CN104862206B - Apparatus system for multiple multi-target nucleic acid whole-process closed sample injection - Google Patents
Apparatus system for multiple multi-target nucleic acid whole-process closed sample injection Download PDFInfo
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- CN104862206B CN104862206B CN201410059786.4A CN201410059786A CN104862206B CN 104862206 B CN104862206 B CN 104862206B CN 201410059786 A CN201410059786 A CN 201410059786A CN 104862206 B CN104862206 B CN 104862206B
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to an apparatus system for multiple multi-target nucleic acid whole-process closed sample injection. The apparatus system is characterized in that the apparatus system comprises a mixing uniformizer connected with a centrifuge and achieving uniform material mixing, sample separators and sample taking devices, the mixing uniformizer, the sample separators and the sample taking devices are sequentially assembled from inside to outside to form a round disk structure having radial line, the mixing uniformizer has a round disk shape, the upper end is provided with at least a sample injection port, a plurality of mixing dividing grooves are arranged in the mixing uniformizer, a plurality of dispensing ports are distributed on the outer periphery, the dispensing ports are provided with through fine pores and are connected with the sample separating ports at the front ends of the sample separators, the rear ends of the sample separators are connected with the sample taking devices, capillary channels are arranged in the sample separators, one end of the capillary channel is communicated with the sample separating port, and the other end is communicated with the reaction cell of the sample taking device. The apparatus system of the present invention has characteristics of simple and reasonable structure, good sealing property, low cost, flexible operation, simpleness, and convenience, and is suitable for industrial production, wherein the process comprising heating, thermal combination, cutting, and separation does not exceed 5 min.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of experiment closed for nucleic acid molecules overall process adds
Sampling device, can be adapted to all kinds of nucleic acid molecules detection reactions, be particularly well-suited to detect the scene or field detection that environmental requirement is harsh
Supporting needs.
Background technology
In recent years, with the progress of social development and biomedical technology, the several samples detection of nucleic acids number for carrying out every year
Amount is all being significantly increased, the requirements more and more higher to field quick detection processing speed, and execute-in-place requires that sensitivity is high.
Simple and efficient demand is also increasingly urgent.With ring mediated isothermal nucleic acid amplification（Loop-mediatedisothermal
Amplification, LAMP) detection as a example by.LAMP is invented earliest by Japanese Scientists Lotomi, by for the 6 of target gene
Individual region and design 4 species-specific primers, using with strand-displacement activity BstDNA polymerases, under constant temperature（60-65
℃）Efficiently（0.5-1h）Amplification target dna.Compared with current pandemic PCR nucleic acid amplification technologies, the technology has disobeys
Bad temperature cycler（PCR）High Deng expensive instrument, and sensitivity, specificity is good, the advantages of response speed is fast
Paid much attention to by academia and industrial circle in recent years, polytype isothermal nucleic acid amplification is developed in succession.
And it is used for field quick detection, the animal embryo of clinical disease diagnosis, popular antibacterial or virus as a kind of emerging technology
The field such as sex identification and gene chip exploitation.In field of virus detection, LAMP technology has been relied on to be developed for quickly at present
Diagnostic detection includes the various popular virus including Hepaitis B virus, influenza virus, sars coronavirus, herpes simplex virus
Method（Hongetal.,2004;Enomotoetal.,2005;Kanekoetal.,2005;）.In Bacteria Detection field, should
Technology is also widely used in the quick detection of Mycobacterium tuberculosiss, escherichia coli, streptococcus pneumoniae, Shigella dysenteriae
（Iwamotoetal.,2003）.Based on the difference of distinguished sequence on Y chromosome, Hirayama etc.（2004）Using LAMP technology
The quick detection to cattle early embryo sex is carried out, LAMP technology has been applied to into a brand-new field.In aquaculture
Defect inspection field, has been employed successfully at present taura syudrome（Tarurasyndrome virus,TSV）, prawn white spot disease
Poison（Whitespotsyndromevirus,WSSV）, infectivity muscle necrosis virus
（Infectiousmyonercrosisvirus,IMNV）Deng the detection of virus causing disease（Kiatpathomchaietal.,2008;
Jaroenrametal.,2009;Puthawibooletal.,2009）.It is growing with the technology, two hinder its
The bottleneck problem of the application of basic unit is also gradually highlighted；First it is that the Aerosol Pollution that brings of susceptiveness of LAMP is asked
Topic, is to tackle this problem because LAMP method is sensitive for the extension of target sequence and amplification amount big, current for LAMP technology
Typically " wanting strict partition, standard operation " is required in actual application, i.e. divide into experiment according to LAMP operating processes
" sample treatment area, solution preparation area, template addition area, detection zone ", and it is last in strict accordance with template in operation
Add, first negative, the rear positive, concentration from low to high is operated.For the requirement of such stringent, make in actual basic unit
It is difficult to accomplish with during, and then causes the technology to be difficult to promote in basic unit's Site Detection.
