CN108603221A - Comprehensive sample processing system - Google Patents

Comprehensive sample processing system Download PDF

Info

Publication number
CN108603221A
CN108603221A CN201580085776.0A CN201580085776A CN108603221A CN 108603221 A CN108603221 A CN 108603221A CN 201580085776 A CN201580085776 A CN 201580085776A CN 108603221 A CN108603221 A CN 108603221A
Authority
CN
China
Prior art keywords
sample
filter
nucleic acid
cell
container
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201580085776.0A
Other languages
Chinese (zh)
Inventor
克里斯托弗·G·库尼
丽贝卡·霍尔姆伯格
菲利普·贝尔格雷德尔
彼得·Q·曲
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Akonni Biosystems Inc
Original Assignee
Akonni Biosystems Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Akonni Biosystems Inc filed Critical Akonni Biosystems Inc
Publication of CN108603221A publication Critical patent/CN108603221A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0099—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor comprising robots or similar manipulators

Abstract

A kind of comprehensive Sample purification system includes shell, sample container frame, filter mounting and cylindrical magnet.The sample container frame and filter mounting are arranged in the shell.The sample container frame is designed for keeping one or more sample containers, and filter mounting is designed for keeping one or more filters.Cylindrical magnet is close and is attached to sample container frame, and the central longitudinal axis by the motor in shell around magnet is rotated with lytic cell.It allows the interested molecule of people to be purified in lytic cell, allows the filter core of the interested molecule of people using being specifically bound to thus.The system can be easy to automate and suitable for allowing the fast purifying and analysis of the interested molecule of people such as nucleic acid and protein.

