CN109295183A

CN109295183A - A kind of method and system of quick detection sample of nucleic acid

Info

Publication number: CN109295183A
Application number: CN201811283288.2A
Authority: CN
Inventors: 罗海贝; 王臣
Original assignee: Shanghai Qianlu Gene Technology Co Ltd
Current assignee: Shanghai Qianlu Gene Technology Co Ltd
Priority date: 2018-07-12
Filing date: 2018-10-31
Publication date: 2019-02-01

Abstract

The invention discloses a kind of method and system of quickly detection sample of nucleic acid, a kind of methods of quick detection sample of nucleic acid, comprising the following steps: S1: sample extraction is to be detected nucleic acid-templated, and PCR reaction system is made；S2:PCR amplification；S3:PCR product detection, which is characterized in that the S2:PCR amplification includes the following steps: S21: driving the PCR reaction system to flow through start-up temperature area by actuator, completes the thermal starting and nucleic acid-templated denaturation to be detected of archaeal dna polymerase；S22: the actuator, which continues the PCR reaction system is driven to circulate between denaturation temperature area and annealing temperature area, to be expanded；S23: after amplification, the actuator continues that the PCR reaction system is driven to flow to PCR product detection zone.By PCR reaction system liquid or the heating and cooling process repeatedly of drop circulated instead of common in-situ temperature module, efficient, quick, Budget PC R is realized, and provide the device of the quick detection sample of nucleic acid to match with set PCR mode.

Description

A kind of method and system of quick detection sample of nucleic acid

Technical field

The invention belongs to field of nucleic acid detection, and in particular to a kind of method and system of quickly detection sample of nucleic acid.

Background technique

Polymerase chain reaction is a kind of for amplifying the Protocols in Molecular Biology for expanding specific DNA fragmentation, is a kind of The special DNA replication dna process of in vitro, the maximum feature of PCR are that micro DNA can be significantly increased.Nineteen eighty-three, the U.S. Mullis advances an idea first, has invented polymerase chain reaction, i.e. simple DNA amplification by it within 1985, it is meant that round pcr Real birth.2013 by now, PCR had developed to third-generation technology, i.e. the digital pcr technology of absolute quantitation.

In general, PCR (polymerase chain reaction) is that 95 DEG C of high temperature time variations become single-stranded in vitro using DNA, low temperature (is moved back Fire) (usually 55-60 DEG C) when primer in conjunction with the single-stranded principle by base pair complementarity, then temperature regulating to archaeal dna polymerase most Suitable reaction temperature (72 DEG C or so), archaeal dna polymerase are mutual by Material synthesis of dNTP along the direction of phosphoric acid to pentose (5'-3') Mend chain.Practical PCR instrument based on polymerase manufacture is exactly a temperature control device, can be in denaturation temperature, renaturation temperature, elongating temperature Between carry out conversion and control well.As long as Taq enzyme can be in gradient of temperature in fact, having enough dNTP and annealing primer Make primer extend in the process.If only the sequence of 35~100bp of synthesis or so, the claimed range of primer annealing temperature can be high In or be lower than 5~10 DEG C of theoretical temperatures, guarantee specificity precursor under, merge annealing and elongating temperature, can with dual temperature PCR Than can more improve reaction speed with stringent three temperature transition.

PCR be current molecular diagnosis field it is most mature be also most widely used technology, from technological layer, state at present Interior round pcr has basically reached international most advanced level, such as the round pcr based on semiconductor refrigerating.But the clinic of PCR at home Still there is very large space using the promotion of detection limit, industry is interior, and there is also a large amount of resource consolidation chances.Currently, most often applied The universal working efficiency of quantitative fluorescent PCR product is not high, and usual process is the PCR of complicated nucleic acid extraction process, heating and cooling repeatedly Process, fluorescence detection program, usually single testing process needs 5 more than hour.Full Automatic Liquid body running based on paramagnetic particle method It stands and is gradually taken seriously in recent years, but its instrument and mating consumptive material are at high price, it is difficult to adapt to domestic PC R molecular diagnosis market.

Summary of the invention

The object of the present invention is to provide a kind of method and system of quickly detection sample of nucleic acid, pass through PCR reactant It is the heating and cooling process repeatedly of liquid or drop circulated instead of common in-situ temperature module, realizes efficient, quick, low Valence PCR, and the device of the quick detection sample of nucleic acid to match with set PCR mode is provided.

In order to achieve the above object, major technique solution of the invention is to provide a kind of side of quickly detection sample of nucleic acid Method, comprising the following steps: S1: sample extraction is to be detected nucleic acid-templated, and PCR reaction system is made；S2:PCR amplification；S3:PCR Product detection, which is characterized in that the S2:PCR amplification includes the following steps:

S21: it drives the PCR reaction system to flow through start-up temperature area by actuator, completes the thermal starting of archaeal dna polymerase With nucleic acid-templated denaturation to be detected；

S22: the actuator continues that the PCR reaction system is driven to circulate in denaturation temperature area and annealing temperature area Between expanded；

S23: after amplification, the actuator continues that the PCR reaction system is driven to flow to PCR product detection zone.

Transition region is equipped between the denaturation temperature area and annealing temperature area, the PCR reaction system is successively by denaturation Humidity province, transition region and annealing temperature area complete a PCR cycle, and the step S22 includes 1-100 PCR cycle.

Invention additionally discloses a kind of systems of method for implementing above-mentioned quick detection sample of nucleic acid, including sample extraction to fill It sets, PCR amplification device and PCR product detection device, which is characterized in that the PCR amplification device includes PCR reaction system chip With linear PCR instrument, the PCR reaction system chip is liquid flow chip, and the liquid flow chip includes the first chip body, institute The first chip body is stated equipped with the first winding shape circulatory flow, first winding shape circulatory flow one end is sequentially communicated with the One winding shape starting runner, the first preparation runner and first sample hole, the first winding shape circulatory flow other end are communicated with First collects runner, and the first collection runner is connected with colloidal gold strip, and the linear PCR instrument is single channel PCR instrument, Including temperature components, at least two temperature controllers and detection components, the temperature components include the upper heating group being oppositely arranged up and down Part and lower heating component, and there are the first of accommodating PCR reaction system chip between the upper heating component and lower heating component Gap, the upper heating component and lower heating component include the first heating module and the second heating module arranged side by side, institute Two there are the second gap, in the upper heating component and lower heating component are stated between the first heating module and the second heating module A first heating module is connect with a temperature controller, two the second heating modules in the upper heating component and lower heating component It is connect with another temperature controller, the detection components include the first heating module and the second heating module of the upper heating component Between the fluorescence detection device and/or Scan device that are equipped with, on first heating module and the second heating module respectively Through a drive hole is equipped with, and two drive holes are respectively connected with an air pump as actuator, the PCR product detection device Instrument is read including fluorescence detection device, test strip and drop.

The PCR reaction system chip is drop stream chip comprising the second chip body, on second chip body Equipped with the second winding shape circulatory flow, second winding shape circulatory flow one end is sequentially communicated with the second winding shape starting stream Road, the second preparation runner and the second sample aperture, the second preparation runner are communicated with third sample aperture, and the second winding shape is followed The circulation road other end is communicated with detection channel, and the detection channel is connected with the second collection runner.

The linear PCR instrument is the multichannel PCR instrument comprising controller, turntable, at least two temperature controllers, multiple Temperature components and drop read instrument, and turntable includes fixing seat and the rotating member that is articulated in fixing seat, and multiple temperature components are fixed On rotating member, drop reads that instrument is fixed on the fixing seat, the first heating module in the multiple temperature components with One temperature controller connection, the fixing seat are equipped with the PCR reaction system chip accommodated in the temperature components for will test The touch push rod of release, the second heating module in the multiple temperature components are connect with another temperature controller, the touch Push rod, temperature controller, drop read instrument and connect with controller.