There is this to can be seen that when detecting that the harsh scene of environmental requirement or field carry out nucleic acid molecules quick detection, such as
What reasonably avoids the Aerosol Pollution in detection process, while and the high flux in nucleic acid molecules sample detection can be solved, it is many
The problem of target spot, become can so that nucleic acid molecules detect that soon preferably serving basic unit's detection one of unit can't steer clear of,
Also the problem that can not escape.
The content of the invention
The technical problem to be solved is to provide a kind of simple and reasonable, good airproof performance for nucleic acid molecules
The apparatus system of the closed sample-adding of overall process, efficiently solves the Aerosol that sample-adding process is caused, and by detachable
Riffle sampler realizes multisample, Mutiple Targets sample-adding, whole device flexible operation, convenient and swift.
The present invention solve the technical scheme that adopted of above-mentioned technical problem for：One kind is used for multiple Mutiple Targets nucleic acid overall process
The apparatus system of closed sample-adding, it is characterised in that：Described device system includes being connected with centrifuge and realizes the mixed of uniform batch mixing
Homogenizer, riffle sampler and sampler are closed, from the inside to the outside successively assembling forms a band radiation for mix homogeneously device, riffle sampler and sampler
The disc-shaped structure of line, mix homogeneously device is disc, and its upper end is provided with least one adding mouth, and multiple mixing point are provided with it
Groove is cut, its peripheral distribution there are multiple distribution openings, distribution openings are provided with the pore of insertion, and distribution openings are by joint and riffle sampler front end
Sample-distributing port be connected, the rear end of riffle sampler is connected with sampler, is provided with capillary channel in riffle sampler, the one of capillary channel
End is connected with sample-distributing port, and the other end is connected with the reaction tank of sampler.
Used as improvement, the mix homogeneously device is involuted by discoidal upper lid and discoidal base, adding mouth
It is arranged on lid, if being provided with the concentric segmentation annulus of dried layer in base, mixing slot segmentation is formed between segmentation annulus, in segmentation
Annulus is provided with the breach that breach flows to water jacket for feed liquid from inside groove, the radial outermost layer for being uniformly arranged on base of distribution openings
Segmentation annulus on.
Used as improvement, the segmentation annulus is three layers, and the adding mouth is multiple, the internal diameter size and liquid-transfering gun of adding mouth
Pipette tips match, and on each adding mouth seal cap is respectively equipped with, adding mouth centered on one of them, and the center adding mouth is arranged on
The center of upper lid, its endoporus is located at the top that internal layer is split in annulus, and remaining adding mouth is evenly arranged in respectively center and adds
Around sample hole, its endoporus is located at the top that the second layer is split in annulus.
Used as improvement, the breach on the segmentation annulus is 3～4 arc notch, arc notch in it is equidirectional deviously
It is disposed in an evenly spaced relation on segmentation annulus.