Description

Comprehensive sample processing system
Technical field
This patent disclosure relates generally to one kind for detaching and/or purifying particularly from reluctant sample matrix and/or difficulty The synthesis sample processing system for allowing the interested molecule of people such as nucleic acid and protein of the organism of fragmentation, and drawn using magnetic force Hair is vortexed is easy to the method for automatically detaching and/or purifying the nucleic acid from sample in conjunction with solid-state Integral filtering part.
Background technology
Molecular test becomes the gold standard for certain diagnostic tests because of its speed, sensibility and specificity.Experiment Room development experiments (LDT) are now in one of the link of most rapid growth in the market in-vitro diagnosis (IVD).Sample preparation is to experiment It is credible most important, but usually become the bottleneck of clinical molecule biology workflow and diagnostic test.Although there are many molecules Detection mode, but only a small amount of automated sample preparation work Flow Policy.Around these sample preparation strategies and chemical building Existing instrument is spent within the scope of 17~$ of $ 150k, but they can not still provide one kind and be difficult to resolve such as life of sample matrix for handling The integrated approach of phlegm and/or the organism such as gram-positive bacteria and acid-fast bacilli (i.e. mycobacteria) of difficult fragmentation.
Including the acid-fast bacilli of mycobacterium strain is usually separated from the phlegm of infected patient.They are referred to as " anti- Acidfast bacilli " is because of its rich lipocyte film, relatively impermeable various basic dyes, unless the dyestuff is combined with phenol. Phlegm is thick and is difficult to handle.Most of analysis sputum samples contain various different amounts of organic debris and various dirty normal The bacterium colony of state or transient state.Chemical decontamination dye/processing is generally used to reduce viscosity and kills pollutant, while allowing to withdraw branch Bacillus.But because its unique cell membrane contains the fat of mycolic acid and high-content, therefore cell is hydrophobic and tends to roll into a ball It gets together.This makes their impermeable common colorants, such as gram colorant.Two kinds of antiacid colorants are common , i.e. carbolfuchsin and fluorchrome such as auramine or auramine rhodamine.Once by colouring, the anti-acid organic solvent of these cells Fade, therefore be referred to as antiacid.But they keep pinkish red after sequentially or simultaneously with acid and ethanol postincubation or auramine Color.
Antiacid smear for microscopic examination is poor to the sensibility of Mycobacterium.Microscope inspection sensibility is influenced by factors, Such as the popularity degree and seriousness of disease, sampling type, sampling quality, existing mycobacterial cells quantity, place in sampling Reason method (direct or concentration), centrifugal separation method, dye technology and inspection quality.It has been proposed that only should be at least 100 The microscope of (in low income country) and preferably 300 (in industrialized country) submerges the inspection in the visual field (or equivalent fluorescence visual field) Negative findings are just reported after testing.Therefore, when correctly carrying out microscope inspection, it may be time-consuming and laborious.
Mycobacterium strain is the bacillus slowly grown, and the common generation time is 12 to 18 hours.Flora only at 1 week or It can be just seen after 8 weeks repoductive times.Sample containing low concentration mycobacterial cells is further such that squamous subculture becomes several times To be necessary.Mycobacteria culture on defined medium can allow to identify specific point contained by biological sample Ramibacterium.But it is time-consuming that this, which is only in particularly with those for the patient at course of infection initial stage,.
Nucleic acid hybridization assay is had been developed for detect the mycobacterium strain in biological sample.Initial experiment utilizes Direct probe hybridization.But it is typically too low in the concentration for picking up from mycobacterial cells contained in the sample of patient so that can not produce Raw positive hybridization signal.Therefore the experiment using PCR amplification is had been developed for.For example, being commercialized as " AmplifiedTMTuberculosis Mycobacteria straightway testing " kit or MTD test kitsKit (Gen-Probe companies, California, USA 92121, Santiago) the exclusive rRNA amplifications (transcript mediated amplification) of MtbC are used, it is followed by root It is detected according to the amplicon of Gen-Probe HPA methods (hybridization protection assay).
In view of above-mentioned restrictive condition, people need a kind of system being simple and efficient, be integrated with sample homogenize, difficult fragmentation Cracking and the polynucleotides of microorganism the needs of purifying to meet clinical labororatory and similar user.
Invention content
In one aspect, the application provides a kind of comprehensive Sample purification system, including shell, sample container frame, filter frame And cylindrical magnet.The sample container frame and filter frame are arranged in the shell.Sample container erection is calculated as keeping one A or multiple sample containers, filter erection are calculated as keeping one or more filters.The cylindrical magnet is close and is attached to Sample container frame, and the motor drive that can be located in shell and rotated around the central longitudinal axis of magnet.
In some embodiments, which includes one or more reagent racks equipped with one or more reagents.
In some embodiments, which includes multiple sample containers, multiple filters and one or more examinations Agent frame.
In certain embodiments, which has the magnetic pole being arranged symmetrically around magnet longitudinal axis.In other implementations In example, which has the opposite magnetic pole being arranged on the longitudinally opposed both ends of magnet.In other embodiments, The cylindrical magnet is electromagnet.
In one embodiment, one or more sample containers are sealed and are designed to injecting one or more reagents The system remained closed after solution.
In one embodiment, which further includes the reagent rack being arranged in the shell, and thus the reagent rack includes depositing The reagent being placed in the hole of the sealing of the separation in this.
In use, which includes magnetic stirring part and multiple beads, be designed to include when the sample container Cell material and magnetic stirring part spin and agitation bead is to undergo cell material when the cylindrical magnet is rotated around its longitudinal axis Mixed and disorderly mixing so that sample homogenizes and cell fragmentation.
In one embodiment, bead includes glass, plastics, ceramic material, mineral, metal or combinations thereof.In specific reality It applies in example, bead is silica bead.
In one embodiment, diameter possessed by bead is in 10~1000 micron ranges.
In one embodiment, which includes metal or alloy.In a particular embodiment, the magnetic stirring part Including stainless steel.In another embodiment, which includes the alloy core for being coated with polymer.In specific embodiment In, which includes the alloy core for being coated with polymer, and thus the alloy core includes neodymium iron boron or SmCo and/or the polymerization Object is PTFE or Parylene.
On the other hand, automatic nucleic acid purification system includes features described above, further includes aspirating system and one or more automatically A manipulator, manipulator design are for distributing reagent automatically according to predetermined way to one or more sample containers and handling sample This material and reagent.In use, which includes that multiple sample containers (are each equipped with stirring parts and pearl Grain), multiple filters and one or more reagent rack.
In another aspect, a method of for purifying the target molecule from sample, include the following steps：(a) it provides According to the Sample purification system of this paper；(b) sample is placed on together with magnetic stirring part and multiple beads in sample container；(c) The sample container is placed on sample container frame；(d) cylindrical magnet is made to be rotated around its longitudinal axis, to the magnetic stirring part It spin and stirs bead and forms the degree of cell lysate to being enough to homogenize sample and broken sample inner cell；(e) make to Few a part of cell lysate flows through the first opening of filter, to which the target molecule in cell lysate is attached to the filter in filter On core；(f) the unbonded part of cell lysate is discharged from filter through the first opening, wherein filter is being left in unbonded part It is preceding through filtration core at least twice；And (g) by making elution buffer flow through the first opening of filter come elution of bound to filter core Target molecule and through first opening by elution buffer from filter be discharged, wherein the elution buffer before leaving filter through filtering Core is at least twice.
In some embodiments, which is polynucleotide molecule.In one embodiment, sample includes phlegm.In spy Determine in embodiment, sputum sample sheet is doubtful containing mycobacterium tuberculosis (MTB), and this method further comprises the following steps：With special Expand the polynucleotide molecule of elution in the primer of MTB, and determine the polynucleotide molecule whether DNA containing MTB.
In another embodiment, target molecule purification process includes using automatic purification system, further includes aspirating automatically System and one or more manipulators, manipulator design is at automatic distribution reagent in a predefined manner to one or more sample containers In and handle sample material and reagent.In the case, each above-mentioned steps are in the middle quilt of each of the multiple sample container It repeats, the filter using equivalent amount and one or more reagent racks.
Description of the drawings
Be described in detail will with reference to the following drawings, wherein：
Fig. 1 is to show for lytic cell and the therefrom flow chart of one embodiment of the integrated approach of purification of nucleic acid.
Fig. 2 shows the illustrative single channel nucleic acid purification systems according to one embodiment.
Fig. 3 shows that magnet cracks the exemplary riding position of chamber relative to sample.
Fig. 4 shows exemplary to aspirate filter.
Fig. 5 A and 5B are the schematic diagrames for describing automatic 8 channel nucleic acid purification system according to another embodiment.
Fig. 6 shows the disposable transport device according to another embodiment.
Fig. 7 shows (the Magnetically-induced Vortexing mangnetos whirlpools MagVor for the nucleic acid from phlegm Rotation)/filter purifying illustrative steps sequence.
Specific implementation mode
When describing the preferred embodiment of the present invention, for the sake of clarity concrete term is used.But the present invention is not beaten It calculates and is confined to selected concrete term.It should be understood that each specific parts include being worked in a similar manner to complete phase Like all technical equivalents of purpose.
Fig. 1 is to describe a kind of for lytic cell and to purify and allow the interested molecule of people nucleic acid for example therein or protein The flow chart of the exemplary process steps of integrated approach.Method 10 includes：Will be equipped with liquid sample suspended substance, magnetic stirring part and The sample tube of cell cracking bead is placed in the (step 11) on the sample rack of magnet；By being split in magnetic stirring part and cell Solve makes magnet under the participation of bead is rotated with the speed for being enough the cell in lysed sample suspended substance come the sample suspensions that homogenize Body (step 13)；So that the sample suspensions body to homogenize is flowed through filter core matrix, the interested molecule of people is allowed to be bound to filter core base at this time Body (step 15)；Cleaning filter core matrix (step 17) and elution of bound allows the interested molecule (step of people from filter core matrix 19).In some embodiments, which is preinstalled with magnetic stirring part and/or cell cracking bead and/or promotes cell cracking And/or retain target molecule integrality reagent.
The liquid sample suspended substance is suspended in the sample in liquid cracking medium.Illustrative sample may include biology Imitate sheet, environmental samples or non-natural sample.Illustrative biological sample may include tissue samples, biological fluid sample Sheet, cell sample, fungi sample, protozoan sample, bacteria sample and Virus Sample.Tissue samples include from any animal or The tissue isolated in plant.Biological sample include but not limited to blood, Cord blood, blood plasma, buffy coat, urine, saliva, Phlegm, NALC processing phlegm, Nasopharyngeal swabs (NPS), nasopharynx Extract (NPA), gastric juice, concentration cough acquisition object, celiolymph, oral cavity Liquid, irrigating solution (such as bronchial), liquor pleurae, excrement and leucocyte removal sample.Cell sample further includes culture cell, comes From the fresh of any cell source including fixed packet wax (FFPE) tissue or the cell and tissue that freeze.Bacteria sample packet It includes but is not limited to culture bacterium, separation of bacterial and the bacterium in any said biological sample.Virus Sample includes but unlimited In culture virus, isolated viral and the virus in any said biological sample.Environmental samples include but not limited to air sample Sheet, water sample, soil sample, rock specimens and any other sample obtained from natural environment.Artificial sample includes not Any sample being present in natural environment.Material that is that the example of " artificial sample " including but not limited to purifies or isolating, Culture materials, synthetic material and any other artificial material.
It can be isotonic, hypotonic or hypertonic that liquid, which cracks medium,.In some embodiments, liquid cracking medium is Aqueous.In certain embodiments, liquid cracking medium contains the composition of buffer solution and/or at least one salt or a variety of salt. In some embodiments, the pH value range of liquid cracking medium is from about 5 to about 8, from about 6 to 8 or from about 6.5 to about 8.5.Various pH buffer solutions can be used to obtain desired pH value.Suitable buffer solution includes but not limited to trihydroxy methyl amino Methane (Tris), MES, bis- (2 hydroxy ethylamine base) Pehanorms, [three hydroxyls of ADA, ACES, PIPES, MOPSO, 1,3- bis- Methyl methylamino] propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, HEPPSO, POPSO, TEA, HEPPS, N- Tricine, (ethoxy) glycine of Gly-Gly, N- bis- and phosphate buffer (such as especially sodium phosphate or Potassium-sodium phosphates).Liquid crack medium can especially include about 10 millimeters to about 100 millimeters buffer solution, about 25 millimeters to about 75 Millimeter buffer solution or about 40 millimeters to about 60 millimeters of buffer solution.The type sum number of the buffer solution used in liquid medium Amount can become according to the difference of the application.In some embodiments, the pH value of liquid cracking medium is about 7.4, this can pass through It is obtained using about 50 millimeters of TRIS buffer.In some embodiments, liquid cracking medium is water.