The sample extraction device includes that the automatic nucleic acid of sample in sample process chip and extraction sample process chip mentions Instrument is taken, the sample process chip includes the substrate equipped with hole, is equipped on the substrate around hole and is sequentially connected to counterclockwise The first arc chambers, the second arc chambers, vertical run, nucleic acid absorption membrane cavity room, lysate chamber and first washing sap cavity Room, nucleic acid absorption membrane cavity room are communicated with eluent chamber, the nucleic acid absorption membrane cavity room other side close to the side of hole It is communicated with template storage chamber, the lysate chamber upper opening, for adding sample to be detected, the lysate chamber, First cleaning solution chamber and eluent chamber are equipped with opening and are respectively equipped with lysate, the first cleaning solution and eluent, described Between nucleic acid absorption membrane cavity room and lysate chamber, between lysate chamber between the first cleaning solution chamber, the nucleic acid inhale Between membrane chamber and eluent chamber and one is respectively connected between nucleic acid absorption membrane cavity room and template storage chamber to fill out Wax pan, nucleic acid absorption membrane cavity room be equipped with nucleic acid absorption film, it is described fill out in wax pan be equipped with heating meltable solid material and Notch is equipped with cover plate.

Between the lysate chamber and the first cleaning solution chamber be equipped with combine sap cavity room, in conjunction with sap cavity room be equipped with opening and Equipped with liquid is combined, one is equipped between the lysate chamber and combination sap cavity room and fills out wax pan.

The first cleaning solution chamber is sequentially communicated with the second cleaning solution chamber, and the first cleaning solution chamber and second are washed It washs and fills out wax pan equipped with one between sap cavity room；Or the first cleaning solution chamber is sequentially communicated with the second cleaning solution chamber and third Cleaning solution chamber, between the first cleaning solution chamber and the second cleaning solution chamber and the second cleaning solution chamber and third washing One, which is equipped with, between sap cavity room fills out wax pan；Or the first cleaning solution chamber is sequentially communicated with the second cleaning solution chamber, third Cleaning solution chamber and the 4th cleaning solution chamber, between the first cleaning solution chamber and the second cleaning solution chamber and second washs Between sap cavity room and third cleaning solution chamber and one, which is equipped with, between third cleaning solution chamber and the 4th cleaning solution chamber fills out wax Slot.

The automatic instrument for extracting nucleic acid includes pedestal, the central axis that can cooperate with substrate hole being connected on pedestal and drive The dynamical system of center axis rotation is moved, corresponding each fill out at the position of wax pan is equipped with an adding thermal resistance on the pedestal, described Dynamical system and adding thermal resistance are connect with the controller, and the pedestal is equipped with for matching simultaneously fixed form storage chamber The fixing groove in room direction.

The single channel PCR instrument includes temperature components, three temperature controllers and detection components, and the temperature components include up and down The upper heating component and lower heating component being oppositely arranged, and there are accommodating PCR between the upper heating component and lower heating component First gap of reaction system chip, the upper heating component and lower heating component include the first heated mould arranged side by side Block, third heating module and the second heating module, there are third space between first heating module, third heating module, There are the 4th gap between the third heating module and the second heating module, in the upper heating component and lower heating component Two the first heating modules are connect with a temperature controller, two the second heated moulds in the upper heating component and lower heating component Block is connect with second temperature controller, two third heating modules in the upper heating component and lower heating component and third temperature Instrument connection is controlled, the detection components include the third space or fluorescence detection device and/or band that the 4th gap is equipped with Scanning means is extended through equipped with a drive hole, and two drive holes on first heating module and the second heating module An air pump is respectively connected with as actuator, the PCR product detection device includes fluorescence detection device, test strip and liquid Drop reads instrument.

The beneficial effects of the present invention are:

1) Standard PCR or digital pcr can be used in the present invention, realizes fabulous compatibility.

2) present invention successfully realizes the full-automatic process from sample to data in a manner of easy, while can be with single channel With multichannel unrestricted choice, suitable for the various application scenarios based on molecular diagnosis；

3) sample nucleic acid of the invention automation extraction element and the assembly easy to accomplish of linear PCR detection device, can also divide Other independent operating, and various situations can accomplish to greatly improve the detection efficiency of molecular diagnosis with to inspection；

4) present invention only completes function by the design simply and readily realized, significantly reduces the production of instrument consumptive material Processing and testing cost；By liquid/drop stream instead of the Circularly liftable temperature process in situ of Standard PCR, highly shortened The time of single detection.

Detailed description of the invention

Fig. 1 is the embodiment of the flow diagram and each step of scheme of the present invention；

Fig. 2 is the structural schematic diagram of the preferred solution of the invention sample process chip；

Fig. 3 is the structural schematic diagram of the preferred solution of the invention automatic nucleic acid extraction apparatus；

Fig. 4 is the structural schematic diagram of the preferred solution of the invention single channel linear PCR instrument, and top is divided into front view, lower part It is divided into lateral plan；

Fig. 5 is the structural schematic diagram of the preferred solution of the invention liquid flow chip；

Fig. 6 is the structural schematic diagram of the preferred solution of the invention drop stream chip；

Fig. 7 is the structural schematic diagram of the preferred solution of the invention multichannel linear PCR instrument；

Fig. 8 is the structural schematic diagram of the full-automatic drop digital pcr detection system of the preferred solution of the invention；

Fig. 9 is the preferred solution of the invention liquid flow chip+inspection of the single channel linear PCR detection system to CYP2C9 gene Test paper slip result；

Figure 10 is detection Capillary Electrophoresis result of the conventional scheme to CYP2C9 gene；

Figure 11 is that the preferred solution of the invention liquid flow chip+single channel linear PCR detection system tries the detection of HPV viruse Paper slip result；

Figure 12 is that conventional scheme detects Capillary Electrophoresis result to HPV viruse；

Figure 13 is the preferred solution of the invention drop stream chip+single channel linear PCR instrument+drop fluorescence reading device to HBV The result of Viral Quantification detection；

Figure 14 is fluorogenic quantitative detection result of the general measure kit to HBV virus；

Figure 15 is the preferred solution of the invention drop stream chip+single channel linear PCR instrument+drop fluorescence reading device to low frequency The result of the detection of Kras gene mutation；

Figure 16 is result of the conventional fluorescent quantitative approach to the detection of low frequency K ras gene mutation；

Figure 17 is the preferred solution of the invention automatic nucleic acid extraction apparatus+multichannel linear PCR instrument to EGFR micro in ctDNA The testing result of gene mutation.

Specific embodiment

Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on Embodiment in the present invention, those of ordinary skill in the art's every other embodiment obtained belong to what the present invention protected Range.

It will be understood by those skilled in the art that in exposure of the invention, term " longitudinal direction ", " transverse direction ", "upper", The orientation of the instructions such as "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom" "inner", "outside" or position are closed System is to be based on the orientation or positional relationship shown in the drawings, and is merely for convenience of description of the present invention and simplification of the description, without referring to Show or imply that signified device or element must have a particular orientation, be constructed and operated in a specific orientation, therefore above-mentioned art Language is not considered as limiting the invention.

It is understood that term " one " is interpreted as " at least one " or " one or more ", i.e., in one embodiment, The quantity of one element can be one, and in a further embodiment, the quantity of the element can be it is multiple, term " one " is no It can be interpreted as the limitation to quantity.

As shown in Figure 1, the present embodiment is described the purpose of the present invention is to above-mentioned deficiency in the prior art, mention For a kind of linear PCR mode with 2 fixed temperatures, common in-situ temperature is replaced by liquid or circulating for drop The heating and cooling process repeatedly of module realizes a kind of efficient, quick, low price PCR mode, and offer and set PCR mode phase The nucleic acid extraction of the PCR consumptive material, upstream matched and the product detection method (shown in attached drawing 1) in downstream.The present invention also provides a set of excellent The linear PCR mode of the outfit spiral clamping plate of choosing and the corresponding two class cores for being directed to Standard PCR and drop formula digital pcr respectively Piece: liquid flow chip and drop stream chip, and it is preferred for the test strips of Standard PCR and for drop formula digital pcr The detection mode that drop fluorescence is read.Each adjacent component is designed as splicing collocation in system of the present invention, answers for difference Realize the linear PCR detection pattern from semi-automatic to full automatic according to actual needs with scene.

