CN108439297A

CN108439297A - Automatic capping device

Info

Publication number: CN108439297A
Application number: CN201810404699.6A
Authority: CN
Inventors: 杨尚鑫; 胡彬; 刘杰; 蔡媛媛; 陶施芳
Original assignee: Shaoxing Xun Xun Kang Biological Technology Co Ltd
Current assignee: Shaoxing Xun Xun Kang Biological Technology Co Ltd
Priority date: 2018-04-28
Filing date: 2018-04-28
Publication date: 2018-08-24
Anticipated expiration: 2038-04-28
Also published as: CN108439297B

Abstract

The invention discloses automatic capping devices, include the clamp assembly of gland drive component and clamping pipe lid, gland drive component is with clamp assembly or liter or drop or pause；Clamp assembly includes gland portion, clamping portion and the clamping driving portion for making clamping portion opening and closing, and in upper, clamping portion under, clamping portion is made of at least two intermediate plates in gland portion.The present invention is used in instrument for extracting nucleic acid, can be directly to the container automatic sealing of the liquid equipped with nucleic acid, and formation can be directly used for follow-up molecule diagnosis and use product, make nucleic acid extraction and transfer integration.

Description

Automatic capping device

Technical field

The present invention relates to nucleic acid extraction fields, more specifically, it is related to automatic capping device.

Background technology

Nucleic acid, such as DNA or RNA, extraction at present mainly use two kinds of extracting methods of centrifugal column method and paramagnetic particle method.It passes The centrifugation column technology of system also applies film to carry out nucleic acid purification, and the principle of its film belongs to silicon adsorption method, and this film is only to nucleic acid There are stronger affinity and adsorption capacity.Sample is cracked under 56 DEG C of temperature conditions using protease first when operation, so Lysate is added in pillar afterwards and is centrifuged, nucleic acid is adsorbed on film, and other impurities are thrown out of, finally with eluent by nucleic acid It elutes.The method extraction effect is good, but while cracking needs to heat, and repeatedly centrifugation is needed when nucleic acid purification, entire to extract Process takes or so 2 hours, of high cost, complicated for operation, time-consuming, relies on laboratory environment, and is difficult to extract degradable RNA.

Paramagnetic particle method nucleic acid extraction is a kind of novel nucleic acids extractive technique using nano biological magnetic bead as carrier, and nucleic acid molecules can Specific recognition occurs and combines with the silicone hydroxyl of magnetic bead surfaces, aggregation or dispersion occur under the action of external magnetic field, to Nucleic acid is carried out to isolate and purify.Currently, the extraction for DNA, paramagnetic particle method nucleic acid extraction generally comprises cracking, combination, washing, elution Four key steps, each step can alone or in combination be realized again by a variety of different methods.If the extraction for RNA, phase Extraction than DNA, it is increasingly complex, appoint the method for so using column to extract and purify at present, while needing that prevention RNA is added All multienzyme of degradation, time-consuming for the extracting method of traditional RNA, and material used is more, and overall cost is high.

Paramagnetic particle method nucleic acid extracting instrument can be divided into：Liquid relief formula and magnetic bar type.Liquid relief formula nucleic acid extraction is to pass through transfer reaction Solution includes to realize the extraction purification of nucleic acid, step：Lysate is added to sample, blows and beats mixing, repeatedly magnetic bead absorption, shifting Lysate is walked, cleaning solution washing magnetic bead is added, magnetic bead is adsorbed, removes cleaning solution, the operations such as elution buffer is added, to To the nucleic acid of purifying.However there are one fatal defects for liquid relief formula instrument, in order to remove waste liquid as thoroughly as possible, liquid-transfering sucker needs Extend into deep-well plates bottom, if suction nozzle by magnetic bead it is too close, magnetic bead and waste liquid can be made to sop up together, so as to cause every time this Sample has fraction raffinate when drawing waste liquid and stays in bottom of the tube, to influence the amplification of subsequent elution efficiency and nucleic acid, such as The success rate of pcr amplification reaction.

Magnetic bar type nucleic acid extracting instrument uses the Magnetic Isolation mode of bar magnet method, and transfer magnetic is carried out by special bar magnet set Pearl shifts sample simultaneously, and magnetic bead orientation is reunited on the special bar magnet set on bar magnet surface, and magnetic is realized by accurately moving Pearl, to eluent, automation extraction process is surveyed to complete nucleic acid from sample lysate to cleaning solution.Specific behaviour's process is as follows：

Step 1, sample-adding are originally：Sealer is torn, is added in liquid sample to cracking fluid apertures；

Step 2, cracking/combination：Lysate lysed sample (reaction a period of time, during which bottom, which can heat, accelerates cracking), Period bar magnet set (there was only bar magnet set, without bar magnet), which is inserted under liquid level, to carry out vibrating mixing up and down, and nucleic acid is made to be suspended in lysate In；After the completion of cracking, the bar magnet set with bar magnet stretches into magnetic bead hole and picks up magnetic bead, is transferred in cracking fluid apertures, bar magnet takes out, magnetic Oscillation mixing makes magnetic bead is evenly spread to adsorb nucleic acid in lysate to stick set up and down, and after standing a period of time, bar magnet is inserted into right It stands afterwards or upper and lower oscillation is all adsorbed on bar magnet to magnetic bead and puts on；

Step 3, salt are washed：It will be transferred in the hole that salt is washed from absorption magnetic bead in lysate, bar magnet takes out, and bar magnet set shakes up and down Swinging mixing makes magnetic bead evenly spread in salt washing lotion, stand a period of time after, bar magnet insertion be then allowed to stand or vibrate up and down to Magnetic bead is all adsorbed on bar magnet and puts on；

Step 4, washing：Magnetic bead after salt is washed takes out, and is transferred in cleaning solution, and oscillation mixing makes magnetic to bar magnet set up and down Pearl evenly spreads in cleaning solution, and after standing a period of time, bar magnet insertion, which is then allowed to stand or is vibrated up and down to magnetic bead, to be all adsorbed on Bar magnet is put on；

Step 5, elution：Magnetic bead after washing is taken out, bar magnet takes out, and oscillation mixing so that magnetic bead is uniform to bar magnet set up and down It is distributed in eluent, is generally heated to that 65 DEG C/5min is stood, heating makes the disengaging of nucleic acid and magnetic bead, at a higher temperature, magnetic Stick insertion, which is then allowed to stand or is vibrated up and down to magnetic bead, to be all adsorbed on bar magnet and puts on, and magnetic bead is recycled, in this way, the work of instrument for extracting nucleic acid It completes.

Later, reaction plate is taken out from middle together, craft pipettor shifts the eluent containing nucleic acid.Bar magnet method carries Take the problem is that：1, nucleic acid eluents after purification need to be transferred in PCR reaction tubes by hand, and transfer process is susceptible to Manual operation error, causes the pollution of human factor, generates false positive.2, nucleic acid eluents are transferred in PCR reaction tubes, are needed Capping by hand, it is inconsistent to be susceptible to seal degree.

Invention content

The purpose of the present invention is to provide a kind of automatic capping devices to the automation capping of PCR reaction tubes.

The technical solution adopted by the present invention to solve the technical problems is：

Automatic capping device includes the clamp assembly of gland drive component and clamping pipe lid, and gland drive component is with folder Have component or liter or drop or pause；Clamp assembly includes gland portion, clamping portion and the clamping driving portion for making clamping portion opening and closing, pressure In upper, clamping portion under, clamping portion is made of cap at least two intermediate plates.

Further, clamping driving portion is that there are two the clamping jaws of movable part for tool, and one intermediate plate of each clamping jaw installation, intermediate plate sets recessed The opening of slot, two grooves is opposite.

Further, groove is opened in clip surface；Alternatively, upper plate and lower plate are installed on intermediate plate, between upper plate and lower plate Distance is adapted to folder portion to be installed, and lower plate holds up folder portion to be installed, and upper plate and lower plate surround the groove of a side opening.

Further, when groove is opened in clip surface, intermediate plate is equipped with extended segment, and extended segment is flushed with the top surface of groove, and two The extended segment of a intermediate plate is opposite, and two extended segments form gland portion；Alternatively, upper plate prolongs compared to lower plate to the direction far from intermediate plate It stretches.

Further, when clamping portion clamping has pipe lid, pipe cover exposes to intermediate plate.

Further, capping apparatus includes pipe cover frame and mobile mechanism, and gland drive component is installed on mobile mechanism, moving machine Structure makes clamp assembly in pipe cover frame and waits for that closure area is reciprocal.

Further, there is pipe cover frame pipe cage, pipe cage to have locating piece, locating piece limitation pipe to cover the rotation on pipe cage Degree of freedom and translational degree of freedom；The quantity of pipe cover frame has one or more.

Further, the base item of protrusion, each locating piece are set on a base item on pipe cage；Locating piece is and pipe lid Coordinate and relative to the protrusion of pipe cage or base item, raised quantity at least two；Alternatively, the quantity of protrusion and pipe lid Sealing quantity is identical；Alternatively, locating piece is coordinated with pipe lid and relative to pipe cage or the pit of base item, the quantity of pit At least two, base item allows the clamping portion of pipe lid exposed or the distance between base item and the clamping portion of pipe lid allow intermediate plate to insert Enter；Alternatively, the quantity in convex hole is identical as the sealing quantity of pipe lid.