Eukaryocyte, prokaryotic cell and/or virus can be suspended with any suitable concentration.It is preferred that the sample includes with simultaneously The concentration that magnetic stirring part moves is not interfered to be suspended in the cell in liquid medium.In some embodiments, eukaryocyte and/or The range of the suspended concentration of prokaryotic cell especially 1~1 × 1010Cells/ml, 1~1 × 105Cells/ml or 1 × 103~1 × 104Cells/ml.In some embodiments, the range of the suspended concentration of virion is 1~1 × 1013Particle/ Milliliter, 1~1 × 1010Particle/milliliter or 1 × 105~1 × 107Particle/milliliter.
In certain preferred embodiments, sample is doubtful to contain MTB.In one embodiment, sample is nasopharynx Extract. In another embodiment, which is Nasopharyngeal swabs.
The term as used herein " cell " refers to eukaryocyte, prokaryotic cell, virus, endospore or any combination thereof. Then, cell can especially include bacterium, bacterial spore, fungi, virion, single celled eukaryotic organism (such as protozoan, Saccharomycete etc.), the separation cell from multi-cell organism or aggregated cells (such as primary cell, culture cell, tissue, entirely Organism etc.) or any combination thereof.
Term " sample " refers to containing target molecule or the doubtful any material containing target molecule.
Term " nucleic acid " refers to individual nucleic acid and nucleic acid polymerization chain, including DNA and RNA, either naturally occur or people (include its analog) or its modifier of work synthesis, especially those be known to occur in nature and there is random length Modifier.
Term " sample container " refers to the container of elongated generally tubular or refers to pipe, is used to fasten and/or handle sample The reagent combined with sample after processing so as to nucleic acid purification or receiving.The sample container needs not be cylindrical and can be whole along its A length or part thereof length tapers slightly.
With the relevant term of cell " cracking " refer at least sub-fraction cell integrality it is broken and from fragmented cell Release intracellular ingredient such as nucleic acid and protein.
Term " homogenizing " refers to blending or is vortexed (different elements such as excrement, tissue, phlegm, saliva) into uniform mixing Object.
Term " closed system " or " be closed container " refer to following pipe or container, are sealed and to be closed substantially (such as Fruit is not closed completely) mode work with interfere or prevent external source or foreign material in processing procedure enter (or leaving) pipe or Container.The building block of closed system or container can be pre-sterilized in manufacturing location, be sterilized when in use using preceding And/or it assembles before use in corresponding closed system and is sterilized after closing.
Term " single use disposable " refers to that the parts are no longer used.That is, being completing its set use After the processing or manufacture of target sample, it is disposed off.
The term as used herein " bulk sorbent agent " or " whole sorptive material " refer to porous three-dimensional sorptive material, It has continuously interconnected pore structure in single-piece, can include the structure of rigidity, self-supporting basic monoblock.Monoblock It prepares for example by cast, sintering or polymeric precursors at the mould with intended shape.Term " bulk sorbent agent " is " whole to inhale Attached property material " is to be different from the aggregate for being encapsulated in bed structure or being embedded into individual absorbability particles in porous matrix, Here, final products include individual absorbability particles.Multicellular monolith polymer is a kind of new material developed nearly ten years. Different from the polymer being made of the bead of very little, monoblock is single continuous polymer member, is prepared by simple moulding Technique.Term " bulk sorbent agent " or " whole sorptive material " are also intended to be different from adsorbent fibers or are coated with the fibre of adsorbent Dimension aggregate such as filter paper or the filter paper for being coated with adsorbent.
In one aspect, the application provides a kind of comprehensive Sample purification system, including shell, sample container frame, filter frame And cylindrical magnet.The sample container frame and the setting of filter frame are in the shell.The sample container frame is designed to be kept for one or more A sample container, filter frame is designed for keeping one or more filters.The cylindrical magnet is close and is attached to sample container frame And the motor drive in shell can be located at and rotated around the central longitudinal axis of magnet.
Fig. 2 shows the example single channel Sample purification systems 100 according to one embodiment.System 100 in Fig. 2 is wrapped Include shell 104, sample container frame 108, filter actuator/frame 112 and cylindrical magnet 116.Sample container frame 108 and filter frame 112 are arranged in shell 104.Sample container frame or sample container seat 108 keep sample container 120.Filter actuating in Fig. 2 Device/frame 112 is designed to remain attached to the filter 124 of syringe 176, and syringe so designs, the injection column in syringe 176 Plug 174 moves up and down that liquid is sucked and distributed through filter 124.Plunger 174 is connected to the control plunger motion in frame 112 Actuator.Cylindrical magnet 116 is close and is arranged except sample container frame 108, and passes through the motor in shell 104 130 around magnet 116 central longitudinal axis rotate.
Each sample container 120 can include one or more cracking chambers for the cell in lysed sample.Preferably, Sample container 120 (and other system units) is sealed and is designed in injecting sample 144 and/or one or more reagent solutions Before and after the system that remains closed.Container 120 can be sealed with lid, cap or cover.Sample container 120 can be with any suitable Material, size and shape manufacture.In certain embodiments, container 120 is by plastic manufacturing.The inner surface of container 120 is preferably It is chemically inert.Sample container 120 for example can be in emiction sampling cup, micro centrifugal pipe (such as Eppendorf pipes), centrifuge tube, refer to pipe, The shape of microwell plate etc..In some embodiments, container 120 has single-chamber/single chamber for keeping cell 180, bead 160 and stirring Part 156 is mixed, as shown in Figure 3.In some embodiments, some container 120 may include multiple discrete chamber/room (such as rows Hole), the mixture of cell 180, bead 160 and magnetic stirring part 156 can be each kept mutually isolatedly.In some realities It applies in example, sample container 120, which is preinstalled to be magnetic, stirring parts and/or cell cracking bead and to be promoted cell cracking and preserve target The chemical substance and/or enzyme of molecular biological activity.
System 100 may include multiple sample containers 120, multiple filters 124, one or more reagent racks 132 or its group It closes.Sample container frame 108 can be designed to keep multiple sample containers 120 and can be placed on the supporting surface of shell 104 so as to Handle more parts of samples 144 simultaneously.Equally, filter frame 112 can be designed to keep multiple filters 124 and can be placed in shell 104 Supporting surface on so as to simultaneously handle more parts of samples 144.Sample container frame 108 can also be used as the holder of sample 144 for Storage.Such as multiple sample containers 120 can be placed on sample container frame 108 and be stored in refrigerator or refrigerator before analysis In.
Referring now to Fig. 3, in use, sample container 120 and cylindrical magnet 116 are designed to：When making cylindrical magnet 116 When being rotated around its longitudinal axis, the magnetic stirring part 156 in sample container spins and to be enough to cause 180 fragmentation of cell and homogenize Power stir bead 160.
Cylindrical magnet 116 can have many magnet shapes or collocation form.In one embodiment, which has edge And surround the magnetic pole (i.e. north and south poles) that magnet longitudinal axis is arranged symmetrically.Magnet can have to be handed over about and along longitudinal axis The multiple opposite magnetic poles replaced, preferably even number, such as 2,4,6,8,10,12,14,16,18,20,22 and 24.In other implementations In example, which has the opposite magnetic pole being arranged on the longitudinally opposed both ends of magnet.In other embodiments, Cylindrical magnet is electromagnet.
Can on sample container 120, under or side make magnet 116 around across 116 center of magnet axis rotation. In certain embodiments, the surface mount that sample container 120 is placed perpendicular to sample container 120, and make magnet 116 around vertical Directly rotated in the axis on the surface that sample container 120 is placed.In other embodiments, sample container 120 is held perpendicular to sample The surface mount that device 120 is placed, and magnet 116 is made to be rotated around the axis for being parallel to the surface that sample container 120 is placed. In other embodiment, the surface mount that sample container 120 is placed perpendicular to sample container 120, and make magnet 116 around with The angled axis rotation in surface that sample container 120 is placed.The angle is more than 0 degree but is less than 180 degree.
Fig. 3 shows relative position of the magnet 116 relative to sample container 120.Magnet 116 is around axis A rotations and causes sample Magnetic stirring part 156 in this container 120 is in the same direction along the axis B rotations for being parallel to axis A.Although Fig. 3 is only shown One axis B, it will be apparent, however, to one skilled in the art, that magnetic stirring part 156 can be around being parallel to other A as shown in the figure Other B axle lines of axis rotate.The magnetic stirring part 156 of rotation collides bead 160 and in the process lytic cell 180.Magnetic Body 116 can with sample container 120 abreast or above, under or diagonally adjacent placement, sample container perpendicular to The chamber of sample container 120 or the surface 190 residing for holder are placed.
Sample container 120 and especially sample 144, bead 160 and magnetic stirring part 156 are located at the effect model of variation magnetic field In enclosing.Such as sample container 120 can be located in the sphere of action of rotary magnetic field, such as by adjacent or close to cylindrical magnet Container 120 is placed at 116.Changing magnetic field especially forces the movement of magnetic stirring part 156 such as rotation, reciprocating motion or combinations thereof, It forces bead 160, cell and liquid medium movement again.In some embodiments, the magnetic stirring part of sample suspensions body 144 156 to be enough to crack the rotating speed of the cell in container 120 and the duration is stirred.Suitable rotating speed and duration regard application Depending on and can rule of thumb be determined by those of ordinary skill in the art.In general, it is sufficient to which the rotating speed of lytic cell is by such as It is lower because usually fixed, such as cell type, the concentration of sample suspensions body 144, the amount of sample suspensions body, magnetic stirring part 156 ruler The size and shape of very little and shape, amount/number, size, shape and the hardness of cell cracking bead 160 and sample container 120.
In certain embodiments, magnetic stirring part 156 is between 1000~6000rpm, preferably about 5000rpm Speed rotates 1~600 second, preferably from about 90~120 seconds time.In certain embodiments, sample container 120 is (such as in urine The shape of analyzer cup or pipe) be placed on the magnetic stirring part in frame, and with highest setting speed (>1000rpm) it is stirred. In other embodiments, sample container 120 is the hole in minitype plate such as elisa plate.In other embodiments, sample container 120 be the cylindrical container with sample inlet and sample exit port.
In certain embodiments, the rotating speed of magnetic stirring part 156 is enhanced to improve lysis efficiency and shorten realization cracking The required time.In certain other embodiments, rotating speed is adjusted to only certain form of cell and is cleaved.Such as Including in the sample suspensions body 144 of 180 type of various kinds of cell, stirring parts 156 can be rotated with First Speed to crack first group Cell is then rotated with second speed to crack second group of cell.In other embodiments, container 120 is coupled to temperature control Molding block controls the temperature of the sample suspensions body 144 before, during and/or after cracking process.In some embodiments In, the temperature of sample suspensions body 144 is maintained at 2~8 DEG C.In some embodiments, sample suspensions body 144 cracking process it Before, among and/or (such as magnetic stirring part rotate during) is heated to 40~80 DEG C, 50~70 DEG C or about 60 later ℃。
Magnetic stirring part 156 can be manufactured by metal or metal alloy.In one embodiment, magnetic stirring part 156 by Stainless steel making.In other embodiments, magnetic stirring part 156 is by being coated with chemical inert material such as polymer, glass or pottery The alloy core of ceramic material (such as porcelain) manufactures.Exemplary alloy core material includes neodymium iron boron SmCo.Exemplary coating polymer packet Include biocompatible polymer such as PTFE and Parylene.
Magnetic stirring part 156 can be any shape and should being small enough to be placed in sample container 120 and Movement or spin or agitation in container 120.Magnetic stirring part 156 especially can be rod-shaped, columnar, criss-cross, V-arrangement , triangle, rectangle, rodlike or plate-like stirring parts.In some embodiments, magnetic stirring part 156 has rectangle shape Shape.In some embodiments, magnetic stirring part 156 has the tuning fork shape of double bifurcateds.In some embodiments, magnetic stirring part 156 have V-arrangement shape.In some embodiments, magnetic stirring part 156 has trapezoidal shape.In certain embodiments, stirring parts 156 longest dimension is slightly smaller than container diameter (such as about 75~95% of container diameter).