The method or apparatus of quick detection sample of nucleic acid of the present invention includes that sample extraction part, fixed temperature (can Adjust) the circulating PCR amplification part of liquid/drop and PCR product detection part.Successively carry out above three step can be completed from Sample to result complete detection process, wherein each step complete independently and can be sequentially completed manually the completion hand of next operation It is dynamic, or each section is smoothly connected by certain way and realizes full automation, or manual plus automation is realized by part linking Etc. modes all fall in scope of the invention as claimed.

Specifically, the sample extraction part be by certain way by sample DNA or RNA release and go Except interfering substances such as albumen therein and carbohydrates to smoothly complete amplification procedure in the case where polymerizeing enzyme effect.The sample is clinic Molecular Detection linked groups or effluent, such as blood, saliva, tissue, paraffin section, saliva, urine, excrement.The mode Comprising it is hands-free take, conventional kit manual extraction and automatic extracting instrument extract, wherein it is hands-free take, traditional extraction kit and business Change the not single range as the claims in the present invention of operations such as automatic extracting instrument, but with subsequent fixed temperature (adjustable) liquid/ The mode that PCR is completed in the combination of the circulating PCR amplification part of drop and PCR product detection part be included in the scope of the invention it It is interior.

Preferably, the sample extraction device in present system, to be connected up to full-automation with following amplification step Effect, special purpose or with subsequent combination mode within the scope of the present invention.The sample extraction device is by sample process Chip and turbine type fixed point circulation extraction apparatus (automatic instrument for extracting nucleic acid) composition, and complete automation according to specific program and extract Process.The sample process chip structure is as shown in attached drawing 2.

Specifically, the circulating PCR amplification part of fixed temperature (adjustable) liquid/drop passes through liquid/drop two It is circulated between a thermal module to complete the PCR mode of amplification procedure.The fixed temperature (adjustable) refers to for certain Specific reaction needs to fix the temperature between two modules, and the adjustable temperature for referring to each module is easy adjusting, thus Amplification instrument is set to adapt to the PCR reaction under all design conditions.Liquid/the drop is directed to two different PCR detections respectively Type, liquid are continuous morphology, and for being opposite regular-PCR type, and drop is discontinuous form, is opposite digital pcr class For type.

Preferably, in present system provide PCR amplification device, by design for regular-PCR liquid flow chip and It is a kind of to realize function for the drop stream chip of digital pcr and linear PCR instrument corresponding with two kinds of chips.The liquid flow For the structure of chip as shown in schematic diagram 5, PCR reaction system flows slowly into chip from left samples hole, flow to opening for top first Dynamic temperature area completes the thermal starting of enzyme and the denaturation of DNA profiling, then flows into formal PCR cycle area, race way is by top The transition region composition in denaturation temperature area, the annealing temperature area of lower section and centre, transition region prevent moving back for denaturation temperature area and lower section Temperature exchange between fiery humidity province.The driving force of liquid flowing is completed by the air pump of sample aperture by the pressurization of continuous-stable, By adjusting the flowing velocity of the strong and weak control liquid of pressure, reach most to adjust flowing time of the liquid in different temperatures area Good PCR condition.Liquid eventually flows to the PCR product detection zone on right side, and completion detection is contacted with test strips reaction zone.The drop The structure of chip is flowed as shown in schematic diagram 6, and PCR reaction system (water phase) flows slowly into chip, oily phase by left lower sample aperture Chip is then flowed slowly by left upper portion oil phase hole, water phase and oil, which interact, to be formed drop after mixing and successively flow into start-up temperature area And race way, complete drop PCR process.The generation of drop and flowing velocity are complete by the air pump for controlling sample aperture and oily phase hole respectively At, by adjust air-flow reach best PCR condition.Drop eventually flows to the drop collecting region on right side and completes to detect, the liquid Drop collecting region can be in runner at a distance, be also possible to a closed connected space, the difference of the two is drop Position and difference of arranging.The structure of the linear PCR instrument is as shown in schematic diagram 4, mainly by upper and lower two different temperatures moulds Block is constituted, and the upper part design of two thermal modules has two pieces and the duplicate thermal module in lower part, has one between two parts A gap, just for filling the liquid flow chip or drop stream chip, there are two gas for one end upper design of thermal module The opening of pump, just on drop stream chip sample aperture and oily phase hole it is corresponding, designing right above the other end of temperature control module has The device read for the reading of drop fluorescence or fluorescent liquid.Particularly, for being detected using colloidal gold strip the case where, is read Device is taken then to should be strip sensor.The linear PCR instrument can be by single group thermal module structure composition single channel, can also be by more Group thermal module structure composition multichannel, as shown in schematic diagram 7, port number be 2 or more arbitrary integer, preferably 8 channels or 16 channels.Further, for multichannel the case where, detection device can be multiple, be also possible to one, preferably 1.It is more The evenly distributed formation of group thermal module is discoid, and disk can drive the multiple groups thermal module being fixed thereon to rotate counterclockwise.

Specifically, the PCR product detection device in the present invention is designed in drop memory block, amplified production memory block or examination Then the upper part in paper slip colour developing area transmits the result to display using specific detector for detecting the amplification of PCR Device realizes function, and the part is corresponding with the job description of the linear PCR instrument specific part.Workflow of the present invention and The optional scheme of each section is by attached illustrated in Figure 1.

According to the above-mentioned description for each section, the present invention provides the systems of quick detection sample of nucleic acid, can be directed to Different application scene and actually detected demand can specific aim elect and be easy apolegamy and complete specific function.According to different samples and Detection project, the present invention preferably two sets of solutions.

For simple tractable sample and qualitative class Molecular Detection project, preferred scheme takes+liquid flow core to be hands-free Piece+single channel linear PCR instrument+test strips detection.The detection program of the preferred embodiment one are as follows: add the good sample of simple process Enter in the well of liquid flow chip and mixed with the PCR Mix of optimization, chip is put into single channel linear PCR instrument, after starting Two parts thermal module is heated to set temperature and keeps constant, and liquid flows slowly into channel under air pump effect and forms liquid Stream, by the completion thermal starting of start-up temperature area and by the complete unwinding of template, liquid flow continues to race way and completes to exist liquid flow Denaturation temperature area and circulating for annealed zone complete PCR amplification, and the sample area that liquid flow continues to flow into test strips is developed the color, The variation of the sensor sensing test strips of detection zone simultaneously shows result by signal transimiison analysis and over the display.

For quantitative class Molecular Detection project, preferred embodiment is automation sample nucleic acid extraction+drop stream chip+multi-pass The linear PCR instrument in road+drop fluorescence reading device.The detection program of the preferred embodiment two are as follows: extract automation sample nucleic acid Instrument is integrated with multichannel linear PCR instrument assembly, and sample is added to lysate chamber and completes cracking, fills out wax in conjunction with sap cavity room downstream Adding thermal resistance starts and completes to circulate at slot, and the rotation of extraction apparatus dynamical system starting disc counter-clockwise will once be split in conjunction with liquid injection It solves sap cavity room and completes mixing；Lysate chamber downstream fills out adding thermal resistance at wax pan and starts and complete to circulate, extraction apparatus dynamical system Mixed liquor is once passed through nucleic acid absorption membrane cavity room completely and enters waste chamber by starting disc counter-clockwise rotation, and nucleic acid is integrated to On nucleic acid absorption film；First cleaning solution chamber downstream fills out adding thermal resistance at wax pan and starts and complete to circulate, extraction apparatus dynamical system Start disc counter-clockwise rotation once by the first cleaning solution by combining sap cavity room and lysate chamber and inhaling completely by nucleic acid Membrane chamber simultaneously enters waste chamber, completes to wash for the first time；Second cleaning solution chamber downstream is filled out adding thermal resistance at wax pan and is started And complete to circulate, extraction apparatus dynamical system starts disc counter-clockwise rotation and the second cleaning solution is once passed through the first washing sap cavity Room passes through nucleic acid absorption membrane cavity room in conjunction with sap cavity room and lysate chamber and completely and enters waste chamber, and completion is washed for the second time It washs；It fills out adding thermal resistance at wax pan on the outside of eluent chamber to start and complete to circulate, extraction apparatus dynamical system starts disc counter-clockwise Low speed rotation once injects eluent on nucleic acid absorption film, and then adding thermal resistance starting and complete is filled out at wax pan in vertical run distal end At circulation, extraction apparatus dynamical system starts disc counter-clockwise high speed rotation and once injects the solution after eluting on nucleic acid absorption film Template storage chamber, automation extraction apparatus continue the extraction operation that process as described above completes next sample.Completion mentions The sample process chip taken is moved to the position of drop stream chip, template storage chamber thereon just with fall in drop stream chip Right above sample aperture, push the piston of template storage chamber that will mix in the sample aperture of DNA profiling injection chip with PCR system. Drop stream chip is pushed into an amplification position of multichannel linear PCR instrument, and two air pumps push PCR anti-respectively according to specific speed System (water phase) and the oil-based system of prepackage is answered to flow slowly into chip, water phase and oil, which interact, to be formed drop after mixing and successively flow into Drop PCR process is completed in start-up temperature area and race way.When the turntable of multichannel linear PCR instrument is moved to drop detection area, Drop flows to the drop detection area of rear side just, and turntable stopping certain time completing to detect and will test result to be transmitted to computer It is realized with initial sample number unified corresponding.It is pushed further into down a piece of drop stream chip at this time, continues to complete next sample Detection realize automation scheme.All kinds of chips are displaced through that mechanical transmission is easy to accomplish, and the control of each step passes through Design specific software is easy to accomplish, and the dynamical system can be motor.