Automatic sealing method, includes the following steps：

Pipe lid, uplink are clamped by step 1, clamping portion, and pipe lid is transferred to alignment sample transition range；

Step 2, clamp assembly downlink, when precompressed of pipe lid, precompressed, wait for that the precompressed space of splenium enters PCR reaction pores position, Clamp assembly suspends downlink；

Step 3, clamping portion discharge pipe lid, clamp assembly downlink, gland portion by pipe lid to wait for that splenium is completely forced into corresponding Container or vessel, pipe lid seal PCR reaction tubes；

Step 5, clamp assembly reset.

Further, automatic sealing method, in step 1, elder generation of gland portion contacting pipe lid, later clamping portion close up, clamp pipe lid.

Advantage is the present invention compared with prior art：1, automatic capping device is used in instrument for extracting nucleic acid, can be directly right The container automatic sealing of liquid equipped with nucleic acid, formation can be directly used for follow-up molecule diagnosis and uses product, make nucleic acid extraction With transfer integration.

2, automation capping is carried out to PCR reaction tubes, avoids and is manually in direct contact with the PCR reaction tubes equipped with nucleic acid, Prevent to pollute.

Description of the drawings

Fig. 1 is the overall structure figure of the present invention.

Fig. 2 is the overall structure figure with door.

Fig. 3 is the schematic diagram of cracking region.

Fig. 4 is the schematic diagram of heating load.

Fig. 5 is the structural schematic diagram of bar magnet-bar magnet set of modules and drive transmission device.

Fig. 6 is the structural schematic diagram of bar magnet-bar magnet set of modules.

Fig. 7 is the schematic diagram in pre-treatment region.

Fig. 8 is the structural schematic diagram that the magnetic patch in pre-treatment region is placed.

Fig. 9 is the schematic diagram of capping apparatus.

Figure 10 is the schematic diagram of intermediate plate.

Figure 11 is the schematic diagram of pipe cover frame.

Figure 12 is the schematic diagram of townhouse PCR reaction tubes.

Figure 13 is the schematic diagram of townhouse pipe lid.

Figure 14 is the schematic diagram of ultraviolet lamp.

Figure 15 is the sample distribution figure of first group of anti-pollution test of DNA reference materials.

Figure 16 is the sample distribution figure of second group of anti-pollution test of DNA reference materials.

Figure 17 is the sample distribution figure of the anti-pollution test of RNA reference materials.

It is identified in figure：Operating space 1, exhaust fan 11, ultraviolet lamp 12, babinet 14, door 141, top plate 142, bottom plate 143, side Plate 144；Bar magnet-bar magnet set of modules 2, bar magnet 21, bar magnet set 22, bar magnet frame 211, bar magnet stock 221, baffle 23, through-hole 231, Slot 222；Drive transmission device 3；Cracking region 4, heating load 41 heat cooling piece 42, accommodating chamber 411, and sleeve 412 passes Hot plate 413, temperature control plate 421；Pre-treatment region 5, pipe support 51, pedestal 511, jack 512, base item 513；Capping apparatus 6, gland drive Dynamic component 61, clamp assembly 62, gland portion 621, clamping portion 622, intermediate plate 6221, groove 6222, extended segment 6223, clamping driving Portion 623, clamping jaw 6231, upper plate 62211, lower plate 62212；Pipe cover frame 8, pipe cage 81, locating piece 82, base item 83, pipe lid 84, lid Plate 841, lid 842 of dashing forward, folder portion 841 to be installed.

It is described with reference to the accompanying drawings

Structure of the present invention or these used technical terms are described further below.These explanations are only Only it is how to be realized in such a way that way of example illustrates the present invention, any limit can not be constituted to the present invention System.

Sample

Include biofluid (such as casing fluids or clinical sample with the sample that can detect of detection device of the present invention This).Either fluid sample can derive from the sample of solid-state or semisolid, including excreta to liquid sample, biological tissue and Food samples.The sample of solid-state or semisolid can be converted to liquid sample using any method appropriate, such as mix, smash It is broken, macerate, be incubated, dissolving or in a suitable solution (such as water, phosphate solution or other buffer solutions) using enzymolysis make With digestion of solid sample." biological sample " includes deriving from animal, plant and food samples, such as including deriving from human or animal Urine, saliva, blood and its ingredient, vaginal fluid, sperm, excrement, sweat, secretion, tissue, organ, tumor, tissue and organ Culture, cell culture and medium.

Method for extracting nucleic acid

In some embodiments, method for extracting nucleic acid, including cleavage step and transfer step, cleavage step complete nucleic acid The nucleic acid of thermal cracking and magnetic bead absorption thermal cracking release, cleavage step are completed in cracking region；The magnetic bead for being adsorbed with nucleic acid is turned It moves on in pretreatment liquid, complete transfer step；Pretreatment liquid is located at pre-treatment region, and cracking region and pre-treatment region are mutually only It is vertical, as shown in Figure 1.As long as in this way, nucleic acid extraction can be realized in step transfer.Cracking region and pre-treatment region refer to independently of each other Be not intersect between cracking region and pre-treatment region, cracking region is the isolated area cracked, and pre-treatment region is Place the isolated area of pretreatment liquid.Magnetic bead is transferred to after pretreatment liquid, and the solution in the PCR reaction tubes in pre-treatment region is made To be the solution that can directly carry out molecule diagnostic process.Such as quantitative fluorescent PCR, regular-PCR, digital pcr and constant temperature PCR etc..

The volume of each cracking station is 100~200 microlitres；The volume of each pre-treatment station is 100~200 microlitres, The container and the container of pre-treatment station for cracking station are made of independent thin-wall tube respectively, and multiple thin-wall tubes are embarked on journey or in column It is linked to be townhouse pipe.For example, independent PCR reaction tubes or townhouse PCR reaction tubes using specification for 100 microlitres or 200 microlitres, such as scheme Shown in 12.

In some embodiments, there is distance between cracking region and pre-treatment region, as shown in figure 12.In this way, cracking zone The temperature change in domain does not interfere with pre-treatment region, and pretreatment liquid is avoided unnecessary evaporation or volatilization occur, place before keeping The liquid measure for managing liquid is accurate.

In some embodiments, in cleavage step, cracking region can accommodate multiple hole positions and carry out thermal cracking simultaneously, each Hole position contains lysate；There are multiple jacks 512, each jack 512 can accommodate a container in pre-treatment area, cleavage step Each Kong Weijun is corresponding with a hole in pre-treatment region 5 position；The magnetic bead of 4 all holes of cracking region position is transferred to preceding place simultaneously Manage the corresponding hole site in region.

In some embodiments, each cracking station and pre-treatment station are made of independent thin-wall tube respectively.Can be An individual thin-wall tube is placed in each cracking station (or each pre-treatment station), can also be that multiple thin-wall tubes are linked to be Townhouse pipe, it is each to crack when in station thin-wall tube one of in townhouse pipe.Such as existing 8 townhouse PCR reaction tubes or 12 It is also multi-hole position container to arrange PCR reaction tubes.Each hole position is independent thin-wall tube and refers to：Adjacent hole position not share common sidewalls. Therefore, it reduces intersection heat transfer to the greatest extent between the position of adjacent hole, improves the temperature accuracy of each hole position, make the cracking of all hole positions The temperature consistency of liquid is good.

The magnetic bead for being adsorbed with nucleic acid is transferred to from lysate in pretreatment liquid by the present invention using bar magnet method, primary to shift Nucleic acid extraction is completed, without to magnetic bead repeatedly wash or elute.Pretreatment liquid refers to using for molecule diagnosis anti- Answer liquid.Here nucleic acid can be RNA or DNA.

Cracking step

In some embodiments, lysate and sample are contained in cracking station.Preferably, preset magnetic bead in lysate, Or it is added in pyrolytic process in magnetic bead or the temperature-fall period of thermal cracking and magnetic bead is added.Optimal mode is：Preset magnetic in lysate Pearl, thermal cracking is completed, temperature declines, before protein renaturation, and magnetic bead is combined with nucleic acid, when temperature drops to protein renaturation When (room temperature), magnetic bead is strong bonded with nucleic acid, reduces influence of the protein to nucleic acid, improves adsorption rate of the magnetic bead to nucleic acid, Be conducive to obtain enough, complete nucleic acid.

When thermal cracking, heating temperature needs to reach 80 DEG C or so or more of high temperature, liquid evaporation will occurs and generates Aerosol, to avoid polluting between thin-wall tube, in some preferred schemes, in thermal cracking temperature-rise period, guiding gas is molten Glue discharges.Preferably, guiding aerosol discharges upwards or above side.

In some embodiments, container is inserted into heating load.The abundant wrapping container of heating load can keep entire The temperature consistency of thin-wall tube avoids occurring aerosol because of the temperature difference of hole position inner wall in cooling and condensing the drop to be formed, from And prevent to lead to the possibility of pollution because of aerosol.