Cell cracking bead 160 can be any granular and/or pearl structure, and hardness is more than cell hardness. Bead 160 can be by plastics, glass, ceramics, mineral, metal and/or any other suitable material manufacture.In some embodiments In, bead 160 can be manufactured by non-magnetic material.Bead 160 can be rotational symmetry (such as ball about at least one axis Shape, circle, oval, oval, egg-shaped and drops particle).In certain embodiments, bead 160 has polyhedron-shaped. In other embodiments, bead 160 is irregular shape particle.In some embodiments, bead 160 is the particle with protrusion. Diameter possessed by bead 160 especially can be in the range of 10~1000 microns, 20~400 microns or 50~200 microns. 160 quantity of bead for being added to each cracking container especially can be in about 1~10000 milligram, 1~1000 milligram, 1~100 milli Gram, in 1~10 nanogram range.
After cell has cracked, cell lysate is inhaled into the filter to allow nucleic acid to be incorporated therein in suitable filter 124 Core matrix 126 (see Fig. 4).Generally, lysate before unbonded part is discharged 124 the same end of filter through filtration core matrix 126 at least twice.This moment, the combination nucleic acid in filter 124 can be stored in a sealed container for further when other Analysis.Alternatively, can be eluted using suitable such as elution buffer further described below from filter in conjunction with nucleic acid.
Fig. 4 shows exemplary filter.Filter 124 includes the multicellular monolith combination filter core matrix in embedded pipetting head 127 126.Monoblock combination filter core matrix 126 includes bulk sorbent agent or whole sorptive material.Multicellular monolith material and nucleic acid specificity Property combine and being made of in whole structure the basic of the self-supporting of rigidity.In some embodiments, multicellular monolith material does not wrap Containing the additional materials for generating nucleic acid compatibility.In some preferred embodiments, multicellular monolith material is the integral material of glass base Such as glass frit.In certain embodiments, glass frit is sintered glass frit.Multicellular monolith material such as glass frit or sintering The porosity of glass frit can be different according to application.In general, the porosity that multicellular monolith material should have allows The nucleic acid that has desired sample flow for specific application and can be maintained within the scope of desired size.In some embodiments, Monoblock combination filter core matrix 126 is glass frit, is made of two sections (126a and 126b) with Different porosities.
In some embodiments, multicellular monolith material is glass frit or sintered glass frit, and (be averaged porosity hole Size) 2~~400 microns, 2~300 microns, 2~220 microns, 2~200 microns, 2~180 microns, 2~160 microns, 2 ~140 microns, 2~120 microns, 2~100 microns, 2~80 microns, 2~60 microns, 2~40 microns, 2~20 microns, 2~16 Micron, 2~10 microns, 2~5.5 microns, 4~400 microns, 4~300 microns, 4~220 microns, 4~200 microns, 4~180 Micron, 4~160 microns, 4~140 microns, 4~120 microns, 4~100 microns, 4~80 microns, 4~60 microns, it is 4~40 micro- Rice, 4~20 microns, 4~16 microns, 4~10 microns, 4~5.5 microns, 10~400 microns, 10~300 microns, it is 10~220 micro- Rice, 10~200 microns, 10~180 microns, 10~160 microns, 10~140 microns, 10~120 microns, 10~100 microns, 10 ~80 microns, 10~60 microns, 10~40 microns, 10~20 microns, 10~16 microns, 16~400 microns, 16~300 microns, 16~220 microns, 16~200 microns, 16~180 microns, 16~160 microns, 16~140 microns, 16~120 microns, 16~ 100 microns, 16~80 microns, 16~60 microns, 16~40 microns, 40~400 microns, 40~300 microns, 40~220 microns, 40~200 microns, 40~180 microns, 40~160 microns, 40~140 microns, 40~120 microns, 40~100 microns, 40~ 80 microns, 40~60 microns, 100~400 microns, 100~300 microns, 100~220 microns, 100~200 microns, 100~ 180 microns, 100~160 microns, 100~140 microns, 100~120 microns, 160~400 microns, 160~300 microns, 160 ~220 microns, 160~200 microns, 160~180 microns, 200~400 microns, 200~300 microns or 200~220 microns In the range of.In other embodiments, multicellular monolith material is glass frit or sintered glass frit, with porosity difference Two sections (126a and 126b).Porosity possessed by each section can (such as 4~10 micron sections and 16 within the above range ~40 micron sections or 16~40 micron sections and 100~160 micron sections).
In some embodiments, the thickness of filter core 1~30 millimeter, 1~25 millimeter, 1~20 millimeter, 1~15 millimeter, 1 ~10 millimeters, 1~8 millimeter, 1~6 millimeter, 1~4 millimeter, 2~30 millimeters, 2~25 millimeters, 2~20 millimeters, 2~15 millimeters, 2 ~10 millimeters, 2~8 millimeters, 2~6 millimeters, 2~4 millimeters, 4~30 millimeters, 4~25 millimeters, 4~20 millimeters, 4~15 millimeters, 4 ~10 millimeters, 4~8 millimeters, 4~6 millimeters, 6~30 millimeters, 6~25 millimeters, 6~20 millimeters, 6~15 millimeters, 6~10 millimeters, 6~8 millimeters, 8~30 millimeters, 8~25 millimeters, 8~20 millimeters, 8~15 millimeters, 8~10 millimeters, 10~30 millimeters, 10~25 Millimeter, 10~20 millimeters, 10~15 millimeters, 15~30 millimeters, 15~25 millimeters, 15~20 millimeters, 20~30 millimeters, 20~25 Millimeter or 25~30 millimeters in the range of.
In some embodiments, multicellular monolith material can have with one or more to allowing the parent of the interested molecule of people It is modified with material such as polynucleotides, protein, lipid or the polysaccharide of property.In some embodiments, the multicellular monolith material It can be modified with one or more materials with nucleic acid compatibility.
In some embodiments, filter core is manufactured by cellular glass monoblock, porous glass ceramics or multicellular monolith polymer. In some embodiments, the manufacture of cellular glass monoblock is utilized such as United States Patent (USP) US4,810,674 and US4, described in 765,818 Sol-gel method, the document, which is hereby cited, to be included in.The manufacture of porous glass ceramics can be by cellular glass monoblock can Control crystallization.In a preferred embodiment, cellular glass monoblock, porous glass ceramics or multicellular monolith polymer be not coated or embedding The binding affinity for burying any additional materials such as polynucleotides or antibody to improve it with nucleic acid.
In some preferred embodiments, which is manufactured by the fine porous glass frit that liquid sample can be allowed to pass through.It is more Hole glass frit is uncoated or is not embedded with any additional materials such as polynucleotides or antibody to improve it to nucleic acid or people is allowed to feel The compatibility of other molecules of interest.Base material suitable for purification of nucleic acid includes the cellular glass frit manufactured by sintered glass, It is formed by crushing bead in hot press with forming single overall structure.The homogeneous texture of frit provides in frit Predictable liquid flow and allow eluent have fluid dynamics similar with sample flow.Predictable liquid flow is visibly moved Perhaps the high-recovery in elution process.
Although filter core matrix 126 is generally placed in pipetting head 127, it can also be loaded into different volumes and shape In the column of shape, syringe or other shells.Various instruments can be utilized to make solution through filtration core matrix 126, including manually or certainly Dynamic pipettor, syringe, syringe pump, hand held injector or for making other classes of the liquid motion through filtration core matrix 126 The automated process or manual method of type.
As shown in Figure 5 A and 5B, which can further include one or more reagent racks 132 being arranged within the casing.Reagent Frame 132 is designed to keep one or more reagents.Reagent rack 132 can be in hypocrateriform, and reagent can be when being ready to use It is introduced into wherein.In the case, the reagent can be introduced into pallet with promote reagent be inhaled into multiple pipetting heads so as to It is sent in 144 processing procedure of sample to multiple sample apertures.Alternatively, reagent rack 132 can be in block or include the porous plate in multiple holes 152 (such as 24 holes, 96 holes) form, thus each in this some holes be designed to keep any reagent so as to handle it is a individually Sample 144.In certain embodiments, hole 152 can be pre-filled with reagent and be sealed in frame 132.In some embodiments, Frame 132 is located near sample container frame 108 and/or filter frame 112 (Fig. 5 B).
Sample purification system 100 can with manual operation or it can be designed to it is semi-automatic or complete by programmable logic Automatically run.In certain embodiments, which may further include aspirates system 136 (Fig. 5 A) and one or more automatically A manipulator (not shown), manipulator design at scheduled computer control mode automatically by reagent from one or more reagents Frame 132 is assigned in multiple sample containers 120 and handles in sample material and agents useful for same to suitable waste canister 172 (figure 5B)。
In an operation mode, reagent rack 132 is in multiwell plate format (such as 24 holes, 96 holes).Preferably, mixture is logical It crosses and is disposed using automated fluid movement to mix, because this prepares the work completed required for mixture to be investigated by reducing It measures.Robot can also execute automatic sampling experiment operating process using device and method known in the art.
Any suitable machine or equipment can be used to make sample 144 to be passed through automatic purification system 100 and its each Different processing steps.Such as system 100 used herein can be come from using various robots known in the art The movement of dynamicization sample 144, reagent and other system units.Illustrative robot system can make sample in axis, two It is moved on a axis or three axis and/or keeps sample dynamic around an axis, two axis or three shaft rotations.Illustrative robot is in track Upper movement, track can be located at workpiece on, under or side.Generally, robot components include functional component such as manipulator, It can be captured and/or travelling workpiece, is inserted into pipette, distribution reagent, suction etc.." manipulator " used herein refers in this way Device, preferably by microprocessor control and physics transmission sample 144, container 120, filter 124, sample container frame 108, filter Head frame 112 and reagent rack 132 are from one place to another place.Each place can be one in automatic purification system 100 A unit.Software for manipulator control can usually obtain from robot manufacturers.
Robot can be made to translate in orbit, such as onto a working area by side, lower section or side, and/or may include hinge Socket part section allows manipulator to reach the different location of workspace.Robot can be driven by motor known in the art, motor It may, for example, be electronic, pneumatic or hydraulic-driven.Any suitable driving control system can be used to control machine People, such as standard PLC programming or other methods known in the art.Optionally, robot includes position feedback system, with light It learns or mechanical system measures position and/or power and robot is allowed to be directed to desired location.Optionally, robot also includes position Assurance mechanism such as mechanical stop, cursor or guiding laser are set, allows to reach specific position repeatedly.
Illustrative automation sampling experiment operating process can for example using Eppendorf epMotion5070, EpMotion5075, Hamilton STARlet, STAR and STAR+Liquid movement robot.Such experimental implementation flow can To be modified to detach RNA, separation genomic DNA and cycle dissociative DNA from entire blood, tissue, saliva, swab Such as Circulating tumor DNA and the cycle foetal DNA extract from Maternal plasma and enriched substance.
Nucleic acid purification method
On the other hand, a method of for purifying the nucleic acid from sample, include the following steps：(a) it provides according to this The nucleic acid purification system of text；(b) by sample, magnetic stirring part and multiple bead injecting sample containers；(c) make cylindrical magnet around it Longitudinal axis rotates, and mixed and disorderly is mixed to being enough to homogenize with undergo cellular content to which magnetic stirring part spins and stirs bead The sample and cell in broken sample and the degree for forming cell lysate；(d) make at least part of cell cracking logistics First opening of filtering head, to which the nucleic acid in cell lysate is bound to the filter core in filter；(e) it is open cell through first The filter is discharged in the unbonded part of lysate, wherein the unbonded part before leaving filter through filtration core at least twice； (f) by making elution buffer flow through the first opening of filter and elution buffer being ejected elution from filter through the first opening Be bound to the nucleic acid of filter core, wherein the elution buffer before leaving filter through filtration core at least twice.
Any mode for executing the present processes, including complete manual, semi or fully automatic reality may be used Test operating process.But the feature of filter, adaptability, simplicity and work flow admission its be easy to adjustment, automation and it is effective In multiple clinical sample matrixes, input sample amount and liquid transporting system.Then, in a preferred embodiment, the operation side Formula includes certain automation.In one embodiment, which includes aspirating system and one or more machines automatically Tool hand, the manipulator design for distributing in reagent to one or more sample containers and according to predetermined way by sample material automatically Material and reagent are disposed to suitable disposable container.In the case, each above-mentioned steps are in the multiple sample container It is repeated using the filter of equivalent amount and one or more reagent racks in each.
The doubtful sample containing MTB is potential danger to user.Therefore, sample can be by heating and/or being added examination Agent is pretreated, and the reagent is suitable for inactivating the microorganism being present in sample to alleviate the danger.Microorganism such as MTB's goes out Work can execute (such as 90 DEG C, 5 minutes) so as to reactive protein denaturation, the enzymic digestion of cell wall structure, machine by heating Tool is broken so that physics is broken or killed cells, chemical treatment.