The structure and working principle of the automatic instrument for extracting nucleic acid of embodiment 1 and sample process chip

Automatic nucleic acid extraction scheme provided herein is a preferred embodiment of the present invention, wherein the knot of sample process chip Fig. 2 is shown in structure signal, and Fig. 3 is shown in the structural representation of automatic instrument for extracting nucleic acid.Sample process chip be it is discoid, center be with gear Hole 201 is scattered with 4 chambers centered on sawtooth hole 201, respectively lysate chamber 202, in conjunction with sap cavity room 203, The one the first cleaning solution chambers 204, the second cleaning solution chamber 205, the connection of lysate chamber 202 combines sap cavity room 203, in conjunction with liquid Chamber 203 is connected to the first cleaning solution chamber 204, and the first cleaning solution chamber 204 is connected to the second cleaning solution chamber 205, constitutes circular arc Shape arrangement.Wherein aperture above 202 volume of lysate chamber, for adding sample to be detected.It is connected to lysate chamber 202 The other end is nucleic acid absorption membrane cavity room 210.The also another channel being connected to nucleic acid absorption membrane cavity room 210, two chambers point Cloth has an eluent chamber 206 at two sections, by center-side, is template storage chamber 207 far from center-side, is located at entire disk Outside.One vertical run of design in the middle part of runner, connection are connected to template storage chamber 207 in nucleic acid absorption membrane cavity room 210 One and half arc chambers 208 are being connected to another half arc chambers 209, and the two is symmetrical on disk.Eluent chamber 206 outsides, vertical run distal end, 202 downstream of lysate chamber, in conjunction under 203 downstream of sap cavity room, the first cleaning solution chamber 204 Trip and 205 downstream of the second cleaning solution chamber, which are designed, fills out wax pan, and structure is by 211 signals.Not essential, the cleaning solution in conjunction with sap cavity room Chamber can be arranged multiple or one according to the difference of sample.

Instrument for extracting nucleic acid be it is discoid, be slightly larger than sample process chip, center is designed as the protrusion axle 301 with gear, can It is exactly matched with the center hole 201 of sample process chip, the central axis 301 of instrument for extracting nucleic acid matches completion rotation with dynamical system Turn.6 adding thermal resistances 302 are distributed on instrument for extracting nucleic acid, position can fill out wax pan 211 relatively on sample process chip just It answers, what Position Design one of the corresponding instrument for extracting nucleic acid of the template storage chamber 207 of sample process chip matched is used to fix The slot 303 in direction.With nucleic acid absorption film chamber there are also another channel, two chambers are distributed in two sections, lean on center-side There is an eluent chamber for placing eluent, be template storage chamber far from center-side, positioned at the outside of entire disk, uses In the nucleic acid-templated of placement purifying completion and it is easily accomplished automation transfer process.In nucleic acid absorption membrane cavity room and template storage chamber Room, which is connected in the middle part of runner, designs a vertical run, connects one and half arc chambers, then be connected to another half arc chambers, and two Half arc chambers symmetrically, keep the balance of entire disk and very big on disk for storing discarded solution, the two Waste reflux is prevented in degree.Wherein filling heats meltable solid material for example but is not limited to paraffin, and paraffin is in fire-bar Be easy to melt under part to get through corresponding chamber, solid paraffin be for prevent fluid exchange and control the liquid of each chamber according to Specific program circulate is in mixing to smoothly complete automatic nucleic acid extraction process.The turbine type fixed point circulates extraction apparatus and is For instrument for extracting nucleic acid, whole heat is completed with the sequence of centrifugation and harmony by the software selective laser sintering in controller.Automatically The working principle of nucleic acid extraction are as follows:

A. lysate chamber 202 is added in sample to be detected and completes cracking；

B. it combines 203 downstream of sap cavity room to fill out adding thermal resistance (302) at wax pan (211) to start and complete to circulate, nucleic acid extraction Instrument dynamical system, which starts disc counter-clockwise rotation, once will complete mixing in conjunction with liquid injection lysate chamber 202；

C. 202 downstream of lysate chamber fills out adding thermal resistance (302) at wax pan (211) and starts and complete to circulate, and extraction apparatus is dynamic Mixed liquor is once passed through nucleic acid absorption membrane cavity room 210 completely and enters waste chamber by Force system starting disc counter-clockwise rotation 208 and 209 (half arc chambers), nucleic acid are integrated on the nucleic acid absorption film in nucleic acid absorption membrane cavity room 210；

D. 204 downstream of the first cleaning solution chamber fills out adding thermal resistance (302) at wax pan (211) and starts and complete to circulate, and extracts Instrument dynamical system starts disc counter-clockwise rotation once by the first cleaning solution by combining sap cavity room 203 and lysate chamber 202 And pass through nucleic acid absorption membrane cavity room 210 completely and enter waste chamber 208 and 209, it completes to wash for the first time；

E. 205 downstream of the second cleaning solution chamber fills out adding thermal resistance (302) at wax pan (211) and starts and complete to circulate, and extracts Instrument dynamical system starts disc counter-clockwise rotation once by the second cleaning solution by the first cleaning solution chamber 204, in conjunction with sap cavity room 203 and lysate chamber 202, pass through nucleic acid absorption membrane cavity room 210 completely and entering waste chamber 208 and 209, completes second Secondary washing；

F. it fills out adding thermal resistance (302) at wax pan (211) on the outside of eluent chamber 206 to start and complete to circulate, extraction apparatus is dynamic Force system starts disc counter-clockwise low speed rotation and once injects eluent on nucleic acid absorption film；

G. vertical run distal end fills out adding thermal resistance (302) at wax pan (211) and starts and complete to circulate, extraction apparatus dynamical system Start disc counter-clockwise high speed rotation and the solution after eluting on nucleic acid absorption film is once injected into template storage chamber 207, completes Whole extraction process.