Improve again, the outermost segmentation circle ring inner wall on the inside of the distribution openings is provided with the accommodating groove of parabolic shape,
Pore arranges the bottom of accommodating groove, and pore is connected by distribution openings with the sample-distributing port of riffle sampler.
Improve again, the riffle sampler is a sleeve pipe structure, recessed being provided with the front end of riffle sampler be connected sample-distributing port with joint,
One end of joint is set in distribution openings, and the other end is plugged in the sample-distributing port of riffle sampler fixed.
Improve again, the front portion of the riffle sampler is provided with point sample hole matched with pipettor gun head sealing, is provided with point sample hole
The self-locking screens of pipettor gun head can be pinned.
Further improve, the rear end of the riffle sampler is provided with the connecting portion being connected with sampler, sampler is that front end is close
Envelope is provided with the container of capillary channel interface, and the inner end of capillary channel interface is connected with reaction tank, and the front end of sampler is plugged on
In the connecting portion of riffle sampler, it is connected with the capillary channel in riffle sampler by capillary channel interface.
Further improve, the upper lid is provided with female thread, and base is provided with corresponding external screw thread, and upper lid passes through with base
Threaded connection is fixed, and is lined with sealing ring between upper lid and base.
Compared with prior art, it is an advantage of the current invention that：Tied using fluid mechanics principle, material science and theory of mechanics
The problems such as closing biotechnology, the effectively solving Aerosol Pollution that causes of sample-adding process and high flux Mutiple Targets, it is convenient to operation
The research and development and popularization and application of LAMP technology need not be conducive to by other expensive instruments.In addition all corollary equipments are all adopted
With existing routine experimental facilitiess and consumptive material, using convenient, it is adapted to classes of agents detection reaction, is particularly suitable for scene and field
The supporting needs of detection.Present configuration advantages of simple, good airproof performance, low cost, and it is flexible operation, simple and convenient, from sample-adding
Separation is cut off to heat seal to complete less than 5min, layman just can operate by simple demonstration, entirely be designed with mark
Standardization, it is possible to achieve industrialized production.
Description of the drawings
Fig. 1 is the structural representation of the experiment sample adding device of the present invention；
Fig. 2 is the structural representation of mix homogeneously device in Fig. 1, and wherein a is the structural representation of upper lid, and b is the knot of base
Structure schematic diagram；
Fig. 3 is the structural representation of point sample sampler in Fig. 1
Specific embodiment
The present invention is described in further detail below in conjunction with accompanying drawing embodiment.
As shown in Figures 1 to 3, the apparatus system for the closed sample-adding of nucleic acid molecules overall process of the present embodiment, including with from
Scheming is connected and realizes mix homogeneously device 1, riffle sampler 2 and the sampler 3 of uniform batch mixing, mix homogeneously device 1, riffle sampler 2 and takes
From the inside to the outside successively assembling forms a disc-shaped structure with lonizing radiation to sample device 3, and mix homogeneously device 1 is disc, is by disk
The upper lid 11 and discoidal base 12 of shape is involuted, wherein upper lid 11 is provided with female thread, base 12 is provided with corresponding
External screw thread, upper lid 11 is threaded connection fixation with base 12, and is lined with sealing ring between upper lid 11 and base 12；Upper lid 11
Three adding mouths 111 are provided with, nucleic acid, enzyme, buffer can be separately added into, adding mouth centered on one of them, the center sample-adding
Mouth is arranged on the center of lid 11, in addition the both sides of two Ge centers wells, and three layers concentric point is provided with base 12
Cyclotomy ring 123,124 and 125, three mixing slot segmentations 122 are formed on base 12 by segmentation annulus 123,124 and 125, wherein
The endoporus of the center adding mouth of upper lid 11 is located at the top that internal layer is split in annulus 123, during the endoporus of remaining adding mouth 111 is located at