In some embodiments, lysate refrigeration is cooled down in cleavage step, after the completion of heating.It is sample hot tearing when heating Solution is just combined with magnetic bead when nucleic acid low temperature (or room temperature), thus refrigeration cooling can shorten combined with magnetic bead after nucleic acid cleavage when Between, accelerate the speed of nucleic acid extraction, shortens extraction time.Entire nucleic acid extraction Row control is completed within half an hour, is improved normal Easy in inactivation, the success rates of degradable material such as the lower extraction RNA of temperature.

In some embodiments, heating load 41 is covered by heating refrigeration module, therefore all holes position quilt of cracking region The temperature of Synchronous Heating, all hole positions rises unanimously.Preferably, heating refrigeration module is by the seamless spliced shape of multiple heating refrigeration units At.In this manner it is ensured that entire cracking region synchronizes heating or cooling, keep all PCR reaction pores position temperature consistent.

The operation of the magnetic bead in fully absorption lysate is covered using bar magnet and bar magnet is：Make the bar magnet set 22 with bar magnet 21 Slowly from up and down motion.The upper limit position of 22 movement of bar magnet set is that bar magnet covers 22 bottoms arrival liquid level, 22 movement of bar magnet set Lower position is the bottom wall and side wall that bar magnet set 22 does not touch container.Lower position can be obtained through experiment.This way be because： The suction of bar magnet set 22 concentrates on end, therefore, allows the end of bar magnet set 22 to move up and down in the whole region of lysate, fills Absorption is divided to be suspended in the magnetic bead in lysate.It, will not be because of liquid when the 22 slow movement of bar magnet set refers to bar magnet 22 movement of set Tension and cause magnetic bead with respect to bar magnet cover 22 displacements.

Bar magnet set 22 and hole position clearance fit, the upper and lower translation in liquid regions of bar magnet set 22.That is, bar magnet set 22 It can move freely hole position is not rebuffed, but the suction of bar magnet set 22 can cover the cross section of entire thin-wall tube substantially, therefore When bar magnet 22 movement of set, the magnetic bead of each sectional position can be adsorbed onto.Alternatively, bar magnet set 22 and the tube wall of hole position have distance, Bar magnet set 22 had both done upper and lower translation in liquid regions or had done left and right translation or formula movement of drawing a circle.For example it draws circle or draws oval equal Belong to formula movement of drawing a circle.

Magnetic bead transfer process

Magnetic bead is transferred to the corresponding aperture position in pre-treatment region using bar magnet method from cracking station, all magnetic beads turn simultaneously It moves.

In some embodiments, in cleavage step, bar magnet 21 and bar magnet set 22 is located at except aerosol discharge path.To Aerosol is avoided to be attached to bar magnet set 22 and pollute.Preferably, lysate is restored to room temperature, carries the bar magnet set of bar magnet 21 22 are inserted into lysate, adsorb magnetic bead.Preferably, the liquid without attaching liq or attachment is only positioned at bar magnet set 22 on bar magnet set 22 Surface is not dripped, then bar magnet set 22 is slowly moved to above the PCR reaction tubes for containing pretreatment liquid.Bar magnet set 22 is slow Slowly the speed moved is not fallen with magnetic bead, bar magnet covers the liquid that 22 surfaces are adhered to and do not drip is advisable.

In some embodiments, it when bar magnet set 22 takes out from cracking station, is equipped in the path that bar magnet set 22 takes out Baffle 23, baffle 23 block container, container are avoided to be taken away by bar magnet set 22.

Magnetic bead discharges process

Magnetic bead is transferred to pretreatment liquid from lysate using bar magnet method, before the magnetic bead taken out in lysate is discharged into In treatment fluid.

In some embodiments, as shown in Figure 7 and Figure 8, there is pedestal 511, the setting of pedestal 511 to accommodate in pre-treatment region 5 The jack of container, each jack 512 are a pre-treatment station, and the quantity of jack 512 has multiple.Preferably, around jack 512 Magnet is set；Each jack 512 corresponds to a bar magnet 21 and bar magnet set 22.Therefore, each pre-treatment station corresponds to a cracking All magnetic beads of station, cracking region 4 can be covered by bar magnet-bar magnet while be transferred to pre-treatment region 5.Pedestal 511 has Support stiffness, pedestal 511 are installed on operating space 1.

In some embodiments, pedestal 511 is equipped with base item 513 outstanding, and jack 512 is arranged along base item 513.Pedestal There are multiple base items 513 on 511, there is distance, each base item 513 there are the multiple jacks of setting between base item 513, each base item 513 Jack 512 forms a pipe support unit.Base item 513 all has support stiffness.Pedestal 511 and 513 one of base item or base item 513 with 511 fixing assembling of pedestal.Preferably, base item 513 holds the container being put into it completely, such as PCR reaction tubes.

In some embodiments, magnet is set under jack 512, alternatively, magnet is set between adjacent jack 512, Or each jack 512 is surrounded with magnet.Jack 512 is cylinder, alternatively, the cylindrical section of jack 512 and PCR reaction tubes Match, alternatively, the shape of jack 512 reacts tube shape matching with the PCR that need to be placed.

Magnetic bead is transferred in pretreatment liquid, and bar magnet 21 22 is withdrawn from bar magnet set, and bar magnet 21 is statically placed in pretreatment liquid, after standing Bar magnet set 22 is axially vibrated.The vibration of bar magnet set 22 is not splashed out outward with pretreatment liquid to be advisable.Bar magnet set 22 vibration, together with The liquid tension of pretreatment liquid makes magnetic bead be discharged into pretreatment liquid from 22 surfaces of bar magnet set.Magnet attracts bar magnet to cover 22 surfaces Magnetic bead, accelerate bar magnet set 22 on magnetic bead release rate and efficiency.

In some embodiments, magnetic patch be set to 512 bottom of jack either magnetic patch be set to jack 512 side or insert Magnetic patch is all arranged in the bottom and side in hole 512.When bar magnet is stood, the suction of magnetic patch attracts magnetic bead to be detached from bar magnet set 22.Bar magnet set 22 vibration when, liquid tension, bar magnet set 22 whipping and magnetic patch suction make jointly magnetic bead with respect to bar magnet set 22 generation displacements, It falls into PCR pretreatment liquids.

Bar magnet 21 is permanent magnet or electromagnet, and bar magnet 21 from bar magnet set 22 when withdrawing, 21 loss of excitation of bar magnet.In this way, magnetic Stick 21 will not attract magnetic bead displacement, magnetic bead to be concentrated mainly on bottom when withdrawing bar magnet set 22, be conducive to magnetic patch and attract magnetic bead, magnetic Pearl fully discharges.

The capping in pre-treatment area

After the transfer step, closing step covers all thin-wall tubes of the same pipe support unit to closing step simultaneously. Capping is made of cover board and setting sealing on the cover board, such as the capping of PCR reaction tubes, has multiple prominent lids on cover board, lid of dashing forward As sealing, lid of dashing forward is identical as the arrangement of PCR reaction tubes to be sealed, and prominent lid reacts seal of tube cooperation (such as mistake with PCR Be full of cooperation etc.).

In some embodiments, each pipe lid of closing step corresponds to a pipe support unit, and pipe, which covers, to be had and thin-wall tube One-to-one sealing；When capping, sealing first part is pressed into corresponding thin-wall tube in advance, in advance enter positioning after, again will be all close Envelope portion is pressed into thin-wall tube simultaneously.

In some embodiments, the jack 512 in pre-treatment area is set on pedestal 511, under pedestal 511 supports when covering Pressure.Before capping, pipe lid 84 is taken out, shifted and is aligned corresponding pipe support unit, 8 positioning pipe lid of pipe cover frame from pipe cover frame 84。

Instrument for extracting nucleic acid

In some embodiments, instrument for extracting nucleic acid has operating space 1, has bar magnet-bar magnet cover die in operating space 1 Block 2 and drive transmission device 3, referring to Figures 5 and 6, drive transmission device 3 keeps bar magnet-bar magnet set of modules 2 reciprocal or mobile, operation There are cracking region 4 and pre-treatment region 5 in space, cracking region 4 and pre-treatment region 5 are mutual indepedent, bar magnet-bar magnet set of modules 2 is reciprocal in cracking region 4 and preceding processing region 5.The magnetic bead for being adsorbed with nucleic acid is transferred to by bar magnet and bar magnet set from cracking region Nucleic acid extraction is completed in pre-treatment region.

Cracking region

In some embodiments, cracking region 4 can accommodate multi-hole position container, and lysate is contained in hole position, one or more Multi-hole position is carried out at the same time thermal cracking；Pretreatment liquid is contained in multi-hole position container, each Kong Weijun and the preceding place of cracking region The hole position for managing region is corresponding；The magnetic bead of all hole positions of cracking region is transferred to the corresponding hole site in pre-treatment region simultaneously.It splits Solution region 4 refers to independently of each other with pre-treatment region 5：Cracking station and the hole position in pre-treatment region do not intersect.In this way, The temperature change of cracking region does not interfere with pre-treatment region, and pretreatment liquid is avoided unnecessary evaporation or volatilization occur, protects The liquid measure for holding pretreatment liquid is accurate.