The structure and working principle of embodiment 2 single channel linear PCR instrument and liquid flow chip

Single channel liquid-flow linear PCR amplification scheme provided herein, wherein the structural representation of single channel linear PCR instrument is shown in Fig. 4, wherein top is plan view, lower section is side view；Fig. 5 is shown in the structural representation of liquid flow chip.Single channel linear PCR instrument It is made of in terms of planar structure upper and lower two different thermostat temperature module the first heating modules 403 and the second heating module 404, There is the second gap of a gap 406 therebetween, prevents temperature between thermal module from transmitting；Two thermal modules first in terms of side structure The design of the upper part of heating module 403 and the second heating module 404 has 2 pieces and the duplicate thermal module in lower part, two parts Between have a gap the first gap of the first gap 407, accommodate PCR reaction system chip.The first heating module of thermal module 403 The drive hole 401 and 402 there are two air pump is separately designed with one end top of the second heating module 404, is separately connected two gas Wherein drive hole 401 is for driving oily phase for pump (408 signal), and 402 for driving water phase, 408 corresponding temperature module of air pump it is another There are a fluorescence detection device and Scan device 405 in one end.Oily phase drive hole 401 is only in fluid,matching drip chip (figure 6) use when digital pcr is carried out, this preferred embodiment is to guarantee that platform, will be whole to the compatibility of Standard PCR and digital pcr Accessory is equipped on same linear PCR instrument.The aperture 501 for thering is a sample-adding and drive hole 402 to drive on the left of liquid flow chip, chip The upper a plurality of runner of design, including the first preparation runner 502, including the first winding shape starting runner 503 and the first winding shape circulation Runner 504, runner 505 is collected in design first after runner, connects colloidal gold strip 506.Liquid flow chip linear PCR test paper The working principle of item colour developing are as follows:

A. the first heating module of thermal module 403 and the second heating module of thermal module 404 are adjusted respectively to specific temperature, The air velocity for adjusting air pump 408 is appropriate, and chip first sample hole 501 is added in PCR reaction system to be amplified, and chip is whole Body completes insertion linear PCR instrument along the first gap of gap 407, and first sample hole 501 is just complete with water phase drive hole 402 at this time Coincide, the first winding shape starting runner 503 and the first winding 504 top half runner of shape circulatory flow just with two pieces of isothermal temperature Spend the first heating module of module 403 completely be bonded, the first winding 504 lower half portion runner of shape circulatory flow just with another two pieces The second heating module of isothermal temperature module 404 is bonded completely；

B. under the driving of air pump 408, water phase forms liquid flow in runner and slowly moves along the first preparation runner 502 Start runner 503 to the first winding shape, completes the thermal starting of enzyme and the denaturation of DNA profiling；

C. liquid flow continues to flow into the first winding shape circulatory flow 504, respectively by the denaturation temperature area of top, intermediate The annealing temperature area of short transition time area and lower section completes a PCR cycle, and liquid flows through several runners (usually 20-50) Complete entire PCR process；

D. the first collection runner 505 that liquid flow eventually flows to right side is completed in amplification, anti-with colloidal gold strip 506 It answers area to contact, completes process color, Scan device 405 detects that band changes in test strips 506, transfers signals to electricity Whole detection process is completed on sub-display.

In some embodiments, pcr needs three temperature, so the single channel PCR instrument includes temperature components, three temperature Control instrument and detection components, the temperature components include the upper heating component being oppositely arranged up and down and lower heating component, and it is described on There are the first gaps of accommodating PCR reaction system chip between heating component and lower heating component, and the upper heating component is under Heating component includes the first heating module arranged side by side, third heating module and the second heating module, first heating There are third spaces between module, third heating module, and there are the 4th between the third heating module and the second heating module Gap, third space and the 4th gap prevent string temperature, two the first heated moulds in the upper heating component and lower heating component Block is connect with a temperature controller, two the second heating modules and second temperature control in the upper heating component and lower heating component Instrument connects, and two third heating modules in the upper heating component and lower heating component are connect with third temperature controller, described Detection components include the third space or fluorescence detection device and/or Scan device that the 4th gap is equipped with, described It is extended through on first heating module and the second heating module equipped with a drive hole, and two drive holes are respectively connected with a gas Pump is used as actuator, and the PCR product detection device includes that fluorescence detection device, test strip and drop read instrument.

The structure and working principle of embodiment 3 single channel linear PCR instrument and drop stream chip

Single channel drop cleanliness PCR amplification scheme provided herein is one of present system preferred embodiment, Fig. 4 is shown in the structural representation of middle single channel linear PCR instrument, is described in detail in example 2；The structural representation of drop stream chip See Fig. 6.There are a water phase well i.e. the second sample aperture 601 and an oily phase hole i.e. third sample aperture on the left of drop stream chip 602, after two runners formation T-types cross, the winding shape starting runner 603 of runner connection second and the second winding shape circulatory flow 604, detection channel 605 and the second collection runner 606 are designed thereafter.The work that drop stream chip linear PCR drop is read is former Reason are as follows:

A. the first heating module of single channel linear PCR instrument (Fig. 4) thermal module 403 and the second heated mould of thermal module are adjusted For block 404 respectively to specific temperature, it is appropriate to water phase and oily phase air velocity respectively to adjust two air pumps at 408, will be to be amplified The second sample aperture of water phase well 601 of chip is added in PCR reaction system, will be used for the mineral oil (or silicone oil etc.) of drop formation The oily phase well third sample aperture 602 of chip is added, chip is whole along the gap first of single channel linear PCR instrument (Fig. 4) Insertion is completed in gap 407, and the second sample aperture of well 601 fits like a glove with water phase drive hole 402 just at this time, tank filler sleeve third Sample aperture 602 fits like a glove with oily phase drive hole 401 just, the second winding shape starting runner 603 and the second winding shape recycle stream 604 top half runner of road is bonded with two pieces of first heating modules of isothermal temperature module 403 completely just, the second winding shape circulation 604 lower half portion runner of runner is bonded with the second heating module of another two pieces of isothermal temperature modules 404 completely just；

B. at 408 under the driving of two air pumps, water phase and oil mutually slowly move along runner and cross to form drop stream, liquid Drip continues to run to the second winding shape starting runner 603, completes the thermal starting of enzyme and the denaturation of DNA profiling；

C. drop stream continues to flow into the second winding shape circulatory flow 604, respectively by the denaturation temperature area of top, intermediate The annealing temperature area of short transition time area and lower section completes a PCR cycle, and drop flows through several runners (usually 20-50) Complete the PCR process of whole drops；

D. drop stream eventually flows to the detection channel 605 on right side after the completion of expanding, and arrives and second collects in runner 606 Have when liquid (by the adjusting of the pressure to air pump, control the speed of drop, thus it is known after a certain period of time, drop stream To detection channel 605), two air pumps stop driving at 408, and drop reads the liquid in the quick Scanning Detction runner 605 of instrument 405 The fluorescence signal that drop expands is transferred to completion whole detection process on electronic console by drop after computer is analyzed.

The structure and working principle of 4 multichannel linear PCR instrument of embodiment

Multichannel linear PCR instrument provided herein is a preferred embodiment of the present invention, and Fig. 7 is shown in structural representation, according to The drop drive part of single channel linear PCR instrument (shown in Fig. 4, structure and working principle are described in detail in example 2) 401 and 402 and temperature-controlled portion the first heating module 403 and the second heating module 404 be a temperature unit, that is, temperature unit 703, multichannel linear PCR instrument is then by turntable 701 and multiple fixations and the temperature unit 703 that is evenly distributed on turntable 701 And drop reads instrument 704 and constitutes.The working principle of multichannel linear PCR instrument progress multisample detection are as follows: at temperature unit It is initial bit in 702 position, is stop bit when temperature unit is in drop to read the position of instrument 704, setting program instruction is When each temperature unit is located exactly at initial bit 702, turntable 701 stops certain time, in the manner described in Example 4 will at this time Add the drop stream chip insertion PCR reaction member of sample.During turntable 701 stops, the chip for being located exactly at stop bit 704 is completed PCR amplification reads instrument scanning one detection of drop detection area completion through the drop at 704, and touches push rod will test 704 The drop stream chip at place is released, the position revert to vacancy continue to rotate to initial bit 702 enter lower chip piece amplification and Program is detected, and so on, multichannel linear PCR instrument cycle operation can be completed multi-pass amount and accomplish with to inspection.Control The revolving speed of turntable 701 is exactly completed PCR process, drop is entirely located in when the chip into initial bit being made to turn to stop bit Detection channel 605 shown in drop stream chip (Fig. 6), second collection runner 606 can then regulate and control different temperatures unit there may be A small amount of error.