Chemical ablation, which is brought, reduces or eliminates the potentially possible of the demand to heat.When handling sample to cultivate, letter Single reagent is used to digest the phlegm and sterilizes the sample.In order to cultivate, the other groups inactivation that will be present in sputum sample sheet is Important, to which MTB can be grown without being overwhelmed by other bacteriums more rapidly grown.It is preferably selected and reagent example may be used Decontamination or inactivation step such as sodium hydroxide (such as 3~5%) or cetylpyridinium chloride inactivate all other bacterium, but It keeps the MTB cells with thicker firmer cell wall intact and lives.But according to method used, 20~ 90% MTB cells can be killed in the process.But about nucleic acid purification, MTB cells need not to be living or excellent, As long as bacterial genomes DNA remains to be amplified.
Inactivation reagent preferably allow for limiting dilution and/or the low ph value of sample with obtain combined with silica it is compatible Property.These reagents can be added into sample in acquisition.In some embodiments, hydrogen peroxide, alcohol such as ethyl alcohol and neighbour Phenylphenol (such as 0.2~0.5%) is used as main active.Hydrogen peroxide (H2O2) can be according to 6~25% concentration It is used as chemosterilant and highly stable in the solution.When being mixed with 0.85% phosphoric acid, H2O2It is activity at low ph values 's.Only ethyl alcohol (such as 95%) can inactivate the MTB in phlegm or water in 15 seconds.O-phenyl phenol as agricultural fungicides According to 0.1~0.41% together with ethyl alcohol or isopropanol in PHENO-CEN, SRAYPAK and CLIPPERCIDE spraying fungicide To use.In addition, o-phenyl phenol can according to the ratio between low reagent and sample room temperature used in 15 minutes and it It can be used in ethyl alcohol or isopropanol or be combined in low ph value Phenolic 256 with 6.65% 2- benzyl -4- chlorophenols It uses (50%~100%).
The volume ratio for inactivating reagent and sample volume generally will be about 0.1:1 to 3:In the range of 1.
Bacteria inactivation rate in primary sample container, which is important offer BSL-1 compatibility workflows, (to work Stream does not need Biohazard Safety Equipment).Many experimental implementation flows involve the transfer of the sample before disinfection, and such operation may Generate aerosol and infection user.Therefore, BSL-1 compatibilities need the extremely carefully sample transfer before disinfection, especially It is the sample transfer that those will produce aerosol and infect user.
According to sample properties, sample can be liquefied at the beginning with reduce its viscosity and it is heterogeneous so as to consistent sample at Reason.Sputum sample is a special challenge.MTB in phlegm is one of the cell and sample type of most processing challenge, because antiacid Bacillus has the sticky heterogeneity of the hydrophobic cell wall of rich fat and phlegm.Standard extraction methods for phlegm are generally opened with infall process Begin, it is typically involved in is handled with N-acetyl-L-cysteine (NALC) and sodium hydroxide (NaOH), is followed by centrifugation point From, clarification and settling flux.Therefore, in the processing sticky sample such as phlegm of height, sample can receive to be chemically treated to reduce viscosity, So that it is unaffected to be followed by subsequent processing step (such as MagVor).Exemplary slime solubilising reagent for being added to sample include but It is not limited to NALC, benzyl dimethyl alkylamine-tertiary sodium phosphate (Z-TSP), benzalkonium and Primestore (Longhorn Vaccines＆Diagnostics, San Antonio Tekes), containing be useful for bacteria lysis and RNA and DNA stablize it is specific Formula.In one embodiment, it is liquefied by the chemically treated sample by mucolysis reagent and is carried out 20 minutes at 60 DEG C. Patient's sputum sample can generally be acquired according to the volume in 1~10 milliliter of range, 5~10 milliliters of ranges or bigger.
In 90s-1Under shear rate, viscosity ranging from about 100~6000cP (mPa.s) of phlegm.It is measured with mPas glutinous Degree is determined by shearing strength divided by shear rate.The sample is preferably liquefied for phlegm viscosity being reduced at least 50%, extremely Few 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.
When handling sample, at least one magnetic stirring part and multiple cell cracking beads are present in sample container.Make Can simply sample container be added in sample suspensions body by user, sample container is placed close to cylindrical magnet, and by making the magnetic Body be enough to allow rotary magnetic field cause magnetic stirring part rotate and be enough to homogenize and lytic cell stirred in sample container The speed of cell cracking bead stirs sample suspensions body.
Sample suspensions body, cell cracking bead and magnetic stirring part can be placed into sample container in any sequence. In some embodiments, sample suspensions body is added into before cell cracking bead and magnetic stirring part in sample container.At it In its embodiment, cell cracking bead and/or magnetic stirring part are placed into sample container before sample collection.
In certain embodiments, the cracking of specific cell type can be by that will add before and/or during whipping step Add agent that sample suspensions body is added to facilitate.The example of additive include enzyme, detergent, surfactant and other chemical substances such as Alkali and acid.It has been found that alkaline condition (such as 10mMNaOH) can improve cracking in whipping process for certain form of cell Efficiency.Sample suspensions body can also or alternatively be heated to enhance lysis efficiency in whipping process.But additive may It is harmful to the downstream processing stages comprising nucleic acid amplification and detection and should be eliminated when possible to simplify this process.
Stirring parts/bead combination brings many advantages relative to traditional cleavage method.Stirring parts/bead is than chemistry and enzyme Rush mode is quickly many, and improved cell or virolysis is provided relative to the physical disruption method of many other types. Stirring parts/bead method is also easy to automate using robot and/or microflow control technique.Cylindrical magnet is reusable, does not need It precisely aligns container and multiple chambers can be driven.Magnetic stirring part it is of low cost, allow its single disposable.
After MagVor steps, the suitable combination buffer containing one or more chaotropic agents is added into sample container To promote nucleic acid to be bound to filter core matrix 126.When solution ph is less than 7, BOOM chemistry or rush of the nucleic acid to silica Cementing conjunction is most efficient.In the case, the high inonic strength solution of chloride containing lithium or sodium chloride or guanidine radicals ion generally with Aliphatic alcohols such as ethyl alcohol or isopropanol combination to salt out DNA and nucleic acid promoted to combine respectively.Suitable combination buffer is with one Determine concentration to be used, to which when sample, caused volume is in filter range of capacity after it is added into processing.This is reduced It inhales and puts number of cycles and and then shorten total processing time.
In certain embodiments, combination buffer is added into sample and is cultured 10 points at 60 DEG C after MagVor Clock.In other embodiments, in MagVor steps before, chaotropic agent and fatty alcohol are added into liquefaction step.In other realities It applies in example, the inactivation, the step of homogenizing and cracking are completed in the form of single step being short in 15 minutes.
Illustrative chaotropic agent includes but not limited to dissolution salt such as guanidine thiocyanate, guanidinium isothiocyanate, guanidine hydrochloride, chlorination guanidine Urea, thiocarbamide, lauryl sodium sulfate (SDS), cetylpyridinium chloride(CPC), sodium chloride, lithium chloride, potassium chloride, sodium perchlorate, high chlorine Sour lithium, sodium iodide and potassium iodide；Fatty alcohol such as butanol, ethyl alcohol, propyl alcohol and isopropanol；Phenol and other oxybenzene compounds.
In some embodiments, in order to promote high molecular weight (HMW) nucleic acid to bind selectively to filter core matrix, with 0% to About 10%, fatty alcohol such as isopropanol preferably is provided in about 4% to about 6% (most preferably=4.7%) range, and extremely with 1.0M 4.0M, preferably about 3.0M to about 4.0M ranges provide dissolution salt such as guanidinium isothiocyanate and/or guanidine hydrochloride.
In some embodiments, in order to promote low molecular weight (LMW) nucleic acid to the combination (concentration) of filter core matrix, with about 10% to about 25%, preferably from about 15% to about 20% (best=17.7%) range offer fatty alcohol such as isopropanol.
In other embodiments, in order to promote to isolate MTB DNA from phlegm, can about 20% to about 60%, preferably from about 30% to about 50% (best=44%) range offer fatty alcohol such as ethyl alcohol.
After above-mentioned inactivation and homogenization step, the pH value of solution should be as needed adjusted to obtain less than 7 PH value.In the case where pH value is higher than 7, solution can be with weak acid such as potassium acetate or sodium phosphate by neutralisation.This using pH >= To be required when 11 NaOH or o-phenyl phenol.Low ph value phenol reagent and hydrogen peroxide agent are intrinsic acid and will Most unlikely need additional buffering liquid.
In one embodiment, nucleic acid be bound to filter core matrix can by by therebetween luer lock connect Filter 124 is attached to syringe 176 to realize.In another embodiment, filter core matrix is in syringe 176.Fig. 4 is shown Illustrative filter 124.Filter 124 includes the porous silica silicon substrate 126 being embedded in filter body 127, for preventing Pollution user and the aerosol filtration part 128 being exposed in face of user and the filter cap 129 for assisting in keeping closed system. In one embodiment, cap 129 is connected to the filter 124 with chipware.In some embodiments, cap is common Falcon pipes Cap 208.Filter 124 is designed that liquid sample to suck and be distributed through matrix every time with filter 124.Utilize syringe 176 or other suitable instruments, the cell lysate in container 120 pass upward through the distal end of filter 124 so that cell lysate In nucleic acid be bound to the filter core matrix 126 in pipetting head 127.Generally, cell lysate is pulled past filter core matrix up and down 126 so that lysate and unbonded part are discharged to suitably once in unbonded lysate part through 127 distal end of pipetting head Property container 172 before through filtration core matrix 126 at least twice.
The combination of sample and dissolution reagent makes DNA and Silica dehydration to promote to be adsorbed onto porous silica silicon substrate On 126.The suction of subsequent cleaning buffer solution puts the period (2~3 times) and removes impurity from matrix 126.This moment, containing being bound to filter core The filter 124 of the nucleic acid of matrix 126 can be stored in the container 120 of sealing further to analyze when other.Or Person can utilize suitable elution buffer to be as further described below eluted from filter in conjunction with nucleic acid.
Nucleic acid be indicated in the solid supports including silica be it is extremely stable, especially when without When being stored under dewatering state to additional stability agent.Therefore, on the other hand, the application provides a kind of stable purification of nucleic acid to transport The form of defeated method, this method is the disposable transport device 200 being intended for single use, which includes being attached to suitably Keep the Luer lock splice grafting head 204 of the top side of the cap 208 of (such as the 50 milliliters of conical pipes) of pipe 212 so that filter 124 is attached to The bottom side (Fig. 6) of pipe cap 208.
Before use (i.e. Nucleic Acid Elution is for analysis), the filter 124 for being attached to cap 208 is taken from holding pipe 212 Down and it is attached to syringe 176 as shown in Figure 2.In the case, user can be easy to be inserted by Luer lock splice grafting head 204 With remove filter 124, while holding to pipe cap 208 so that keep pipe 212 to protect user and filter 124 in order to avoid pollution.When this At the end of operation order, it can be dried with the porous silica filter core matrix 126 in conjunction with nucleic acid, the filter 124 with cap It is swirled on empty holding pipe 212 to transport.During transportation, pipe 212 is kept to protect filter 124 from pollution.Stablize The nucleic acid of change can then with elution buffer by rehydrated and be eluted in storage pipe to freeze to store for a long time, or using with Similar automated system used in clinic or be simply directly eluted to using disposable syringe 176 test and analyze device or In person's sample tube.In the latter cases, syringe 176 may be used as passing through silica substrate 126 for elution buffer Inhale the mechanism for putting the period.
When preparing analysis of molecules, the suction of elution buffer is put the period (2~3 times) and is removed in conjunction with nucleic acid from matrix 126. The nucleic acid purification resulted in PCR compatible buffered solutions that finishes of the process.This way allows the flexibility for specification, It can be used in conjunction with liquid transporting system for high flow rate application to it or be used in conjunction for low stream with simple pipetting head Rate application.
Fig. 7 shows the illustrative steps sequence for carrying out MagVor/ filter purifying to the nucleic acid from phlegm.It is doubtful containing The sputum sample of MTB is collected with sample container.Chemical reagent mixture containing inactivation reagent and mucolysis reagent is added (step It is rapid 1).Sample container is then placed in extraction seat (or frame) and sample content object be subjected to mangneto be vortexed (MagVor) up to 2~ 15 minutes, preferably from about 10 minutes (steps 2).After cell cracking, bead pellets are allowed 1~2 minute and combination buffer quilt Container (step 3) is added.Filter is attached to the bottom side of the pipe cap in Fig. 6/Luer lock splice grafting head and utilizes Luer lock by user Syringe is connected to the top side (step 4) of pipe cap/Luer lock splice grafting head by fixed connection.Filter is perforated through cap by user from top And it is pierced into container, and cell lysate is sucked into filter and syringe stem is made to move up and down into filtering core matrix 2~3 times to promote It is bound to filter core matrix, unbonded part is thus made to return to (step 4) in pipe.After combining step herein, sample container is with being equipped with The new pipe of cleaning reagent is replaced, and filter core matrix collects the cleaning buffer solution in pipe and is cleaned (step 5).It is walked in cleaning After rapid, pipe is subsequently lifted out liquid.In some embodiments, filter is further dried by passing air through filter core matrix (step 6).In some embodiments, being air-dried to reduce remaining cleaning reagent for several bouts is carried out.Then, user makes Filter/cap adaptation connector is detached from syringe, filter is put back into the new holding pipe equipped with drier, and filter/cap is adapted to Connector, which is attached to, keeps pipe to store (step 7).Nucleic acid in filter is stable for transport or can be from filter Nucleic acid is purified by flash for PCR analyses etc..The elution of nucleic acid can be by making elution buffer through filtration core matrix 2 before collection It is completed to 3 times.