Heating load

Heating load to containing sample lysate heat up, make sample discharge nucleic acid, nucleic acid at low temperature (or room temperature) with Magnetic bead combines.Heating load accommodates thin-wall tube and carries out hot transmission to thin-wall tube.

In some embodiments, as shown in figure 4, heating load 41 has heat transfer plate 413 and accommodating chamber 411, accommodating chamber is set In heat transfer plate 413.When nucleic acid extraction, first to lysate heat up, after cracking nucleic acid discharge, nucleic acid release after will low temperature (or often Temperature) when, nucleic acid just with magnetic bead combineing with, when cooling down, temperature is consistent everywhere in the thin-wall tube in heating load 41, and steam is avoided to condense Inner wall in hole position, prevents from polluting.Multiple accommodating chambers 411 form a heating unit；Heating load is heated with one or more Unit, it is independent between adjacent heating unit.It is independently referred between adjacent heating unit between adjacent heating unit not Share common sidewalls reduce intersection heat transfer between adjacent heating unit to the greatest extent, improve temperature accuracy, improve all Kong Weizhong cracking The temperature consistency of liquid.Each accommodating chamber 411 is surrounded by a sleeve 412, independent between the sleeve 412 of each heating unit, Sleeve is connected with heat transfer plate.As shown in Figure 5.Each hole position is independent thin-wall tube and refers to：Adjacent hole position not share common sidewalls. Therefore, it reduces intersection heat transfer to the greatest extent between the position of adjacent hole, improves the temperature accuracy of each hole position, make the cracking of all hole positions The temperature consistency of liquid is good.

Heat cooling piece

Heating cooling piece heats up or cools down under the control of temperature control plate, and the temperature for heating cooling piece passes to heating load.

In some embodiments, as shown in figure 4, heating load 41 is completely covered in heating cooling piece 42.Heat cooling piece 42 Directly each accommodating chamber 411 of heating load 41 is heated, improves the consistency of temperature.Heat cooling piece 42 (such as Peltier) After the completion of heating, carry out refrigeration cooling.The cooling procedure of thermal cracking just has the heating refrigeration cooling of cooling piece 42, keeps lysate fast Speed is cooled to room temperature, shortens cracking and takes, and then entire nucleic acid extraction Row control is completed within half an hour.Heat cooling piece Between it is seamless spliced.Heating cooling piece 42 heats cooling piece by multi-disc, seamless spliced to refer to adjacent two panels heating cooling piece Between without apparent gap or distance.Heating cooling piece is corresponding with radiator fan.

Independent temperature plate

In some embodiments, heating cooling piece 42 is equipped with temperature sensing by independent temperature controller temperature control, heating load 41 Device, temperature sensor are connect with temperature controller.As shown in Fig. 2, temperature control plate 421 and customer-oriented control end communication, temperature control plate 421 Receive the real time temperature that temperature sensor transmits.Independent temperature control plate 421 can realize the accurate temperature controlling to heating cooling piece 42. Temperature control plate 421 should control heating, also control refrigeration.

It is vented colloidal sol

The volume of thin-wall tube (such as PCR reaction tubes) is small, and it is gentle molten that sample and lysate generate steam at a high temperature of cracking Glue guides steam and aerosol discharging operation space to avoid steam and Aerosol Pollution operating space.

In some embodiments, as shown in Fig. 2, operating space 1 sets exhaust fan 11, exhaust fan 11 is directed at cracking region 4；Row Fan 11 is located above the top or side of cracking region 4.During cracking, steam that exhaust fan 11 generates high temperature and Or aerosol discharging operation space, it avoids polluting in operating space.Preferably, when exhaust fan 11 works, bar magnet-bar magnet set Module 2 is suspended in outside cracking region 4.Exhaust fan 11 by steam and or aerosol guide the side far from bar magnet-bar magnet set of modules 2 into To avoiding bar magnet set contaminated.

Bar magnet-bar magnet set of modules

Bar magnet is inserted into bar magnet set, and bar magnet set is inserted into lysate, and magnetic bead is adsorbed in bar magnet set under magnetic fields.

In some embodiments, as shown in figure 5, bar magnet-bar magnet set of modules 2 include equipped with bar magnet 21 bar magnet frame 211, can The bar magnet stock 221 and baffle 23, baffle 23 of detachable installation bar magnet set 22 are located under bar magnet stock 221, and baffle 23 is higher than Heating load 41, each heating unit have a region stopped by baffle 23, baffle 23 be equipped with allow bar magnet cover 22 by Through-hole 231；When bar magnet set 22 is located at pause region, the first ultraviolet lamp is directed at baffle 23；Top plate 142 or side plate 144 are equipped with second Ultraviolet lamp.First ultraviolet lamp carries out sterilization to the irradiation from bottom to top of baffle 23 and other component, and the second ultraviolet lamp is to behaviour Make the component in space 1 and irradiates progress sterilization from the top down.

Each bar magnet is inserted into a bar magnet set, each corresponding thin-wall tube of bar magnet set.In order to ensure bar magnet and bar magnet Set can obtain magnetic bead in cracking region, and magnetic bead is transferred to pre-treatment region, position arrangement and the cracking region of bar magnet The position of thin-wall tube is arranged and the arrangement of the position of the thin-wall tube in pre-treatment region is identical.

In some embodiments, there are one or more bar magnet units, each bar magnet unit, which has, embarks on journey on bar magnet frame 211 Or the multiple bar magnets 21 arranged in column, it is mutually parallel between bar magnet 21, is mutually aligned between bar magnet unit, is parallel；Bar magnet 21 1 End is on bar magnet frame 211, and the other end of bar magnet 21 is as insertion end；The insertion end of all bar magnets 21 is located on bar magnet frame 211 Approximately the same plane.Each bar magnet 21 corresponds to a bar magnet set 22, and each bar magnet unit corresponds to a bar magnet and covers unit；Bar magnet stock 221 are equipped with the slot 222 of bar magnet set unit, and slot 222 keeps the jack of bar magnet set 22 exposed, as shown in Figure 5.All bar magnet sets 22 bottom end is generally aligned in the same plane.

The pipe support in pre-treatment region

Pipe support is used to place the thin-wall tube (such as PCR reaction tubes) in pre-treatment region, and the structure of pipe support will determine thin-wall tube It arranges position.

In some embodiments, as shown in fig. 7, pipe support includes pedestal 511, pedestal 511 is equipped with jack 512, jack 512 Quantity has multiple；Each jack 512 corresponds to a bar magnet 21 and bar magnet set 22.A thin-wall tube can be accommodated in each jack 512. The arrangement of jack 512 is identical as the arrangement of accommodating chamber 411 of heating load 41, all magnetic bead energy of cracking region 4 Enough while being transferred to pre-treatment region 5.Base item 513 and pedestal 511 can bear lower pressure, have support stiffness.

Spacing between adjacent pipe support unit is identical as the spacing between the heating unit in heating load 41, therefore, In multiple heating units thermal cracking simultaneously, all magnetic beads can be disposably transferred in pretreatment liquid with bar magnet method simultaneously, complete At nucleic acid extraction.Heating unit and pipe support unit only need to be extended, you can realize container expansion, the scalability of nucleic acid extraction ability It is good, and hole Bits Expanding has substantially no effect on nucleic acid extraction and takes.The entire no matter how many hole positions of extraction apparatus carry out thermal cracking, use Same set of bar magnet-bar magnet set of modules.

In some embodiments, pedestal 511 is equipped with base item 513 outstanding, and jack 512 is arranged along base item 513.Pedestal There are multiple base items 513 on 511, there is distance, each base item 513 to have the multiple jacks 512 of setting, each base item 513 between base item 513 Jack 512 formed a pipe support unit.Base item 513 all has support stiffness, referring to Fig. 7.513 one of pedestal 511 and base item, Or base item 513 and 511 fixing assembling of pedestal.Preferably, base item 513 holds the PCR reaction tubes being put into it completely.

Magnetic frame

It is inserted into magnetic bead in bar magnet set and is discharged into pretreatment liquid.In order to make all magnetic beads rapidly from bar magnet set as possible It is discharged into pretreatment liquid, so that pretreatment liquid is located in magnetic field, is acted on using magnetic-adsorption, magnetic bead is made to be detached from bar magnet set.

In some embodiments, each jack 512 of pipe support is located in magnetic field.Preferably, magnet is set to 512 bottom of jack Portion or side or magnet surround jack 512, and the number of magnets near each jack is 1 or gives more.Magnetic bead is transferred to When pre-treatment region 5, bar magnet 21 is statically placed in from 22 extraction of bar magnet set, bar magnet set 22 in pretreatment liquid, and bar magnet covers the absorption of 22 bottoms Magnetic bead is detached from bar magnet set 22 under the suction of magnet, and magnet accelerates the rate of release of magnetic bead and magnetic bead is made to be filled from bar magnet set 22 Divide release.