The structure and working principle of the full-automatic drop digital pcr detection system of embodiment 5

Full-automatic drop digital pcr system provided herein is a preferred embodiment of the present invention, and figure is shown in structural representation 8, mainly realized by the automatic instrument for extracting nucleic acid (Fig. 3) on right side and the multichannel linear PCR instrument (Fig. 7) in left side and by the two The component of mechanical connection is constituted.It realizes full automatic working principle are as follows:

A., sample to be detected is added to the lysate chamber 202 of sample process chip (Fig. 2), respectively scanned samples and sample Processing chip keeps the two corresponding.Then by sample process chip centre axis 811, all sample process chips are successively grasped Make, is successively stacked on central axis 811；

B. the sample to be tested processing chip of position 801 is fallen into 802 treatment region of position and completes automatic nucleic acid extraction, position Set the central axis 301 of the automatic instrument for extracting nucleic acid of 803 corresponding diagrams 3, the structure of automatic instrument for extracting nucleic acid and sample process chip Structure and working principle are described in detail in embodiment 1.

C. control rotates nucleic acid-templated area 805 (2 template storage chamber 207 of corresponding diagram) to the leftmost side after extracting, Second sample aperture 601 of the hole just with the drop stream chip 807 (Fig. 6 schematic construction) of lower section of drawing a design matches；

D. scanning device 806 carries out barcode scanning to sample process chip 802 and drop stream chip 807 simultaneously, so that the two is corresponding, Starting propels bar 804 and injects nucleic acid-templated (propelling bar is the piston rod in syringe-like) 805 in chip 807 simultaneously；

E. the chip handled is pushed into multichannel linear PCR instrument (Fig. 7 schematic construction) turntable 810 (turn of corresponding diagram 7 Disk 701) initial bit 702, the multichannel linear PCR instrument working principle being then described in detail according to embodiment 4 detected, directly To providing result；

F. the chip 802 detected is abandoned by the place of pushing away, and the sample process chip of 801 new positions is pushed at position 802 Reason area completes new round detection according to the above process, to successfully realize full-automatic detection, and accomplishes with to inspection.

Application of 6 liquid flow chip of the embodiment+single channel linear round pcr in terms of Drug Discovery (is used with warfarin For medicine CYP2C9 genetic test)

It using mouth epithelial cells as sample, quickly handles through one-step method and is uniformly mixed with PCRMix, which is based on north The full formula gold biological production in capitalAnimal Tissue PCR Kit (article No.: AD201) carry out sample at and Amplification, substantially process are as follows:

1) it takes the 80 μ l of AD1 in kit to mix with 20 μ l of AD2, is gently wiped with disinfecting cotton swab and take a small amount of oral epithelium thin 80 μ l of AD3 is added into system and is uniformly mixed spare, splits for what is handled well by born of the same parents, and being inserted into above-mentioned mixed liquor about 10 minutes Solve object.

2) it is searched online using NCBI and the primer for designing CYP2C9 gene is as follows:

Upstream primer: 5 '-CTGAATTGCTACAACAAATGTG-3 ' carry out test strips detection, 5 ' label lifes for cooperation Object element.

Downstream primer: 5 '-GATACTATGAATTTGGGACTTC-3 ' carry out test strips detection, 5 ' label ground for cooperation Gaoxin.

3) preparation of PCR amplification system is carried out according to following dosage:

After system is mixed, it is divided into two groups of A and B, every group of 40 μ l.

4) wherein liquid flow chip is added in A group, is based on liquid flow chip of the present invention+single channel linear PCR instrument+examination The method of paper slip is expanded and is detected, and the principle of this method and substantially process are described in detail referring to the embodiment of the present invention 2.B group is then It is put into Standard PCR instrument, carries out product detection according to regular-PCR program and using 2100 bioanalyzer of agilent.Two kinds Method completes PCR amplification according to following temperature program:

94℃ 5min

94℃ 20s

58℃ 20s(35cycles)

5) testing result of A group method is as shown in attached drawing 9, and the testing result of B group method is as shown in attached drawing 10.As a result table Bright, the integral method based on the detection of liquid flow chip of the invention, linear PCR mode and test strips is for CYP2C9 gene Detection and conventional method indifference, it was demonstrated that effectiveness of the invention.

The testing principle (existing technology) of the test strips are as follows: expand in the system of completion and be marked with respectively comprising both ends The target amplification product and free upstream and downstream primer of biotin and digoxin, system is first in the combined area of test strips and embedding Colloidal gold mixing with marked by streptavidin, the biotin end of target amplification product and the Streptavidin terminal specific of colloidal gold Property combine, system continuously moves to detection line area (T line), the target product other end digoxin of association colloid gold and T line area mark The anti digoxin antibody of note is specifically bound, and is formed colloidal gold and is detected band, the remaining colloidal gold with marked by streptavidin after Continuous to be moved to line of reference area (C line), the Streptavidin for the colloidal gold that dissociates and the biotin of C line area label are specifically bound, shape At colloidal gold internal reference band.

7 liquid flow chip of embodiment+single channel linear round pcr is in cervical carcinoma screening using (with high-risk 16 type HPV For genetic test)

Using the patient's Cervical scrapes for making a definite diagnosis 16 type HPV infections as sample, quickly handles through one-step method and mixed with PCR Mix Uniformly, the process, detection method, comparison scheme and embodiment 6 described in complete unanimously, difference be using primer not Together, it is searched online using NCBI and the primer for designing 16 type HPV viruse specific regions is as follows:

Upstream primer: 5 '-CGTAACCGAAATCGGTTGAAC-3 ' carry out test strips detection, 5 ' label lifes for cooperation Object element.

Downstream primer: 5 '-CAGCCTCTACATAAAACCATC-3 ' carry out test strips detection, 5 ' label ground for cooperation Gaoxin.

The testing result of basic the method for the invention is as shown in attached drawing 11, and the testing result of conventional calibration method is by attached drawing Shown in 12.The result shows that the integral method pair based on the detection of liquid flow chip of the invention, linear PCR mode and test strips Detection and conventional method indifference in 16 type HPV viruses, it was demonstrated that effectiveness of the invention.

8 drop stream chip of embodiment+single channel linear round pcr is in pathogen quantitative detection using (with HBV gene For quantitative detection)

To make a definite diagnosis the blood samples of patients for carrying HBV virus as sample, it is utilized respectively the QIAamp DSP of Qiagen company production (article No.: 61704) carrying out viral nucleic acid extraction to Virus Spin Kit, then cooperates single-pass using drop stream chip of the invention The linear PCR instrument in road and drop fluorescence reading device carry out HBV viral copy number quantitative detection.The method of comparison is to utilize Hunan sage The hbv nucleic acid immue quantitative detection reagent box (state's tool (standard) word 2013 the 3401888th) of Hunan company production carries out HBV Then viral copy number quantitative detection compares the result of two methods.Utilize QIAamp DSP Virus Spin Kit The substantially process of extracts kit combination the method for the invention are as follows:

1) 200 μ l serum samples are taken, 25 μ l QIAGEN Protease, 200 μ l Buffer AL, 6 μ l are sequentially added Carrier RNA is vortexed after mixing and places 56 DEG C of incubations 15 minutes；

2) 250 μ l dehydrated alcohols are added, is vortexed after mixing and is placed at room temperature for 5 minutes；

3) mixture is added on centrifugal column, 6000g is centrifuged 1 minute on centrifuge, abandons lower layer's solution；

4) 500 μ l Buffer AW1 are added on centrifugal column, 6000g is centrifuged 1 minute on centrifuge, abandons lower layer's solution；

5) 500 μ l Buffer AW2 are added on centrifugal column, 6000g is centrifuged 1 minute on centrifuge, abandons lower layer's solution；

6) 500 μ l dehydrated alcohols are added on centrifugal column, 6000g is centrifuged 1 minute on centrifuge, abandons lower layer's solution；

7) continue to dally 3 minutes in 20000g, centrifugal column is 3 minutes dry as 56 DEG C；

8) 50 μ l Buffer AVE are added on centrifugal column, are placed at room temperature for 5 minutes, 20000g is centrifuged 1 point on centrifuge Clock collects lower layer's solution for standby, is DNA profiling to be checked；

9) it is searched online using NCBI and the primer/probe for designing the virus-specific region HBV is as follows:

Upstream primer: 5 '-ATGTGTCTGCGGCGTTTTATC-3 '

Downstream primer: 5 '-ACAMACGGGCAACATACCTT-3 '

Probe: FAM-CATCCTGCTGCTATGCCTCATCTTCT-HBQ1

10) QuantStudio of Thermo company production is utilized^TM3D Digital PCR Master Mix (article No. A26358 digital pcr) is carried out, the DNA profiling that step 8 is obtained dilutes 1000 times, and carries out PCR amplification body according to following dosage The preparation of system:

11) the water phase hole of drop stream chip is added after the completion of PCR system mixing, and 20 μ l mineral is added into oily phase hole Oil is then based on drop stream chip of the present invention+single channel linear PCR instrument+drop fluorescence reading device method and is expanded Increase and detect, the principle of this method and substantially process are described in detail referring to the embodiment of the present invention 3.And it is completed according to following temperature program PCR amplification:

Its testing result is as shown in attached drawing 13.

