valves, and controls the start and stop of the peristaltic pump 8 and the opening and closing of the first valve, the second valve, the third valve and the fourth valve through a controller, the controller can be a PLC, the full-automatic operation can be realized by setting a corresponding program, the complex manual external operation is not needed, the use convenience is obviously improved, it should of course also be provided to provide a power source, which may preferably be a battery 16, for use in the field. Specifically, the battery 16 may be disposed under the entire system, and the battery 16 is partitioned using the partition plate 15 to prevent liquid from entering the battery 16.

In addition to the above embodiments, an oil inlet 33 for inputting oil is provided at an inlet 34 of the circulation pipe, so that the oil and the mixed liquid are merged and then flow into the circulation pipe 35. The detection sensitivity is improved by the way that the liquid drops wrap the mixed liquid, and the detection requirement of higher accuracy of the biological warfare agent is met. In the embodiment, the qPCR analyzer is upgraded to dPCR, so that the detection sensitivity is improved, and the requirement of higher accuracy detection of biological warfare agents is met.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The nucleic acid extraction pretreatment amplification system provided by the invention is described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims

1. A nucleic acid extraction pretreatment amplification system is characterized by comprising a filtering device (4), a DNA extraction device (3), a uniformly mixing device (10), a PCR amplification device (11) and a driving device for driving liquid to flow;

the filtering device (4) comprises: the buffer solution filter comprises a first pipeline (17) for inputting a sample, a second pipeline (18) for inputting a buffer solution and a third pipeline for communicating the middle part of the first pipeline (17) with the middle part of the second pipeline (18), wherein the third pipeline is provided with an osmotic membrane (19) for filtering, the inlet and the outlet of the first pipeline (17) are respectively provided with a first valve for controlling opening and closing, the inlet and the outlet of the second pipeline (18) are respectively provided with a second valve for controlling opening and closing, and the outlet of the second pipeline (18) is used for discharging filtered waste liquid;

the DNA extraction device (3) comprises: an extraction duct and heating means for heating the extraction duct, the outlet of the first duct (17) being connected to the inlet (23) of the extraction duct;

the mixing device (10) comprises a mixing pipeline for multiple shunting and confluence, one end of the mixing pipeline is provided with a buffer solution inlet (29) connected with the outlet (26) of the extraction pipeline and a reaction solution inlet (31) for inputting a reaction solution, and the other end of the mixing pipeline is provided with a mixed solution outlet (32);

the PCR amplification apparatus (11) comprises: a first heating area for decomposing the double-stranded DNA into the single-stranded DNA, a second heating area for duplicating the single-stranded DNA into the double-stranded DNA, and a circulating pipeline (35) which is arranged on the first heating area and the second heating area in a reciprocating and folding way, wherein an inlet (34) of the circulating pipeline is connected with the mixed liquid outlet (32);

the PCR amplification device (11) comprises a first heat preservation box (37), a second heat preservation box (38) and a third heat preservation box (39) are arranged in the first heat preservation box (37), the second heat preservation box (38) and the third heat preservation box (39) are mutually sealed, oil liquid is filled in the first heat preservation box (37), the circulating pipeline is arranged in the second heat preservation box (38) and the third heat preservation box (39) in a penetrating mode in a reciprocating and turning-back mode, a first heating device is arranged in the first heat preservation box (37), a second heating device is arranged in the second heat preservation box (38), so that oil liquid in the second heat preservation box (38) forms the first heating area, and a third heating device is arranged in the third heat preservation box (39), so that the oil liquid in the third heat preservation box (39) forms the second heating area.

2. The nucleic acid extraction pretreatment amplification system of claim 1, wherein the filtration device (4) comprises a first substrate, wherein a first pipe groove and a second pipe groove penetrating the first substrate and a third pipe groove communicating the middle part of the first pipe groove with the middle part of the second pipe groove are formed on the upper end surface of the first substrate, a first cover plate hermetically attached to the first substrate is covered on the first substrate, the first cover plate and the first pipe groove, the second pipe groove and the third pipe groove respectively form the first pipeline (17), the second pipeline (18) and the third pipeline, and the permeable membrane (19) is arranged at the joint of the second pipe groove and the first pipe groove.