In some embodiments, as shown in figure 8,512 lower section of jack is equipped with pedestal 514, pedestal 514 is equipped with and container bottom The tube seat 5141 of contact, jack 512 is corresponded with tube seat 5141, and is mutually communicated.Preferably, pedestal 514 is provided with receiving magnetic The mounting groove 5142 of body, mounting groove 5142 are arranged along 5141 periphery of tube seat.Preferably, each adjacent two tube seat 5141 is one group of pipe A mounting groove 5142 is arranged in slot unit, every group of tube seat unit side.Preferably, every group of 5141 unit both sides of tube seat are respectively provided with peace Slot 5142 is put, mounting groove 5142 is located at the both sides of tube seat unit midline position, and mounting groove 5142 is symmetrical arranged.Preferably, it places Slot 5142 is diamond shape, and the groove angle of tube seat unit both sides mounting groove 5142 is opposite, and the trough rim of mounting groove 5142 abuts tube seat.It is preferred that , mounting groove 5142 is square.

Automatic capping device

Automatic capping device can be placed directly on follow-up diagnosis to the automatic gland of the thin-wall tube in pre-treatment area, sealing, formation The sample of nucleic acid product handled in equipment (such as PCR equipment).

Automatic capping device can be moved alone, can also be with bar magnet-bar magnet set of modules synchronizing moving.

In some embodiments, automatic capping device 6 includes the clamp assembly 62 of gland drive component 61 and clamping pipe lid, Gland drive component 61 is with clamp assembly 62 or rises or drops or suspend；Clamp assembly 62 includes gland portion 621, clamping portion 622 With the clamping driving portion 623 for making 622 opening and closing of clamping portion, gland portion 621 is in upper, clamping portion 622 under, and clamping portion 622 is by least Two intermediate plates 6221 form.It holds pipe lid when intermediate plate 6221 closes up tightly, realize clamping, pipe lid is discharged when intermediate plate 6221 opens.Intermediate plate 6221 be in release conditions when, gland portion 621 pushes all prominent lids (waiting for closure portion) simultaneously.For example, gland portion 621 is positioned at dress Pressing plate on folder portion 622, pressing plate covering or part cover all prominent lids of pipe lid, and pressing plate can simultaneously apply all prominent lids Power.

In some embodiments, clamping driving portion 623 is that there are two the clamping jaw 6231 of movable part, the peaces of each clamping jaw 6231 for tool An intermediate plate 6221 is filled, as shown in Figure 10, intermediate plate 6221 sets groove 6222, and the opening of two grooves 6222 is opposite.When clamping, pipe The folder portion to be installed of lid enters intermediate plate 6221 from the opening of groove 6222, and the bottom surface of groove 6222 holds up the bottom surface of folder portion to be installed.It is excellent Choosing, groove 6222 is opened in 6221 surface of intermediate plate, such as processes groove 6222 with the mode of wire cutting；Alternatively, intermediate plate 6221 Upper installation upper plate 62211 and lower plate 62212, the distance between upper plate 62211 and lower plate 62212 are adapted to folder portion to be installed, lower plate 62212 hold up folder portion to be installed, and upper plate 62211 and lower plate 62212 surround the groove 6222 of a side opening.

When groove 6222 is opened in 6221 surface of intermediate plate, intermediate plate 6221 is equipped with extended segment 6223, extended segment 6223 and groove 6222 top surface flushes, and the extended segment 6223 of two intermediate plates 6221 is opposite, and two extended segments 6223 form gland portion 621；Alternatively, Upper plate 62211 extends compared to lower plate 62212 to the direction far from intermediate plate 6221.When the extended segment of two intermediate plates 6,221 6223 or on There are the spaces for accommodating prominent lid, intermediate plate 6221 not to squeeze prominent lid 842 when plate closes up, between clamping portion 622.When clamping portion 622 is released When putting folder portion 841 (cover board of pipe lid) to be installed, folder portion 841 to be installed is exited in the bottom of groove 6222, at this point, gland portion 621 (prolongs Stretch section) cover board 841 is still covered, therefore following pressures can be applied to all prominent lids 842.

When 622 clamping of clamping portion has pipe lid, pipe cover exposes to intermediate plate 6221.When capping, 62 clamping of clamp assembly Capping alignment container pushes, and the exposed portion of pipe lid is pressed into container in advance.Then, clamping portion 622 discharges pipe lid, and gland portion is by pipe lid It is completely forced into container, sealing container.

It is then townhouse pipe lid, connection that such as container, which uses townhouse PCR reaction tubes (such as 8 townhouse pipes or 12 townhouse pipes), pipe lid 84, Comb lid includes cover board 841 and multiple prominent lids 842, and lid 842 of each dashing forward seals a PCR reaction tube.When capping, townhouse pipe covers All prominent lids 842 simultaneously be pressed into corresponding PCR reaction tubes.

In some embodiments, capping apparatus includes mobile mechanism, and gland drive component is installed on mobile mechanism, moving machine Structure keeps clamp assembly reciprocal in pipe cover frame and preceding processing region.

Intermediate plate

Intermediate plate is installed on clamping driving portion (such as clamping jaw), and intermediate plate realizes clamping and release to pipe lid.

Intermediate plate includes ontology, and as shown in figs. 9-10, ontology sets gland portion 621 and clamping portion 622, and gland portion 621 is in upper, dress Folder portion 622 is under.At least two intermediate plates realize the clamping of pipe lid, hold pipe lid when intermediate plate closes up tightly, realize that clamping, intermediate plate 6221 are opened Pipe lid is discharged when opening.When intermediate plate 6221 is in release conditions, gland portion 621 pushes all prominent lids 842 (waiting for closure portion) simultaneously.Example Such as, gland portion 621 is all prominent lids 842 of the pressing plate on clamping portion 622, pressing plate covering or part covering pipe lid 84, Pressing plate can simultaneously exert a force to all prominent lids 842.

In some embodiments, ontology sets groove 6222, and the opening of two grooves 6222 is opposite.When clamping, pipe lid waits for Clamping portion 841 enters intermediate plate 6221 from the opening of groove 6222, and the bottom surface of groove 6222 holds up the bottom surface of folder portion 841 to be installed.It is excellent Choosing, groove 6222 is opened in body surface, such as processes groove with the mode of wire cutting；Alternatively, installing upper plate on ontology 62211 and lower plate 62212, the distance between upper plate 62211 and lower plate 62212 be adapted to folder portion 841 to be installed, 62212 support of lower plate Folder portion 841 to be installed is played, upper plate 62211 and lower plate 62212 surround the groove 6222 of a side opening.

When groove 6222 is opened in body surface, ontology is equipped with extended segment 6223, the top of extended segment 6223 and groove 6222 Face flushes, and the extended segment 6223 of two ontologies is opposite, and two extended segments 6223 form gland portion 621.Alternatively, 62211 phase of upper plate Extend to the direction far from ontology than lower plate 622.When the extended segment of two ontologies 6223 or upper plate 62211 close up, clamping portion There are the spaces for accommodating prominent lid, ontology not to squeeze prominent lid between 622.When clamping portion 622 discharges the 841 (lid of pipe lid of folder portion to be installed Plate) when, folder portion 841 to be installed is exited in the bottom of groove 6222, at this point, gland portion 621 (i.e. extended segment) still covers cover board, therefore Following pressures can be applied to all prominent lids.

When capping, 62 clamping of clamp assembly capping alignment thin-wall tube and is pushed, and all prominent lids are pressed into corresponding thin-walled in advance Pipe.Then, clamping portion 622 discharges pipe lid, and prominent lid is completely forced into thin-wall tube by gland portion 621, completes capping.

Pipe cover frame

Before capping, pipe lid is located in stable position with pipe cover frame, mobile mechanism is with clamp assembly in pipe cover frame It is reciprocal between region and pre-treatment region.

In some embodiments, there is pipe cover frame 8 pipe cage 81, pipe cage 81 to have locating piece 82, the limitation pipe of locating piece 82 Rotational freedom and translational degree of freedom of the lid 84 on pipe cage 81.When pipe lid 84 is combined with locating piece 82, pipe lid 84 can not Relative positioning part 82 rotates or translation, and only behind intermediate plate clamping pipe lid 84, pipe lid 84 is removed from locating piece 82.Locating piece 82 Quantity it is identical as the quantity of pipe support unit.There are the base item 83 of protrusion, each locating piece 82 to be set to a base on pipe cage 81 On item 83.The width of base item 83 is matched with 842 size of prominent lid of pipe lid 84, to increase the cover board 841 and pipe cage of pipe lid 84 The distance between 81, cover board 841 is picked up convenient for intermediate plate.

The first positioning method of pipe cover frame

In some embodiments, locating piece 82 be with pipe lid coordinate and relative to the protrusion of pipe cage 81 or base item 83, it is convex The quantity at least two risen.Pipe cover frame 8 includes pipe cage 81 and protrusion, and at least two convex to form a pipe lid positioning list Member.There are the base item 83 of protrusion, protrusion to be set on a base item 83 on pipe cage 81.Pipe cover frame 8 is set to instrument for extracting nucleic acid In operating space 1.The raised quantity of each pipe lid positioning unit is identical as the prominent lid 842 of pipe lid to be positioned, and each protrusion is corresponding One prominent lid 842.There is base item 83 on pipe cage 81, base item 83 protrudes from pipe cage 81, and bump array is raised on base item 83 The center of circle is located on the center line of base item 83.The matched of the height and prominent lid 842 of protrusion.Pipe cover frame 8 has one or more manage Lid positioning unit has spacing between adjacent pipe lid positioning unit, and adjacent pipe lid positioning unit is mutually aligned.