Utilize the substantially process of holy Hunan hbv nucleic acid immue quantitative detection reagent box method are as follows:

1) qualitative reference product A-D and each 200 μ l of sample to be examined are taken respectively, are not had pipe that 100 μ l DNA are added and are extracted solution 2, shake It is stored at room temperature 10 minutes after swinging mixing 15 seconds；

2) after brief centrifugation, centrifuge tube is placed on magnetic separator, slowly absorbs liquid after 3 minutes；

3) every pipe, which is added 600 μ l DNA and extracts the μ l DNA of solution 3 and 200, extracts solution 4, and concussion mixes 5 seconds, instantaneously from Centrifuge tube is placed on magnetic separator after the heart, is slowly absorbed liquid after 3 minutes clean；

4) do not have pipe that 50 μ l PCRMix (+1 μ l enzyme mixation of 49 μ l PCR reaction solution) is added, by brown residue on tube wall It is resuspended completely, it is spare that liquid is transferred to PCR pipe；

5) amplification system is transferred to ABI 7500 and carries out fluorogenic quantitative detection, and complete PCR according to following temperature program Amplification:

Its testing result is as shown in attached drawing 14.

Compare two ways detection, the results showed that based on the method for the present invention it is quantitative HBV copy Particle density be 1.94 × 10⁵Copies/ μ l is 1.97 × 10 based on the quantitative HBV copy Particle density of holy Hunan kit method⁵Copies/ μ l, the two base This is consistent, shows the validity of drop stream chip/linear PCR method of the present invention.

Application of 9 drop stream chip of the embodiment+single channel linear round pcr in specific position tumor screening is (with excrement Just in sample for micro Kras detection in Gene Mutation)

Using excrement as sample, the detection wherein micro Kras mutation of human archeocyte.First with the production of Qiagen company QIAamp Fast DNA Stool Mini Kit (nucleic acid extraction, substantially process 51604) article No.: are carried out to excrement are as follows:

1) fecal sample about 200mg is taken, 1ml InhibitEX Buffer is added, lasting be vortexed is shaken 1 minute, and in 16000g is centrifuged 1 minute；

2) 25 μ l Proteinase K Solutions are added in 600 μ l supernatant of gentle aspiration, and 600 μ l Buffer AL are added, and are vortexed and mix 70 DEG C are placed on to be incubated for 10 minutes；

3) 600 μ l dehydrated alcohols are added, is vortexed and mixes, divides 3 times and crosses centrifugal column, in the case where 16000g is centrifuged 1 minute and is removed Layer solution；

4) 500 μ l Buffer AW1 are added, be centrifuged 1 minute in 16000g and remove lower layer's solution；

5) 500 μ l Buffer AW2 are added, is centrifuged 1 minute and removes lower layer's solution in 16000g, continue in 16000g Sky is fallen 3 minutes；

6) 200 μ l Buffer ATE are added, are centrifuged 1 minute in 16000g, collection lower layer's eluent is spare, is DNA mould Plate；

7) it is searched online using NCBI and the primer/probe for designing mankind's Kras gene G12C site mutation is as follows:

Upstream primer: 5 '-AAATGACTGAATATAAACTTGTGGT-3 '

Downstream primer: 5 '-CTCTATTGTTGGATCATATTCGTC-3 '

Probe: FAM-AGTTGGAGCTTGTGGCGTAGGCAAG-HBQ1

8) it is searched online using NCBI and designs GAPDH gene specific section as internal reference, primer/probe is as follows:

Upstream primer: 5 '-GCTGCTTTTAACTCTGGTAAAGTG-3 '

Downstream primer: 5 '-TAGCACTCACCATGTAGTTGAG-3 '

Probe: VIC-TGATGCATCTATGAACGCTTC-HBQ1

9) QuantStudio of Thermo company production is utilized^TM3D Digital PCR Master Mix (article No. A26358 digital pcr) is carried out, and carries out the preparation of PCR amplification system according to following dosage:

After system is mixed, it is divided into two groups of A and B, every group of 20 μ l.

10) wherein A group takes 20 μ l PCR systems that the water phase hole of drop stream chip is added, and 20 μ l mines are added into oily phase hole Object oil is then based on the progress of drop stream chip of the present invention+single channel linear PCR instrument+drop fluorescence reading device method Amplification and detection, the principle of this method and substantially process are described in detail referring to the embodiment of the present invention 3.B group be then put into ABI 7500 into Row fluorogenic quantitative detection.Two methods complete PCR amplification according to following temperature program:

The testing result of A group method is as shown in attached drawing 15, and the testing result of B group method is as shown in attached drawing 16.The result shows that Integral method based on the detection of liquid flow chip of the invention, linear PCR mode and test strips is prominent for Kras gene G12C Displacement point detects the low frequency mutation of 1.25copies/ μ l, and Kras mutation is not detected in conventional fluorescent quantitative PCR, it was demonstrated that this The method sensitivity of invention is higher.

10 multichannel automatic nucleic acid extraction apparatus of embodiment+multichannel linear PCR technology is in circulating tumor Application of the DNA target into medication guide (by taking EGFR gene rare mutation detection in blood as an example)

The blood sample for choosing 10 operation of lung cancer patients detects the catastrophe in the site its EGFR gene T790M, is target Medication guide is done to medication Afatinib.

Automatic nucleic acid extraction is carried out according to method for extracting nucleic acid described in embodiment 1, sample 1 to sample 10 respectively takes Corresponding sample process chip is added in 1-20 minutes and is put on automatic nucleic acid extraction apparatus at random, at random until work by 1ml After obtain 10 parts of DNA profilings, ABI company production Qubit 3.0 on to each group template carry out concentration mensuration, as a result As shown in the table.

Extract gained DNA sample concentration measurement

It is searched online using NCBI and the primer/probe for designing human EGFR gene T790M site mutation is as follows:

Upstream primer: 5 '-ATGCGAAGCCACACTGACG-3 '

Downstream primer: 5 '-CCAGGAGGCAGCCGAAG-3 '

Probe: FAM-GCAGCTCATCATGCAGCTCATGCCCTTCG-HBQ1

Reference gene, which remains unchanged, uses GAPDH gene, described in primer/tip reference embodiment of the present invention 9.Utilize Thermo The QuantStudio of company's production^TM3D Digital PCR MasterMix (article No. A26358) progress digital pcr, and according to Following dosage carries out the preparation of PCR amplification system:

Continue the detection that 10 parts of samples are carried out according to the detailed operation method of multichannel linear PCR instrument described in embodiment 4, point Amplification reaction system is not added in corresponding drop stream chip, is put into multichannel linear PCR instrument at random, until testing result It is fully completed.It is compared as shown in the table with the result for being initially based on high-flux sequence.

T790M catastrophe result judgement and with initial results compare

The testing result to 10 samples of multichannel linear PCR instrument is anticipated as shown in Figure 17, and the result of statistical analysis is such as Shown in upper table.The present embodiment is successfully completed based on automatic nucleic acid extraction apparatus of the invention and multichannel linear PCR instrument to 10 The automatic detection of sample has very strong convenience and reliability.Its result detected and the result of high-flux sequence are complete Unanimously, the science for further illustrating the method for the invention, based on several preferred embodiments of the invention its advantage of energy system.