Between split annulus 124 in top；12 distribution openings 121 are distributed with the outside circumference of base 12, distribution openings 121 are radial
On the outermost segmentation annulus 125 for being evenly spaced apart to be arranged on base 12, distribution openings 121 are provided with the pore 127 of insertion, point
The outermost inwall of segmentation annulus 125 with the inner side of mouth 121 is provided with the accommodating groove 128 of parabolic shape, and pore 127 is arranged on
The bottom of accommodating groove 128, pore 127 is connected by distribution openings 121 with the sample-distributing port of riffle sampler 2, is divided with middle in inner side
The breach 126 of 3～4 arcs is provided with cyclotomy ring 123 and 124, arc notch 126 is arranged in equidirectional uniform intervals deviously
On segmentation annulus 123 and 124 so that flow to water jacket from inside groove after feed liquid mixing, during first sample-adding, nucleic acid and enzyme by
It is little in the amount of sample-adding, will slowly be blended in buffer after sample-adding, with the centrifugal action of centrifuge, nucleic acid and enzyme and slow
After rushing liquid mix homogeneously, outermost layer is arrived by the breach 126 in segmentation annulus 123 and 124, then by pore 127 from distribution
Mouth 121 is flowed in riffle samplers 2；Riffle sampler 2 is a sleeve pipe structure, and the front end of riffle sampler 2 is arranged with and be connected a point sample with joint 4
Mouthful, one end of joint 4 is set in distribution openings 121, and the other end is plugged in the sample-distributing port of riffle sampler 2 and fixes, before riffle sampler 2
Portion is provided with point sample hole matched with pipettor gun head sealing, and in point sample hole the self-locking screens that can pin pipettor gun head is provided with, with
Realize the requirement for being added separately to reaction tank of different nucleic acid in Multiple experiments；There is capillary channel 21 in the mid portion of riffle sampler 2,
Capillary channel 21 is connected using metal or nonmetallic materials, one end of capillary channel 21 with sample-distributing port, the other end and sampler 3
Reaction tank 31 be connected；The rear end of riffle sampler 2 is provided with the connecting portion being connected with sampler 3, and sampler 3 is removably to block
Put in connecting portion, sampler 3 is provided with the container of capillary channel interface 32 for forward end seal, the inner end of capillary channel interface 32 with
Reaction tank 31 is connected, and the front end of sampler 3 is plugged in the connecting portion of riffle sampler 2, and by capillary channel interface 32 and divides
Capillary channel 21 in sample device 2 is connected.
During sample-adding, nucleic acid, enzyme, buffer etc. are added to into adding mouth on the mix homogeneously device 1 of centre by closed system
In 111, after the mix homogeneously of mixing slot segmentation 122 in mix homogeneously device 1, divide in order under the centrifugal action of centrifuge
Not Tong Guo distribution openings 121, the sample-distributing port and capillary channel 21 for connecting riffle sampler 2 flowed in each reaction tank 31, whole sample-adding
During carry out under air-tight state completely, mix under enzyme and DNA air-tight states, therefore not reacting generation, will not to produce gas molten
Glue stain, completes the overall process for being loaded sample mixing distribution under air-tight state completely, will not produce cross-contamination.Meanwhile, by certainly
The detachable riffle sampler of master realizes multisample, the closed sample-adding of Mutiple Targets.
Expand sample-adding test below with LAMP is carried out further to the closed effectiveness of the experiment sample adding device of the present invention
Checking：
Experiment adopts the capillary channel of Φ 0.3mm internal diameters, and using PTFE material capillary channels, PTFE capillary has its
Coefficient of friction is minimum, but due to fluoro- carbon chain molecules intermolecular forces it is extremely low, relatively wide temperature of the politef at -190 DEG C～280 DEG C
One of excellent mechanical property, the high molecular feature of perfluorocarbon is kept to be non-brittle in low temperature in the range of degree.And with resistance toization
Corrosion and weatherability are learned, politef is hardly corroded by any chemical reagent.Politef is nonhygroscopic, does not fire, to oxygen,
The equal stabilizer pole of ultraviolet, so with excellent weatherability.But not only limit and only use PTFE materials, also can select such as silica gel
The materials such as class, PE classes.