3. The nucleic acid extraction pretreatment amplification system of claim 2, wherein the first valve comprises a first one-way valve (12) provided at an inlet of the first pipe tank and a first solenoid valve (5) provided at an outlet of the first pipe tank, and the second valve comprises a second one-way valve (13) provided at an inlet of the second pipe tank and a second solenoid valve (6) provided at an outlet of the second pipe tank.

4. The nucleic acid extraction pretreatment amplification system of claim 1, wherein the DNA extraction device (3) comprises a second substrate (24), an extraction tube slot folded back and forth is formed at an upper end of the second substrate (24), a second cover plate (22) hermetically attached to the second substrate (24) is covered at an upper end of the second substrate (24), the second cover plate (22) and the extraction tube slot form the extraction conduit, and the heating device is a heating plate (25) attached to a lower end of the second substrate (24).

5. The nucleic acid extraction pretreatment amplification system of claim 4, wherein the second substrate (24) and the second cover plate (22) are further encapsulated with a first heat-insulating layer (21) on the outer side, and the first heat-insulating layer (21) is further encapsulated with a second isolation layer (20) on the outer side for preventing liquid from entering.

6. The nucleic acid extraction pretreatment amplification system of claim 1, wherein the mixing device (10) comprises a third substrate (30), the upper end surface of the third substrate (30) is provided with a mixing pipe groove for multiple shunting and confluence, a buffer liquid pipe groove for inputting buffer liquid, a reaction liquid pipe groove for inputting reaction liquid and a mixed liquid pipe groove for outputting mixed liquid, the buffer liquid pipe groove and the reaction liquid pipe groove are converged and communicated with one end of the mixing pipe groove, the other end of the mixing pipe groove is communicated with the mixing pipe groove, the upper end surface of the third base plate (30) is sealed and jointed with a third cover plate (28), the third cover plate (28) and the mixing pipe groove, the buffer solution pipe groove, the reaction liquid pipe groove and the mixed liquid pipe groove form the mixing pipeline, the buffer solution pipeline, the reaction liquid pipeline and the mixed liquid pipeline respectively.

7. The nucleic acid extraction pretreatment amplification system of claim 1, wherein the PCR amplification device (11) comprises a fourth substrate, a circulation tube groove is formed on the upper end surface of the fourth substrate, a fourth cover plate is hermetically attached to the upper end surface of the fourth substrate, the circulation tube groove and the fourth cover plate form the circulation duct (35), a first heating plate is attached to the lower end surface of the fourth cover plate on one side below the circulation tube groove to form the first heating zone, and a second heating plate is attached to the lower end surface of the fourth cover plate on the other side below the circulation tube groove to form the second heating zone; and a second heat-insulating layer is packaged on the outer side of the PCR amplification device (11), and a fourth isolating layer is packaged on the outer side of the second heat-insulating layer.

8. The nucleic acid extraction, pretreatment and amplification system of any one of claims 1 to 7, wherein the inlet of the second conduit (18) is connected to a buffer storage bag (7) for storing a buffer, the reaction solution inlet (31) is connected to a reaction solution storage bag (14) for storing a reaction solution, the driving device is a peristaltic pump (8) disposed at the outlet (36) of the circulation conduit, the buffer inlet (29) is provided with a third valve, the reaction solution inlet (31) is provided with a fourth valve, and the system further comprises a controller in signal connection with the peristaltic pump (8), the first valve, the second valve, the third valve and the fourth valve.

9. The nucleic acid extraction pretreatment amplification system of claim 8, wherein an oil inlet (33) for inputting oil is further provided at the inlet (34) of the circulation pipeline, so that the oil and the mixed solution are merged and then flow into the circulation pipeline (35).