Additive such as trehalose, 0.1%Triton-X-100 orPlus reagents (Biomatrica) can To be added into elution buffer or be added into elution nucleic acid to enhance its stability.
In certain embodiments, this method further comprises following steps：Nucleic acid is eluted, with the primer for being exclusively used in pre-determined target Whether amplification elution nucleic acid determines sample containing the nucleic acid corresponding to target.The target for being preferred for detection is included in and finds in phlegm Bacterium and viral pathogen, including but not limited to MTB, aurococcus, methicillin-resistant staphylococcus aureus (MRSA), streptococcus pyogenes, streptococcus pneumonia, Streptococcusagalactiae, haemophilus influenzae, haemophilus parainfluenzae, catarrh be not Draw bacterium, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, acinetobacter calcoaceticus, Bordetella pertussis, Neisseria meningitidis, charcoal Subcutaneous ulcer bacillus, Nocard's bacillus, actinomyces, mycoplasma pneumoniae, chlamydia pneumoniae, Legionnella, Ye Shi lungs pityrosporion ovale, Flu-A disease Poison, cytomegalovirus and rhinovirus.
System 100 as described herein can be detected less than 1000 cells/mls, preferably shorter than 100 cells/mls, more The MTB of preferably shorter than 50 cells/mls, most preferably less than 10 cells/mls level.It is assumed that 1 colony forming unit (cfu) is substantially Equal to 10 cells, above system can be used at least 100cfu/ milliliters, 10cfu/ milliliters, 5cfu/ milliliters or even 1cfu/ milliliters of detection.
However, it should be understood that each clinical sample is unique and will be in viscosity, particle, mucus, surface contamination It is different from each other in terms of object, microorganism and/or human gene background.It suppose there is clinical sample ingredient and automatic filter sample preparation be real Test the set performance of expected change used of operating process, it is thus possible to need to change certain steps in filter program it is expected As a result.
For example, although there is filter as described herein larger pore size, sample to homogenize and liquefy for high-efficiency fine Cellular lysate and the step of then being combined with filter core matrix, are of crucial importance.Also can with homogeneous and good liquefied lysate, sample With higher flow rate through filtering head, this shortens total sample process time.Below as shown in a large amount of plasma assay operating processes, Big input sample amount can be effectively treated with following filter, which provides thoroughly to homogenize and liquefy to user and be difficult to The chance of the sample (online or offline) of processing, only needs the concern input sample amount of minimum degree.
In addition it should be appreciated that the relatively slug flow speed during nucleic acid combines or elutes generally results in higher nucleic acid yield, Although having paid the cost of total processing time.DNA shear rates are also reduced into minimum level compared with slug flow speed.
It is recommended that filter core matrix is completely dried to prevent residual solvent from being eluted jointly with purification of nucleic acid sample and inhibit downstream Processing or experiment.Because filter is not dried by centrifugation or vacuum filter, therefore by flow velocity and period in drying steps Both numbers, which are increased to, to be utmostly important.
Because the attachment method of shape, frit material and extremely mechanical channel hand is all only for every apparatus manufacturer Special, therefore each liquid transporting system needs different filters to construct.Filter core matrix size (diameter, thickness and pore size) and core Sour binding ability is related (to elution efficiency), just as to as desired by any solid phase extraction techniques.Although thick (>4 millimeters) Matrix can be embedded into 1 milliliter of filter with enhance for great amount of samples nucleic acid binding ability and/or balance specifically filtering Matrix binding ability between head specification, the flow velocity in filter thickness and initial (existing in former lysate) in conjunction with step Between still have tradeoff.Therefore, advantageously major diameter matrix is embedded in big flux filter for automatic experimental implementation sometimes Initial step (such as 5 milliliters of Hamilton/Akonni for largely extracting of flow).It suppose there is by liquid The specific filter configuration that body movement robot building quotient specifies, but it is expected filter nucleic acid yield in the liquid from different manufacturers All it is identical and unreasonable between body movement platform or between different filter sizes.The clinic of automatic filter experimental implementation flow Assessment and will detailed report elsewhere with the direct comparison of available commercial automatic system.
MagVor/ filter processes have many advantages compared to conventional method.First, this process is compatible with automation.Secondly, Filter core matrix, which is constrained in pipetting head, reduces its easy cross contamination.On the other hand, from the fibrous dioxy on capstan SiClx matrix easily can be crushed and release may be pollution sources fine particle.Similarly, magnetic bead grain activity is relied on Property brings similar danger so as to the technology of purifying.The relatively large porosity of filter core matrix allows full-bodied sample to flow through base Body is without blocking.Multiple suckings and assignment period allow the enhancing compared to the centrifugal separation using sample single pass matrix Target nucleic acid combine.In addition, dissolution chemistry is provided perfects method for remove inhibitor and nuclease, and make it have length Phase stability.
Further it will be not to be read as restrictive example by below and illustrate the present invention.Text of the statement is drawn All bibliography, patent and Patent Application Publication and the content of attached drawing and table, which are cited, to be included in herein.
Example
Example 1：MagVor is homogenized and is cracked
The effect of MagVor systems is tested relative to the MTB in Dipel spore, streptococcus pyogenes and phlegm, thus Utilize the ratio between volume v:V is 1:1 glass beads are cracked with sample, 1 milliliter of total sample volume and 30~120 seconds MagVor. Extraction MTB DNA are extremely challenging for its Mycobacterial cell wall and low (10 bacillus) infective dose from raw phlegm 's.Cracking effect gives birth to the quantitative real-time PCR of sputum sample sheet to estimate by equivalent before and after MagVor is handled.MagVor Processing be found relative to the untreated sample aliquot of i.e. identical sputum sample sheet (but do not handled by MagVor systems) with Detection of nucleic acids is improved by average 2.5 periods (approximate 1log).
Nucleic acid extraction and purifying are may interfere in order to investigate physical disruption kit forms (particle, agitator disk, coating) Degree carries out the analysis to four kinds of beads and three kinds of magnetic plates and is surpassed with identifying to generate in the solution after MagVor cracking Fine grain cracking bead and magnetic plate.NPA samples are assembled, and are identified by real-time PCR to allowing the interested target DNA of people Feminine gender, then with intact methicillin MRSA or MTB target cell tracers.Sample (0.5 milliliter) is handled in duplicate And it is cleaved by the MagVor processing in 10 minutes of 5000rpm.Nucleic acid is used to be filtered using the artificial MagVor/ of guanidine radicals combination buffer Head program is purified, then analyzed by quantitatively real-time PCR (or RT-PCR).Glass beads be easily precipitated to cracking bottom of the tube and The apparent inhibition or degradation to DNA or RNA are not shown in these experiments.
As shown in table 1, compared to untreated, the one of the DNA rate of recovery is obtained using MagVor processing especially under high titre It causes to improve.
Table 1.MagVor/ filters are from tracer NPA to the synthetical recovery of MRSA and MTB DNA：
Example 3：The comparison of comprehensive MagVor/ filters prototype and Qiagen Nucleic acid purification kits
The nucleic acid cleavage and purification efficacy of integrated system are evaluated compared to similar Qiagen Nucleic acid purification kits.Mould Type sample type includes the Flu-A in MRSA, NPS in NPA, the MTB in human gene DNA and NPA from whole blood. Qiagen kits include DNA mini kits (no mechanical lysis, but have the processing of 10 minutes Proteinase Ks), Viral RNA mini Kit (no mechanical lysis, but using RNA carriers) and Mini Blood kits (Proteinase K culture in 10 minutes).Because Qiagen does not have the kit for being exclusively used in MTB extractions, therefore BD GeneOhm lytic reagents boxes and the mini DNA of Qiagen are extracted Kit is applied in combination.BD crack and Qiagen kits also have limited input sample amount, therefore, NPA and NPS samples with 200 microlitres of volumes are handled, and whole blood is handled with 100 microlitres, 10 microlitres and 1 microlitre volumes.For each sample type and drop Degree (every part of sample n=24 extraction) prepares, sealing and processing replicate agent plate, right by quantitative real-time PCR analysis purification of nucleic acid Threshold average period obtained than its threshold value average period (Ct) and from similar Qiagen extractions (every part of sample n=8 extraction) Value.Positive and negative control is carried out to detect potential cross contamination with each plate.
The result of the analysis is summarised in table 2 and shows the similar performance and effect relative to Qiagen kits Power.For both systems, the detection limit condition of the Flu-A in MRSA and NPS in NPA may each be about 103Cell or disease Poison/milliliter.Human DNA is easy to obtain from 1 microlitre of whole blood, and Template Controls show free nucleic acid cross contamination evidence.These numbers According to showing comprehensive autgmentability and effect of the sample preparation prototype relative to available commercial high-quality sample preparation reagent box.
Table 2. is recycled relative to the nucleic acid from MagVor- filters system (n=24) of other DNA extraction kits
Example 4：Sputum
Because most of submit the sample for mycobacteria culture before analysis by energy rapid multiplication mycobacteria Various organic pollutions, therefore breath sample is generally submitted to digestion-decontamination pretreatment.Then, by using reducing or disappearing The operation sequence that the sunken mycobacteria stayed in mucoitin and cell is released while depollution bacterium is come from clinical sample Most preferably withdraw mycobacteria.
NALC-NaOH sedimentations have become for decontamination and digest the sputum sample of non tuberculous Mycobacterium (NTM) Standard.But extract MTB DNA in spontaneous phlegm and be rich in challenge because be on the one hand phlegm high viscosity and it is heterogeneous and On the other hand it is the MTB cell walls of difficult rupture.It is (just cold that the transport of DNA extracts compared to raw phlegm alleviates logistics complexity For transport).Although NALC-NaOH is strictly a digestion process, its activity of NALC rapid loss, it is desirable that restore daily new Fresh reagent.In addition, this program needs to centrifuge, complexity and equipment are added additional.In addition, NaOH exposures cause MTB Cell death and degradation of dna.
Therefore, it is to make people interested to develop following liquifying method, can be used as interesting to the raw phlegm of processing User the optional means of replacement.Because making a living, phlegm is difficult to send to the sample type of sample container, therefore develops molten using enzyme The single stage phlegm liquefaction process of liquid.It is cultivated 15~20 minute to 10 parts of raw phlegm and at 56 DEG C by the way that 1 part of liquid enzyme is added, or even high Heterogeneous and sticky sputum sample is spent also to be liquefied to viscosity similar with 5~10% glycerine.These liquefaction sputum sample easily transfer and employments Work MagVor and filter experimental implementation flow processing, without causing the magnetic plate in cracking tube to stop operating or block filter Head.
Example 5：Spontaneous phlegm extraction MTB DNA
Automatic 8 channel prototype system as shown in Figure 5 A and 5B be used to indicate that from the TB positives life phlegm extract DNA can Row.Using Truant TB fluorescent colorants, the clinical samples of None- identified are confirmed as by smear for microscopic examination or Smear 2+ or Smear 4+.The Smear 2+ life sputum sample sheet and quartering Smear 4+ lifes sputum sample of the quartering this according to the liquid in example 4 Change experimental implementation flow to be handled and be then added into MagVor pipes.Comprehensive MagVor/ filter test operation flows are then led to It crosses and automatically extracts/purify progress.Eluent is analyzed with IS6110qPCR analyzers, finds the concentration of extract for Smear 2+ It is 3.6 ± 0.7pg/ μ L, is 49 ± 8pg/ μ L for Smear 4+.This data supports automatic nucleic acid separator to be used for from the TB positives The feasibility of sample extraction DNA.
Table 3 show dilution series research as a result, its compare by automatic system MTB in real time detect and by people The MTB of work MagVor/ filter systems is detected in real time.MTB cells are impregnated in 500 microlitres of TB feminine genders phlegm and sediment (through NALC- The phlegm of NaOH processing) in, here, 10 cells are roughly equal to 1cfu/ milliliters.Corresponding to acid-fast bacilli (AFB) smear-positive and The corresponding cellular level of AFB smear negatives is included into compare.
Table 3. shows the dilution series research that the MTB DNA by artificial and automatic MagVor/ filter systems are detected in real time
Described above is to instruct how those of ordinary skill in the art realize the present invention, it is therefore intended that is described in detail and all Those are well-known apparent modifications and changes for the those skilled in the art for having read this specification.But intend all Such apparent modifications and changes are covered in the scope of the invention by claims limited below.Claims purport Cover building block claimed and in any sequence the step of, the sequence effectively meets set there Purpose, unless context clearly indicates otherwise.