In addition, sealing gasket and sealing ring all adopt silica gel material, lock nut PE materials, sample adding device adopts medical
Level PVC makes, and check valve adopts diaphragm type check valve, and sample-adding mixing pit is spherical crown concave surface, last gamma ray sterilizing.
Application Example 1
Sample-adding experiment under pure environment
DNA, dNTP, probe, enzyme and buffer are amounted to into 2.5ml by with the present invention's in the laboratory of pure environment
Experiment sample adding device mixing sample-adding amounts to 100 pipes into test tube, is doing LAMP experiments, finds no pollution and affects experimental result
Phenomenon.
Application Example 2
Sample-adding experiment under Aerosol Pollution severe environments
Carry out in laboratory in colleges and universities, because the various DNA extraction work of laboratory is a lot, Aerosol Pollution itself is serious, adopts
With the experiment sample adding device of the present invention, probe is placed in advance in test tube, entire upper band has the lid of capillary channel, uses lock nut
It is connected on sample injector, Microcystin DNA fills sample with one of well, removes pipette tips after pipettor and stays in sample-adding
Close on hole and with closure, the μ l of enzyme 10 of another well filling Dalian treasured biological production, on same closed cover；It is mixed in advance
The reaction buffer containing dNTP for getting togather is closed after the filling of center well with closure.By the experiment with the present invention
Sample adding device mixing sample-adding does altogether ten groups of LAMP experiments into test tube and finds no pollution and affect experimental result phenomenon.
Application Example 3
Aerosol Pollution situation is carried out in inspection and quarantine scientific and technical research institute laboratory after sample-adding experiment, using the present invention
Experiment sample adding device, probe is placed in advance in test tube, entire upper band has the lid of capillary channel, be connected to lock nut plus
On sample device, by ten groups（Vibrio cholera, monokaryon Listerella, vibrio parahaemolytious, shigella, Enterohemorrhagic E.coli (O157:
H7), Vibrio vulnificus, yersinia, vibrio alginolyticus, Vibro harveyi, Salmonella）DNA is filled with one of well
Sample, removes pipette tips after pipettor and stays on well and closed with closure, and another well filling Dalian is precious biological raw
The enzyme of product, on same closed cover；Closed with closure after the filling of center well containing dNTP buffer.By with this
Bright experiment sample adding device mixing is loaded into test tube and carries out after LAMP experiments, and standard control finds no pollution and affects
Experimental result phenomenon, effect is ideal.
Claims (8)
1. a kind of apparatus system for the closed sample-adding of multiple Mutiple Targets nucleic acid overall process, it is characterised in that：Described device system
That uniform batch mixing is realized including being connected with centrifuge mixs homogeneously device, riffle sampler and sampler, mix homogeneously device, riffle sampler and
From the inside to the outside successively assembling forms a disc-shaped structure with lonizing radiation to sampler, and mix homogeneously device is disc, and its upper end sets
There is at least one adding mouth, multiple mixing slot segmentations are provided with it, its peripheral distribution there are multiple distribution openings, and distribution openings are provided with passes through
Logical pore, distribution openings are connected by joint with the sample-distributing port of riffle sampler front end, and the rear end of riffle sampler is connected with sampler,
Capillary channel is provided with riffle sampler, one end of capillary channel is connected with sample-distributing port, the reaction tank phase of the other end and sampler
Connection；
The mix homogeneously device is involuted by discoidal upper lid and discoidal base, and adding mouth is arranged on lid,
If being provided with the concentric segmentation annulus of dried layer in base, mixing slot segmentation is formed between segmentation annulus, be provided with scarce on segmentation annulus
Confession feed liquid flows to the breach of water jacket, the radial outermost segmentation annulus for being uniformly arranged on base of distribution openings from inside groove
On.