Second of positioning method of pipe cover frame

In some embodiments, locating piece 82 be with pipe lid coordinate and relative to pipe cage 81 or the pit of base item 83, it is recessed The quantity at least two in hole, base item 83 allow the clamping portion 622 of pipe lid exposed or the clamping portion 622 of base item 83 and pipe lid it Between distance allow intermediate plate be inserted into.At least two pipe lid positioning units of pit formation one.Pipe cover frame 8 is set to instrument for extracting nucleic acid Operating space in.The pit quantity of each pipe lid positioning unit is identical as the prominent lid 842 of pipe lid to be positioned, as shown in figure 13, Each pit corresponds to a prominent lid 842；Pit is cylinder.There are base item 83, base item 83 to protrude from pipe cage 81 on pipe cage 81, Pit is arranged on base item 83, and the center of circle of pit is located on the center line of base item 83.Pipe cover frame 8 has one or more pipe lid positioning Unit has spacing between adjacent pipe lid positioning unit, and adjacent pipe lid positioning unit is mutually aligned.

Automatic sealing method

Automatic sealing method, includes the following steps：

Pipe lid is transferred to alignment sample transition range by step 1, intermediate plate clamping pipe lid, uplink；

The clamping portion release pipe lid of step 3, intermediate plate；

Step 4, clamp assembly downlink, gland portion wait for that splenium is completely forced into corresponding container or vessel, pipe lid by pipe lid Seal PCR reaction tubes；

Step 5, clamp assembly reset.

Preferably, step 1 clamping pipe lid includes：

Step 1.1, clamping jaw and intermediate plate are moved to alignment pipe lid, make the distance between clamping portion of intermediate plate allow to cover into Enter；

Step 1.2, gland drive component make clamping jaw downlink, and until gland portion touch pipe lid, gland portion is close to the top of pipe lid Face while intermediate plate close up, clamping portion clamping pipe lid.

Operating space

In some embodiments, band can open the inside cabin formation aforesaid operations space 1 of the babinet 14 of door or lid.Preferably, Babinet 14 is made of door 141, top plate 142, bottom plate 143 and side plate 144, cracking region 4, pre-treatment region 5, drive transmission device 3 and mobile mechanism be respectively arranged on bottom plate 143.

In some embodiments, airspace, radiator fan and heating cooling piece are mounted on bottom plate under bottom plate 143 143, heating cooling piece is upper, and radiator fan is under, radiator fan airspace.

In some embodiments, the jack 512 in pre-treatment region 5 is set to pedestal 511, and pedestal 511 is set to bottom plate 143, bottom Pressure when 511 support capping of seat.

In some embodiments, bottom plate 143 is equipped with the first ultraviolet lamp, and the first ultraviolet lamp is located at bar magnet-bar magnet set of modules 2 Suspend region.As shown in figure 14, to bar magnet-bar magnet set of modules 2, radiation sterilization sterilizes ultraviolet lamp 12 from bottom to top.

Embodiment 1

The minimum detection limit of DNA reference materials is tested

Using the present invention method for extracting nucleic acid be manually operated experiment, and using instrument for extracting nucleic acid automatically extract nucleic acid into Row verification experimental verification feasibility：

Experiment is manually operated using the method for extracting nucleic acid of the present invention, steps are as follows：

Step 1, obtaining the PCR reaction tubes equipped with lysate and magnetic bead, (this experiment uses the 8 townhouse PCR pipes, hole position to be sequentially A, B, C, D, E, F, G, H), to the addition sample of PCR reaction tubes；The hole A4, the B4 and C4 position of first PCR reaction tube is added 10²CFU/ml, Bordetella pertussis (DNA reference materials), D4, E4, the holes F4 position are added 10³CFU/ml, (DNA is referred to Bordetella pertussis Product)；The hole G4, H4 position is added 10⁴CFU/ml, Bordetella pertussis (DNA reference materials)；The holes the A5 position of second PCR reaction tube is added 10⁴CFU/ml, Bordetella pertussis (DNA reference materials), the hole B5, C5, D5 position are added 10⁵CFU/ml, (DNA is referred to Bordetella pertussis Product), E5, F5, the holes G5 position are added 10⁶CFU/ml, Bordetella pertussis (DNA reference materials)；Sample is not added for the holes H5 position；

PCR reaction tubes after two sample-addings are put into cracking region and carry out thermal cracking by step 2, in thermal cracking processes, guiding Aerosol discharges outward, and after the completion of cracking, the bar magnet set with bar magnet is inserted into lysate, and bar magnet set and bar magnet stand a period of time Afterwards, slow up-down vibration；

Step 3, bar magnet and bar magnet set take out from lysate, and pre-treatment region is slowly transferred to from cracking region, preceding There is processing region the PCR reaction tubes for containing pretreatment liquid, bar magnet and bar magnet set to be inserted into pretreatment liquid, and bar magnet extraction is preceding The PCR reaction tubes for the treatment of region are placed around magnet, and bar magnet, which is sleeved in pretreatment liquid, to be stood a little while, and bar magnet set shakes up and down later Dynamic, pretreatment liquid will not splash out；

Step 4, bar magnet extract the PCR reaction tubes in pre-treatment area out, complete magnetic bead transfer；To the PCR reaction tubes in pre-treatment area Capping.

Using the instrument for extracting nucleic acid operation experiments of the present invention, steps are as follows：

S1, obtain equipped with lysate and magnetic bead PCR reaction tubes (this experiment uses 8 townhouse PCR pipes, and hole position sequence is A, B, C, D, E, F, G, H), to the addition sample of PCR reaction tubes；The hole A2, the B2 and C2 position of first PCR reaction tube is added 10²CFU/ml, Bordetella pertussis (DNA reference materials), D2, E2, the holes F2 position are added 10³CFU/ml, (DNA is referred to Bordetella pertussis Product)；The hole G2, H2 position is added 10⁴CFU/ml, Bordetella pertussis (DNA reference materials)；The holes the A5 position of second PCR reaction tube is added 10⁴CFU/ml, Bordetella pertussis (DNA reference materials), the hole B3, C3, D3 position are added 10⁵CFU/ml, (DNA is referred to Bordetella pertussis Product), E3, F3, the holes G3 position are added 10⁶CFU/ml, Bordetella pertussis (DNA reference materials)；Sample is not added for the holes H3 position；It is ready to contain The PCR reaction tubes of pretreatment liquid are filled with, and are positioned over the pipe support in pre-treatment area；

S2, two PCR reaction tubes after sample-adding are put into the heating load of cracking region, it is negative that each hole position is put into heating In the sleeve carried；Bar magnet-bar magnet set of modules is located at except cracking region, is heated to lysate, and heating, exhaust fan are opened, After heating, freeze cooling lysate；

S3, cracking are completed, and exhaust fan stops, and bar magnet is inserted into bar magnet set, and bar magnet-bar magnet set is moved to cracking region, often A bar magnet-bar magnet set is inserted into PCR reaction tubes, after standing a period of time, slow up-down vibration, to adsorb the every of lysate The magnetic bead in a section；

S4, bar magnet and bar magnet set are extracted out from lysate, and are slowly transferred to pre-treatment area, are directed at pre-treatment area PCR reaction tubes；Bar magnet and bar magnet set are inserted into pretreatment liquid, and bar magnet moves up, covers and extract out from bar magnet,

S5, bar magnet, which are sleeved in pretreatment liquid, to be stood a little while, and bar magnet covers up-down vibration n times later, and bar magnet set moves up, magnetic Stick-bar magnet set of modules leaves pre-treatment area；

S6, capping apparatus clamping pipe lid and the PCR reaction tubes for being moved to alignment pre-treatment region, fixture is downward, by pipe lid The sealing for being pressed into PCR reaction tubes, completing PCR reaction tubes.Instrument for extracting nucleic acid work finishes, and opens the door, the PCR reaction tubes that will be sealed Carry out sensitivity technique.

The total time-consuming of the instrument for extracting nucleic acid of the present invention is 16~18min, and after the completion of instrumentation, pre-treatment region is taken out PCR reaction tubes can be directly placed into PCR instrument and carry out subsequent analysis.

Experimental result：

Conclusion：The result of manual operation and instrumentation is without significant difference；The kit sensitivity of instrumentation reaches 100CFU/ml meets kit performance verification.