Claims (20)

1. a kind of Sample purification system, including：
Shell；
The sample container frame being arranged in the shell, wherein sample container erection is calculated as keeping one or more samples Container；
The filter mounting being arranged in the shell, wherein the filter mounting is designed as keeping the one or more to include For combine allow the interested molecule of people filtration members filter；With
Cylindrical magnet close to the sample container frame and except the sample container frame, the magnet is by being arranged in the shell Motor around the magnet central longitudinal axis rotate.
2. Sample purification system according to claim 1 further includes the sample container being arranged in the sample rack, wherein the sample This container includes sample suspensions body, magnetic stirring part and multiple beads.
3. Sample purification system according to claim 1 further includes being arranged in one or more of shell reagent rack, described Reagent rack includes multiple reagents in the container for the sealing for being stored in several separations in each frame.
4. Sample purification system according to claim 1 further includes that automation aspirates system.
Further include one or more manipulators 5. Sample purification system according to claim 4, the manipulator design be for Distribute in reagent to one or more of sample containers and handle sample material and reagent automatically according to predetermined way.
6. Sample purification system according to claim 5, wherein the system further includes multiple sample containers, multiple described Filter and one or more reagent racks.
7. Sample purification system according to claim 1, wherein the cylindrical magnet has the longitudinal axis along and about the magnet The magnetic pole being arranged symmetrically.
8. Sample purification system according to claim 1, wherein the cylindrical magnet has the opposed longitudinal direction for being arranged in the magnet Opposite magnetic pole on both ends.
9. Sample purification system according to claim 2, wherein the bead is two of diameter in 10~1000 micron ranges Aoxidize silicon beads.
10. Sample purification system according to claim 2, wherein the magnetic stirring part includes the alloy core for being coated with polymer.
11. Sample purification system according to claim 10, wherein the alloy core includes neodymium iron boron or SmCo, and wherein, The polymer is PTFE or Parylene.
12. a kind of purifying the side for allowing the interested molecule of people from sample using Sample purification system according to claim 1 Method, including：
Sample tube equipped with liquid sample suspended substance, magnetic stirring part and cell cracking bead is placed on the sample rack；
By making the cylindrical magnet in the presence of the magnetic stirring part and the cell cracking bead to be enough to crack the sample The speed of cell in suspended substance rotates come the sample suspensions body that homogenizes；
In the case where allowing the interested molecule of people to be bound to filter core matrix, make the sample suspensions body to homogenize flow through to include The filter of the filter core matrix, wherein the filter is installed on the filter mounting；
Clean the filter core matrix；With
The interested molecule of people is allowed from elution of bound on the filter core matrix.
13. method according to claim 12, wherein the sample tube is preinstalled with selected from the one or more of the following group, the group by Magnetic stirring part, cell cracking bead, promote cell cracking reagent and reservation allow the interested molecule of people integrality examination Agent forms.
14. method according to claim 12, wherein described that the interested molecule of people is allowed to be nucleic acid.
15. method according to claim 14, wherein the liquid sample suspended substance includes phlegm.
16. method according to claim 15, further comprising the steps of：This is expanded with the primer for being exclusively used in mycobacterium tuberculosis The nucleic acid of elution simultaneously determines whether the nucleic acid includes Mycobacterium tuberculosis DNA.
17. a kind of method for purifying the nucleic acid from sample, including：
Sample tube is placed on sample rack, wherein the sample tube is split equipped with liquid sample suspended substance, magnetic stirring part and cell Solve bead, wherein the sample rack is located near cylindrical magnet；
The cylindrical magnet is set to be rotated around its longitudinal axis, to which the magnetic stirring part in each sample tube spins and stirs cell Cracking bead generates the degree of cell lysate to the cell fragmentation for being enough to make in sample suspensions body；
At least part of cell lysate is set to flow through the first opening of filtering pipetting head, to come from the cell cracking The nucleic acid of object is bound to the filter core matrix in the pipetting head；
By the unbonded part for flowing through the filter core matrix in flow step of the cell lysate from described in the filter First opening discharge；
As follows elution of bound to the filter core matrix nucleic acid：Elution is added by first opening through the filter Buffer solution makes the elution buffer flow through the filter core matrix, and is had passed through from filter discharge through first opening The elution buffer of the filter core matrix.
18. method according to claim 17, wherein the sample includes phlegm.
19. method according to claim 17, further comprising the steps of：This is expanded with the primer for being exclusively used in mycobacterium tuberculosis The nucleic acid of elution simultaneously determines whether the nucleic acid includes Mycobacterium tuberculosis DNA.
20. method according to claim 17, wherein the cylindrical magnet has the symmetrical cloth of longitudinal axis along and around the magnet The magnetic pole set.
CN201580085776.0A 2015-12-01 2015-12-01 Comprehensive sample processing system Pending CN108603221A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2015/063232 WO2017095394A1 (en) 2015-12-01 2015-12-01 Integrated sample processing system