2. apparatus system according to claim 1, it is characterised in that：The segmentation annulus is three layers, and the adding mouth is
Multiple, internal diameter size and the liquid-transfering gun pipette tips of adding mouth match, and on each adding mouth seal cap is respectively equipped with, and one of them is
Center adding mouth, the center adding mouth is arranged on the center of lid, and its endoporus is located at the top that internal layer is split in annulus, its
Remaining adding mouth is evenly arranged in respectively around the well of center, and its endoporus is located at the top that the second layer is split in annulus.
3. apparatus system according to claim 2, it is characterised in that：Breach on the segmentation annulus is 3～4 arcs
Breach, arc notch is disposed in an evenly spaced relation in deviously on segmentation annulus in equidirectional.
4. apparatus system according to claim 1, it is characterised in that：Outermost segmentation annulus on the inside of the distribution openings
Inwall is provided with the accommodating groove of parabolic shape, and pore arranges the bottom of accommodating groove, pore by distribution openings and riffle sampler point
Sample mouth is connected.
5. apparatus system according to claim 1, it is characterised in that：The riffle sampler is a sleeve pipe structure, riffle sampler
Recessed being provided with front end be connected sample-distributing port with joint, and one end of joint is set in distribution openings, and the other end is plugged on riffle sampler
It is fixed in sample-distributing port.
6. apparatus system according to claim 5, it is characterised in that：The front portion of the riffle sampler is provided with and pipettor gun head
Point sample hole of sealing matching, in point sample hole the self-locking screens that can pin pipettor gun head is provided with.
7. apparatus system according to claim 6, it is characterised in that：The rear end of the riffle sampler is provided with and is connected with sampler
The connecting portion for connecing, sampler is provided with the container of capillary channel interface, the inner end of capillary channel interface and reaction tank for forward end seal
It is connected, the front end of sampler is plugged in the connecting portion of riffle sampler, by the capillary tube in capillary channel interface and riffle sampler
Road is connected.
8. apparatus system according to claim 1, it is characterised in that：The upper lid is provided with female thread, and base is provided with
Corresponding external screw thread, upper lid is connected with base by thread, and is lined with sealing ring between upper lid and base.
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| CN201410059786.4A CN104862206B (en) | 2014-02-21 | 2014-02-21 | Apparatus system for multiple multi-target nucleic acid whole-process closed sample injection |
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| CN201410059786.4A CN104862206B (en) | 2014-02-21 | 2014-02-21 | Apparatus system for multiple multi-target nucleic acid whole-process closed sample injection |
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| CN104862206B true CN104862206B (en) | 2017-04-12 |
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| CN106119099B (en) * | 2016-06-14 | 2018-07-20 | 西安交通大学 | A kind of totally-enclosed more targeting nucleic acid isothermal amplifications detection integrated apparatus |
| CN110408610A (en) * | 2018-04-27 | 2019-11-05 | 恺硕生物科技(厦门)有限公司 | Nucleic acid extracting reagent item |
| CN111855370A (en) * | 2020-06-28 | 2020-10-30 | 江苏省人民医院（南京医科大学第一附属医院） | Automatic nasopharynx swab specimen sample mixing device for virus nucleic acid non-dilution sample mixing detection |
| CN114324957B (en) * | 2022-03-16 | 2022-05-20 | 天津德祥生物技术有限公司 | Blood type positive and negative shaping sample adding card and sample adding assembly |
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| CN1888902B (en) * | 2006-08-11 | 2011-05-18 | 杭州优思达生物技术有限公司 | Full closed target nucleic amplifier fast testing device |
| CN102199531A (en) * | 2011-03-30 | 2011-09-28 | 复旦大学 | Microfluidic chip for multiple loop-mediated isothermal amplification (LAMP) detection and preparation method thereof |
| CN202808796U (en) * | 2012-07-31 | 2013-03-20 | 中国检验检疫科学研究院 | Loop-mediated isothermal amplification reaction tube |
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