Embodiment 2

The anti-pollution test of instrument for extracting nucleic acid

First group of anti-pollution test of 2.1DNA reference materials

S1, obtain equipped with lysate and magnetic bead PCR reaction tubes (this experiment uses 8 townhouse PCR pipes, and hole position sequence is A, B, C, D, E, F, G, H), to the addition sample of PCR reaction tubes, the arrangement of sample is as shown in figure 15,

The holes the A3 position of first PCR reaction tube is DNA strong positives reference material 10⁶CFU/m (Bordetella pertussis), the holes B3 position are Negative sample, the holes C3 position are DNA strong positives reference material 10⁶CFU/m (Bordetella pertussis), the holes D3 position are negative sample, the holes E3 position For DNA strong positives reference material 10⁶CFU/m (Bordetella pertussis), the holes F3 position are negative sample, and the holes G3 position is that DNA strong positives refer to Product 10⁶CFU/m (Bordetella pertussis), the holes H3 position are negative sample；

The holes the A4 position of second PCR reaction tube is negative sample, and the holes B4 position is DNA strong positives reference material 10⁶CFU/m (hundred Day cough bacillus), the holes C4 position is negative sample, and the holes D4 position is DNA strong positives reference material 10⁶CFU/m (Bordetella pertussis), the holes E4 position For negative sample, the holes F4 position is DNA strong positives reference material 10⁶CFU/m (Bordetella pertussis), the holes G4 position are negative sample, the holes H4 Position is DNA strong positives reference material 10⁶CFU/m (Bordetella pertussis)；

The holes the A5 position of third PCR reaction tubes is DNA strong positives reference material 10⁶CFU/m (Bordetella pertussis), the holes B5 position are Negative sample, the holes C5 position are DNA strong positives reference material 10⁶CFU/m (Bordetella pertussis), the holes D5 position are negative sample, the holes E5 position For DNA strong positives reference material 10⁶CFU/m (Bordetella pertussis), the holes F5 position are negative sample, and the holes G5 position is that DNA strong positives refer to Product 10⁶CFU/m (Bordetella pertussis), the holes H5 position are negative sample；

The holes the A6 position of 4th PCR reaction tube is negative sample, and the holes B6 position is DNA strong positives reference material 10⁶CFU/m (hundred Day cough bacillus), the holes C6 position is negative sample, and the holes D6 position is DNA strong positives reference material 10⁶CFU/m (Bordetella pertussis), the holes E6 position For negative sample, the holes F6 position is DNA strong positives reference material 10⁶CFU/m (Bordetella pertussis), the holes G6 position are negative sample, the holes H6 Position is DNA strong positives reference material 10⁶CFU/m (Bordetella pertussis)；

PCR reaction tubes after four sample-addings is anti-by first PCR reaction tube, second PCR reaction tube, third PCR It should manage and be put in heating load successively with the 4th PCR reaction tube；

It gets out 4 and contains 8 townhouse PCR reaction tubes of pretreatment liquid, and be positioned over the pipe support in pre-treatment area；

S2, four PCR reaction tubes after sample-adding are put into the heating load of cracking region, it is negative that each hole position is put into heating In the sleeve carried；Bar magnet-bar magnet set of modules is located at except cracking region, is heated to lysate, and heating, exhaust fan are opened, After heating, freeze cooling lysate；

S6, capping apparatus clamping pipe lid and the PCR reaction tubes for being moved to alignment pre-treatment region, fixture is downward, by pipe lid The sealing for being pressed into PCR reaction tubes, completing PCR reaction tubes.Instrument for extracting nucleic acid work finishes, and opens the door, the PCR reaction tubes that will be sealed Carry out pollution detection.

Experimental result A3-H3 is first row, and A4-H4 is that second row A5-H5 is third row, and A6-H6 is the 4th row

Conclusion：No cross contamination between the Kong Yukong of the PCR reaction tubes of instrumentation.

2.2, second group of anti-pollution test of DNA reference materials

S1, obtain equipped with lysate and magnetic bead PCR reaction tubes (this experiment uses 8 townhouse PCR pipes, and hole position sequence is A, B, C, D, E, F, G, H), to the addition sample of PCR reaction tubes, the arrangement of sample is as shown in figure 15, the A3 of first PCR reaction tube The hole Kong Wei, B3 position, the holes C3 position, the holes D3 position, the holes E3 position, the holes F3 position, the holes the G3 holes Wei HeH3 position are DNA strong positives reference material (hundred Day cough bacillus)；

The holes A4 position, the holes B4 position, the holes C4 position, the holes D4 position, the holes E4 position, the holes F4 position, the holes G4 position and the H4 of second PCR reaction tube Hole position is negative sample；

The holes the A5 position of third PCR reaction tubes, the holes B5 position, the holes C5 position, the holes D5 position, the holes E5 position, the holes F5 position, the holes G5 position, H5 Hole position is DNA strong positives reference material (Bordetella pertussis)；

The holes the A6 position of 4th PCR reaction tube, the holes B6 position, the holes C6 position, the holes D6 position, the holes E6 position, the holes F6 position, the holes G6 position, H6 Hole position is negative sample；

PCR reaction tubes after four sample-addings are put in heating load successively；

Experimental result：

Conclusion：Sample and lysate, pretreatment liquid use townhouse PCR reaction tubes as container (such as 8 townhouses, 12 townhouses), Cross contamination is not deposited between each group of townhouse PCR reaction tube and other group of townhouse PCR reaction tube.

2.3, the test for contamination of RNA reference materials

The holes the A3 position of first PCR reaction tube is RNA reference materials (Respiratory Syncytial Virus(RSV) reference material), and the holes B3 position is feminine gender Sample, the holes C3 position are RNA reference materials (Respiratory Syncytial Virus(RSV) reference material), and the holes D3 position is negative sample, and the holes E3 position is that RNA is referred to Product (Respiratory Syncytial Virus(RSV) reference material), the holes F3 position are negative sample, and the holes G3 position is that RNA reference materials (join by Respiratory Syncytial Virus(RSV) Examine product), the holes H3 position is negative sample；

The holes the A4 position of second PCR reaction tube is negative sample, and the holes B4 position is that RNA reference materials (join by Respiratory Syncytial Virus(RSV) Examine product), the holes C4 position is negative sample, and the holes D4 position is RNA reference materials (Respiratory Syncytial Virus(RSV) reference material), and the holes E4 position is negative sample This, the holes F4 position is RNA reference materials (Respiratory Syncytial Virus(RSV) reference material), and the holes G4 position is negative sample, and the holes H4 position is RNA reference materials (Respiratory Syncytial Virus(RSV) reference material)；

The holes the A5 position of third PCR reaction tubes is RNA reference materials (Respiratory Syncytial Virus(RSV) reference material), and the holes B5 position is feminine gender Sample, the holes C5 position are RNA reference materials (Respiratory Syncytial Virus(RSV) reference material), and the holes D5 position is negative sample, and the holes E5 position is that RNA is referred to Product (Respiratory Syncytial Virus(RSV) reference material), the holes F5 position are negative sample, and the holes G5 position is that RNA reference materials (join by Respiratory Syncytial Virus(RSV) Examine product), the holes H5 position is negative sample；

The holes the A6 position of 4th PCR reaction tube is negative sample, and the holes B6 position is that RNA reference materials (join by Respiratory Syncytial Virus(RSV) Examine product), the holes C6 position is negative sample, and the holes D6 position is RNA reference materials (Respiratory Syncytial Virus(RSV) reference material), and the holes E6 position is negative sample This, the holes F6 position is RNA reference materials (Respiratory Syncytial Virus(RSV) reference material), and the holes G6 position is negative sample, and the holes H6 position is RNA reference materials (Respiratory Syncytial Virus(RSV) reference material)；

Embodiment 3

3.1, the precision test of DNA reference materials

S1, obtain equipped with lysate and magnetic bead PCR reaction tubes (this experiment uses 8 townhouse PCR pipes, and hole position sequence is A, B, C, D, E, F, G, H), to the addition sample of PCR reaction tubes, the arrangement of sample is as shown in figure 15, gets out 4 and is loaded with cracking DNA reference materials (10 are added in 48 townhouse PCR reaction tubes in 8 townhouse PCR reaction tubes of liquid and magnetic bead⁶CFU/ml, pertussis Bacillus)；

S6, capping apparatus clamping pipe lid and the PCR reaction tubes for being moved to alignment pre-treatment region, fixture is downward, by pipe lid The sealing for being pressed into PCR reaction tubes, completing PCR reaction tubes.Instrument for extracting nucleic acid work finishes, and opens the door, the PCR reaction tubes that will be sealed Carry out precision detection.

Experimental result：

Conclusion：The use of the CV values of instrument for extracting nucleic acid is 2.63% ＜ 5%, instrument essence precision complies with standard.

3.2, the precision test of RNA reference materials

S1, obtain equipped with lysate and magnetic bead PCR reaction tubes (this experiment uses 8 townhouse PCR pipes, and hole position sequence is A, B, C, D, E, F, G, H), to the addition sample of PCR reaction tubes, the arrangement of sample is as shown in figure 15, gets out 4 and is loaded with cracking RNA reference materials (Respiratory Syncytial Virus(RSV)) are added in 48 townhouse PCR reaction tubes in 8 townhouse PCR reaction tubes of liquid and magnetic bead；

Conclusion：The use of the CV values of instrument for extracting nucleic acid is 1.60% ＜ 5%, instrument essence precision complies with standard.

Embodiment 4

The sample that the instrument for extracting nucleic acid of the present invention obtains and the silent winged KingFisher of match^TMDuo Prime magnetic beads for purifying instrument obtains The sample obtained；And two instruments are simultaneously to DNA reference materials (10⁴CFU/ml, Bordetella pertussis) carry out nucleic acid extraction after carry out QPCR is detected.