Publications (1)

Publication Number Publication Date
CN108603221A true CN108603221A (en) 2018-09-28

Family

ID=58797568

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201580085776.0A Pending CN108603221A (en) 2015-12-01 2015-12-01 Comprehensive sample processing system

Country Status (5)

Country Link
EP (1) EP3384042A4 (en)
JP (1) JP2018537676A (en)
CN (1) CN108603221A (en)
CA (1) CA3007019A1 (en)
WO (1) WO2017095394A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021190420A1 (en) * 2020-03-21 2021-09-30 Brain Navi Biotechnology Co., Ltd. Method and system to perform nasal swabbing based on image matching
CN115155148A (en) * 2022-06-09 2022-10-11 北京科技大学 Ultra-precise automatic micro-reaction solution filtering device

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112358965B (en) * 2020-11-09 2023-01-24 中国计量大学 Magnetic bead method nucleic acid extraction equipment

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102686306A (en) * 2009-09-21 2012-09-19 阿科尼生物系统公司 Magnetic lysis method and device
CN103403545A (en) * 2010-12-09 2013-11-20 阿科尼生物系统公司 Sample analysis system
US20150203806A1 (en) * 2014-01-21 2015-07-23 Uw Center For Commercialization Systems for disrupting biological samples and associated devices and methods

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69940647D1 (en) * 1998-05-01 2009-05-07 Gen Probe Inc Automatic diagnostic analyzer
JP5775086B2 (en) * 2009-10-16 2015-09-09 プロメガ・コーポレーション Apparatus for heating, exciting and magnetizing and method of operating the apparatus

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102686306A (en) * 2009-09-21 2012-09-19 阿科尼生物系统公司 Magnetic lysis method and device
CN103403545A (en) * 2010-12-09 2013-11-20 阿科尼生物系统公司 Sample analysis system
US20150203806A1 (en) * 2014-01-21 2015-07-23 Uw Center For Commercialization Systems for disrupting biological samples and associated devices and methods

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
A. BERASALUCE，ET AL.: "Bead beating-based continuous flow cell lysis in a microfluidic device", 《RSC ADV.》 *
段满乐等: "《现代临床实验室管理学》", 30 September 2005, 中国科学技术出版社 *
王庸晋: "《现代临床检验学 第2版》", 30 November 2007, 人民军医出版社 *
王虹等: "核酸纯化对蝎型探针定量A81准确检测结核分枝杆菌=M2的必要性研究", 《中国感染控制杂志》 *
闻曲等: "《PICC临床应用及安全管理》", 30 November 2012, 人民军医出版社 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021190420A1 (en) * 2020-03-21 2021-09-30 Brain Navi Biotechnology Co., Ltd. Method and system to perform nasal swabbing based on image matching
CN115155148A (en) * 2022-06-09 2022-10-11 北京科技大学 Ultra-precise automatic micro-reaction solution filtering device
CN115155148B (en) * 2022-06-09 2023-09-01 北京科技大学 Ultra-precise automatic micro-reaction solution filtering device

Also Published As

Publication number Publication date
JP2018537676A (en) 2018-12-20
EP3384042A4 (en) 2019-05-08
EP3384042A1 (en) 2018-10-10
WO2017095394A1 (en) 2017-06-08
CA3007019A1 (en) 2017-06-08

Similar Documents

Publication Publication Date Title
US11767549B2 (en) Integrated sample processing system
CA2693654C (en) Polynucleotide capture materials, and methods of using same
JP5595415B2 (en) Automated system for lysis of microorganisms present in samples intended for analysis and extraction and purification of nucleic acids of these microorganisms
JP4632262B2 (en) Sample processing
US20080171337A1 (en) Nucleic acid isolation method by heating on magnetic support
US10036054B2 (en) Bead beating tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms
CN105592925B (en) System and method for collecting the sample of nucleic acid
RU2380418C1 (en) Replaceable microfluid module for automated recovery and purification of nucleic acids from biological samples and method for recovery and purification nucleic acids with using thereof
AU2017211983B2 (en) Bead beating tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms
US20070125942A1 (en) Apparatuses, systems and methods for isolating and separating biological materials
JP6817932B2 (en) A device for acquiring biomaterials and / or bioinformation from a sample containing a heterogeneous matrix.
JP2011516075A (en) Automatic purification apparatus, multiwell plate kit, and method for extracting hexane from biological sample
EP0819255A1 (en) System for releasing and isolating nucleic acids
JP2012517020A (en) Nucleic acid purification
US6852495B2 (en) Process of extracting nucleic acid and process of simultaneously carrying out extraction and purification of nucleic acid
CN108603221A (en) Comprehensive sample processing system
AU2018211530B2 (en) Bead beating tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms
WO2018085817A1 (en) Automated method for release of nucleic acids from microbial samples
US20240043941A1 (en) Systems, methods, and apparatuses for concentration and identification of a microorganism from blood
JP2009125033A (en) Method for isolating nucleic acid, nucleic acid extraction device, and method for identifying cell type using them, and method for detecting gene using them
JP2023552237A (en) Devices, kits, and methods for extracting nucleic acids
RU2595374C2 (en) Method for automated extraction with simultaneous purification of nucleic acids from several biological samples
EP3408388A1 (en) Bead beating tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms
AU2018202375B2 (en) Polynucleotide capture materials, and methods of using same
LE TRAN A Disposable, Pressurized, and Self-contained Cartridge for Performing Automated Bacterial Nucleic Acid Extraction

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 1260385

Country of ref document: HK