The above description of disclosed embodiment of this invention makes those skilled in the art can be realized or use the present invention, Meanwhile the foregoing is merely a prefered embodiment of the invention, is not intended to limit the invention, all spirit and original in the embodiment of the present invention Within then, any modification, equivalent replacement, improvement and so on be should be included within the protection scope of the embodiment of the present invention.This Invention is not limited to above-mentioned preferred forms, anyone can obtain other various forms of productions under the inspiration of the present invention Product, however, make any variation in its shape or structure, it is all with technical solution identical or similar to the present application, It falls within the scope and spirit of the invention.

Claims

1. a kind of method of quickly detection sample of nucleic acid, comprising the following steps: S1: sample extraction is to be detected nucleic acid-templated, is made PCR reaction system；S2:PCR amplification；S3:PCR product detection, which is characterized in that the S2:PCR amplification includes the following steps:

S21: driving the PCR reaction system to flow through start-up temperature area by actuator, complete archaeal dna polymerase thermal starting and to Detect nucleic acid-templated denaturation；

S22: the actuator continues that the PCR reaction system is driven to circulate between denaturation temperature area and annealing temperature area It is expanded；

2. the method for quick detection sample of nucleic acid according to claim 1, it is characterised in that the denaturation temperature area and move back Transition region is equipped between fiery humidity province, it is complete that the PCR reaction system successively passes through denaturation temperature area, transition region and annealing temperature area At a PCR cycle, the step S22 includes 1-100 PCR cycle.

3. a kind of system for the method for implementing quickly to detect sample of nucleic acid described in claim 1, including sample extraction device, PCR Amplification device and PCR product detection device, which is characterized in that the PCR amplification device includes PCR reaction system chip and linear PCR instrument, the PCR reaction system chip be liquid flow chip, the liquid flow chip include the first chip body, described first Chip body is equipped with the first winding shape circulatory flow, and it is winding that first winding shape circulatory flow one end is sequentially communicated with first Shape starting runner, the first preparation runner and first sample hole, the first winding shape circulatory flow other end are communicated with the first receipts Mass flow pathway, the first collection runner are connected with colloidal gold strip, and the linear PCR instrument is single channel PCR instrument comprising temperature Spend component, at least two temperature controllers and detection components, the temperature components include the upper heating component being oppositely arranged up and down and under Heating component, and there are the first gap of accommodating PCR reaction system chip, institutes between the upper heating component and lower heating component It states heating component and lower heating component includes the first heating module and the second heating module arranged side by side, described first adds There are the second gap between thermal modules and the second heating module, two first in the upper heating component and lower heating component plus Thermal modules are connect with a temperature controller, two the second heating modules in the upper heating component and lower heating component and another Temperature controller connection, the detection components include the upper heating component the first heating module and the second heating module between be equipped with Fluorescence detection device and/or Scan device, extend through on first heating module and the second heating module and be equipped with One drive hole, and two drive holes are respectively connected with an air pump as actuator, the PCR product detection device includes fluorescence Detection device, test strip and drop read instrument.

4. the system of the method according to claim 3 for implementing quickly to detect sample of nucleic acid described in claim 1, feature It is that the PCR reaction system chip is drop stream chip comprising the second chip body, second chip body are equipped with Second winding shape circulatory flow, second winding shape circulatory flow one end are sequentially communicated with the second winding shape starting runner, the Two preparation runners and the second sample aperture, the second preparation runner are communicated with third sample aperture, the second winding shape recycle stream The road other end is communicated with detection channel, and the detection channel is connected with the second collection runner.

5. the system of the method according to claim 3 for implementing quickly to detect sample of nucleic acid described in claim 1, feature It is that the linear PCR instrument is the multichannel PCR instrument comprising controller, turntable, at least two temperature controllers, multiple temperature Component and drop read instrument, and turntable includes fixing seat and the rotating member that is articulated in fixing seat, and multiple temperature components, which are fixed on, to be turned On moving part, drop reads that instrument is fixed on the fixing seat, the first heating module in the multiple temperature components with one Temperature controller connection, the fixing seat are equipped with the PCR reaction system chip accommodated in the temperature components for will test and release Touch push rod, the second heating module in the multiple temperature components connect with another temperature controller, the touch push rod, Temperature controller, drop read instrument and connect with controller.

6. the system of the method according to claim 3 for implementing quickly to detect sample of nucleic acid described in claim 1, feature It is that the sample extraction device includes the automatic instrument for extracting nucleic acid of sample in sample process chip and extraction sample process chip, The sample process chip includes the substrate equipped with hole, surrounds hole on the substrate and is equipped with first be sequentially connected to counterclockwise Arc chambers, the second arc chambers, vertical run, nucleic acid absorption membrane cavity room, lysate chamber and the first cleaning solution chamber, it is described Nucleic acid absorption membrane cavity room is communicated with eluent chamber close to the side of hole, and the nucleic acid absorption membrane cavity room other side is communicated with mould Plate storage chamber, the lysate chamber upper opening, for adding sample to be detected, the lysate chamber, the first washing Sap cavity room and eluent chamber are equipped with opening and are respectively equipped with lysate, the first cleaning solution and eluent, the nucleic acid absorption Between membrane cavity room and lysate chamber, between lysate chamber between the first cleaning solution chamber, nucleic acid absorption membrane cavity room One, which is respectively connected with, between eluent chamber and between nucleic acid absorption membrane cavity room and template storage chamber fills out wax pan, it is described Nucleic acid absorption membrane cavity room be equipped with nucleic acid absorption film, it is described fill out in wax pan be equipped with heating meltable solid material and notch be equipped with Cover plate.

7. the system of the method according to claim 6 for implementing quickly to detect sample of nucleic acid described in claim 1, feature It is to be equipped between the lysate chamber and the first cleaning solution chamber and combines sap cavity room, be equipped with opening in conjunction with sap cavity room and is equipped with In conjunction with liquid, one is equipped between the lysate chamber and combination sap cavity room and fills out wax pan.

8. the system of the method according to claim 6 for implementing quickly to detect sample of nucleic acid described in claim 1, feature It is that the first cleaning solution chamber is sequentially communicated with the second cleaning solution chamber, the first cleaning solution chamber and the second cleaning solution It is equipped with one between chamber and fills out wax pan；Or the first cleaning solution chamber is sequentially communicated with the second cleaning solution chamber and third washing Sap cavity room, between the first cleaning solution chamber and the second cleaning solution chamber and the second cleaning solution chamber and third wash sap cavity It is equipped with one between room and fills out wax pan；Or the first cleaning solution chamber is sequentially communicated with the second cleaning solution chamber, third washing Sap cavity room and the 4th cleaning solution chamber, between the first cleaning solution chamber and the second cleaning solution chamber and second washs sap cavity Between room and third cleaning solution chamber and one, which is equipped with, between third cleaning solution chamber and the 4th cleaning solution chamber fills out wax pan.

9. the system of the method according to claim 6 for implementing quickly to detect sample of nucleic acid described in claim 1, feature It is that the automatic instrument for extracting nucleic acid includes pedestal, in the central axis and driving that can cooperate with substrate hole that are connected on pedestal The dynamical system of mandrel rotation, corresponding each fill out at the position of wax pan is equipped with an adding thermal resistance on the pedestal, the power System and adding thermal resistance are connect with the controller, and the pedestal is equipped with for matching simultaneously fixed form storage chamber side To fixing groove.

10. the system of the method according to claim 3 for implementing quickly to detect sample of nucleic acid described in claim 1, special Sign is that the single channel PCR instrument includes temperature components, three temperature controllers and detection components, and the temperature components include phase up and down Upper heating component and lower heating component to setting, and there are accommodating PCR is anti-between the upper heating component and lower heating component Answer the first gap of system chip, the upper heating component and lower heating component include the first heating module arranged side by side, Third heating module and the second heating module, there are third spaces between first heating module, third heating module, described There are the 4th gap between third heating module and the second heating module, two in the upper heating component and lower heating component First heating module is connect with a temperature controller, two the second heating modules in the upper heating component and lower heating component with Second temperature controller connects, two third heating modules and third temperature controller in the upper heating component and lower heating component Connection, the detection components include the third space or fluorescence detection device and/or Scan that the 4th gap is equipped with Device is extended through equipped with a drive hole on first heating module and the second heating module, and two drive holes connect An air pump is connected to as actuator, the PCR product detection device includes that fluorescence detection device, test strip and drop are read Take instrument.