S1, obtain equipped with lysate and magnetic bead PCR reaction tubes (this experiment uses 8 townhouse PCR pipes, and hole position sequence is A, B, C, D, E, F, G, H), to the addition sample of PCR reaction tubes, the arrangement of sample is as shown in figure 15, gets out 1 and is loaded with cracking 8 townhouse PCR reaction tubes of liquid and magnetic bead；DNA reference materials (10 are added in A, B, C, D, E, F, G, this 7 Kong Weijun⁴CFU/ml, hundred Day cough bacillus), the holes H position is negative sample, and sample size is 20 microlitres；It is anti-to get out the 8 townhouse PCR that 1 contains pretreatment liquid Ying Guan, and it is positioned over the pipe support in pre-treatment area；

The instrument of the instrument for extracting nucleic acid of the present invention takes as 16min；It is loaded manually and takes about 5min, after the completion of instrumentation The sample of QPCR processing can be directly placed by obtaining.Although this experiment has only used 7 sample inventions can handle 32 simultaneously Kind or more sample.

Use the silent winged KingFisher of match^TMThe operating operation test of Duo Prime magnetic beads for purifying instrument, steps are as follows：

Step 1, sample-adding are originally：96 orifice plates are obtained, sequentially add 96 by lysate, in conjunction with liquid, cleaning solution and eluent manually The Kong Weizhong of orifice plate, 8 Kong Weiyi regions of a usual row, if 8 holes of first row accommodate lysate, 8 holes of second row accommodate In conjunction with liquid, third arranges 8 holes and accommodates the first cleaning solution, and the 4th hole of row 8 accommodates second of cleaning solution, the 5th Kong Rong of row 8 Receive eluent；The position for accommodating lysate is corresponding with heating module；Sample is added in lysate again, sample size requirements are in 5 millis It rises；

Step 5, elution：Magnetic bead after washing is taken out, bar magnet takes out, and oscillation mixing so that magnetic bead is uniform to bar magnet set up and down It is distributed in eluent, is generally heated to that 65 DEG C/5min is stood, heating makes the disengaging of nucleic acid and magnetic bead, at a higher temperature, magnetic Stick insertion, which is then allowed to stand or is vibrated up and down to magnetic bead, to be all adsorbed on bar magnet and puts on, and magnetic bead is recycled；After magnetic bead recycles, instrument behaviour Work terminates.

Instrument is opened, reaction plate is taken out, the eluent containing nucleic acid is transferred to PCR reaction tubes with pipettor by hand In, it is handled being put into QPCR analytical instrument after PCR reaction tubes by hand capping.Sai Mo flies KingFisher^TMDuo Prime magnetic The instrumentation that pearl purifies instrument takes about 45min, and sample-adding takes about 15min by hand, and transfer is for QPCR processing by hand Sample takes about 25min.In the instrument, a reaction plate most multipotency is loaded 12 kinds of samples.

Testing result is as follows；

As a result：

The QPCR analysis samples and Sai Mo that the instrument for extracting nucleic acid of the present invention obtains fly KingFisher^TMDuo Prime magnetic beads It purifies the QPCR that instrument obtains and analyzes sample, the analysis sample that nucleic acid instrument for extracting nucleic acid of the invention obtains is silent better than match to fly KingFisher^TMThe analysis sample that Duo Prime magnetic beads for purifying instrument obtains.

Meanwhile to equipment of the invention and control experiment, for the nucleic acid extraction amount and integrality under identical sample size Investigation, it is found that the amount of the DNA of equipment extraction of the invention is the 2 times or more for the DNA for comparing instrument extraction.Meanwhile for phase The extraction of RNA in the sample of same volume, find the RNA of the equipment extraction of the present invention be 10 times of the DNA extracted than instrument with On, this of the invention may not use many washing process, reduce RNA exposures, to reduce the probability of RNA degradations.

The integrality of DNA and RNA is investigated, finds the nucleic acid of this equipment extraction almost without fragment and fracture, 85% It all keeps complete above, and uses conventional methods and the method for comparative apparatus, nucleic acid only only has that 30-45%'s is complete Property.In this way, in order to effectively carry out following amplification, it is necessary to which more samples extract, and ensure more nucleic acid Integrality can be only achieved the purpose of amplification.In addition, if nucleic acid (such as DNA or RNA) is imperfect, target target nucleic acid The efficiency of amplification just need the longer time.It was found that the nucleic acid extracted using the present invention, in the case of identical sample size, Such as 20 microlitres, the nucleic acid that the present invention extracts can be expanded effectively, and use comparative apparatus extraction nucleic acid cannot be effective Amplification or the result for feminine gender occur.

In the case where lacking any element specifically disclosed herein, limitation, may be implemented illustrated and described herein Invention.Used terms and expressions method is used as the term of explanation and unrestricted, and is not intended in these terms and table Up to any equivalent for excluding shown and described feature or part thereof in the use of method, and it should be realized that various remodeling exist All it is feasible in the scope of the present invention.It is therefore to be understood that although specifically being disclosed by various embodiments and optional feature The present invention, but the modifications and variations of concept as described herein can use by those of ordinary skill in the art, and recognize It is fallen into for these modifications and variations within the scope of the present invention of the appended claims restriction.

It is described herein or record article, patent, patent application and every other document and can electronically obtain The content of information include herein by reference, just as each individual publication by specific and single in full to a certain extent Solely point out by reference.Applicant retains from any of any this article, patent, patent application or other documents And all material and information are incorporated into the right in the application.

Claims

1. automatic capping device, it is characterised in that：Capping apparatus includes the clamp assembly and gland drive component of clamping pipe lid, folder Tool component is installed on gland drive component；Clamp assembly includes gland portion, clamping portion and the clamping of clamping portion opening and closing is made to drive Portion, in upper, clamping portion under, clamping portion is made of at least two intermediate plates in gland portion.

2. the instrument for extracting nucleic acid of automatic sealing as described in claim 1, it is characterised in that：Clamping driving portion is that there are two can for tool The clamping jaw in dynamic portion, each clamping jaw install an intermediate plate, and intermediate plate sets groove, and the opening of two grooves is opposite.

3. the instrument for extracting nucleic acid of automatic sealing as claimed in claim 2, it is characterised in that：Groove opens up and clip surface；Or Person, installation upper plate and lower plate, the distance between upper plate and lower plate be adapted tos with folder portion to be installed on intermediate plate, lower plate picking-up folder portion to be installed, Upper plate and lower plate surround the groove of a side opening.

4. the instrument for extracting nucleic acid of automatic sealing as claimed in claim 3, it is characterised in that：When groove is opened up with clip surface, Intermediate plate is equipped with extended segment, and extended segment is flushed with the top surface of groove, and the extended segment of two intermediate plates is opposite, and two extended segments form gland Portion；Alternatively, upper plate extends compared to lower plate to the direction far from intermediate plate.

5. the instrument for extracting nucleic acid of automatic sealing as claimed in claim 3, it is characterised in that：When clamping portion clamping has pipe lid, pipe Cover exposes to intermediate plate.

6. automatic capping device as described in claim 1, it is characterised in that：Capping apparatus includes pipe cover frame and mobile mechanism, Gland drive component is installed on mobile mechanism, and mobile mechanism makes clamp assembly in pipe cover frame and waits for that closure area is reciprocal.

7. automatic capping device as claimed in claim 6, it is characterised in that：There is pipe cover frame pipe cage, pipe cage to have positioning Part, locating piece limitation pipe cover rotational freedom and translational degree of freedom on pipe cage；The quantity of pipe cover frame has one or more.

8. automatic capping device as claimed in claim 6, it is characterised in that：There is the base item of protrusion on pipe cage, it is each to position Part is set on a base item；Locating piece is with the cooperation of pipe lid and relative to the protrusion of pipe cage or base item, and raised quantity is extremely It is two less；Alternatively, the quantity of protrusion is identical as the sealing quantity of pipe lid；Alternatively, locating piece is and pipe lid cooperation and opposite In pipe cage or the pit of base item, the quantity at least two of pit, base item allows the clamping portion of pipe lid exposed or base item and pipe The distance between clamping portion of lid allows fixture to be inserted into；Alternatively, the quantity in convex hole is identical as the sealing quantity of pipe lid.

9. automatic sealing method, includes the following steps：

Step 1, capping module clamping portion clamp pipe lid, uplink, by pipe lid be transferred to alignment sample transition range；

Step 2, capping module downlink, when precompressed of pipe lid, precompressed, wait for that the precompressed space of splenium enters PCR reaction pores position, capping Module suspends downlink；

Step 3, clamping portion discharge pipe lid, and capping module downlink, gland portion waits for that splenium is completely forced into corresponding container by pipe lid Or vessel, pipe lid seal PCR reaction tubes；

Step 5, capping module reset.

10. automatic sealing method as claimed in claim 9, it is characterised in that：In step 1, elder generation of gland portion contacting pipe lid, later Clamping portion closes up, clamps pipe lid.