CN105039149B - A kind of closed experimental system setup of Rapid identification nucleic acid amplification product and application - Google Patents
A kind of closed experimental system setup of Rapid identification nucleic acid amplification product and application Download PDFInfo
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
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Abstract
The present invention relates to a kind of closed experimental system setup of Rapid identification nucleic acid amplification product, it is characterised in that：Including the reaction tube, sampling sample mixing device and reaction indication box being arranged successively from top to bottom, wherein reaction tube, there is sealable lid；There is the cut substrate that can cut reaction tube in the middle part of sampling sample mixing device, there is sharp protrusion thing in the middle part of reaction indication box, reaction indication box tail end has the cavity volume that can accommodate lateral chromatography test strips, the invention further relates to closed experimental system setup in ring mediated isothermal amplification and the application in nucleic acid lateral f chromatographic flow test strips combination quick detection target nucleic acid amplification product method, compared with prior art, the present invention, which is avoided that in target nucleic acid amplification product release air, forms serious Aerosol Pollution, amplified production can carry out fluoroscopic examination in this completely closed device using the nucleic acid lateral f chromatographic flow test strips reacted in indication box, with visualization directly perceived, quick advantage and convenient, highly promotion and application at the scene.
Description
Technical field
The invention belongs to technical field of biological, is related to the multinomial technology collection such as nucleic acid amplification technologies and lateral chromatography technology
Into biological technical field, more particularly to a kind of closed experimental system setup of Rapid identification nucleic acid amplification product and application.
Background technology
In recent years, with social development and the progress of biomedical technology, the several samples detection of nucleic acids number carried out every year
Amount is all being significantly increased, and to the requirements more and more higher of field quick detection processing speed, execute-in-place requires high sensitivity.
Simple and efficient demand is also increasingly urgent.
After K.B Mullis from PE-Cetus companies of the U.S. in 1985 et al. have initiated round pcr, it is with quite frightened
The speed of people is widely used in the every field such as medical science and molecular biology.Round pcr is in biomedicine etc. in the past 20 years
Great effect has been played in each research field and clinical practice.Now, round pcr has turned into a kind of to specific in sample
DNA fragmentation carries out the important method of analysis and research and Testing and appraisal.Nearly all research for being related to molecular biology nucleic acid is all
PCR is used, phase has all been made in Causal Agent Identification, genetic diseases diagnosis, immunological investigation, oncogene exploration and treatment etc.
The contribution answered.
Whether PCR primer is specific amplification, the result is that no accurately and reliably extremely important, it is necessary to it is carried out strict
Analysis and identification, can just draw correct conclusion.The analysis of PCR primer, can be according to research object with purpose difference and using not
Same analysis method.Gel electrophoresis analysis：PCR detections at present are mainly with agarose, polyacrylamide gel electrophoresis product, agarose electricity
Although swimming detection is fairly simple, EB ethidium bromide stainings are needed, are observed under uviol lamp, the sensitivity of detection is in ng levels, it is necessary to mark
Accurate Marker determines whether the specificity of product；Polyacrylamide gel electrophoresis colloid preparation process is more complicated, afterwards aobvious
Color process is also more complicated, and time-consuming for whole detection time.
LAMP technology earliest by Japanese Scientists Notomi etc. (2000) establish, by 6 regions for target gene and
4 species-specific primers are designed, using the Bst archaeal dna polymerases with strand-displacement activity, (60-65 DEG C) is efficient under constant temperature
(0.5-1h) expands target dna.Compared with current pandemic PCR nucleic acid amplification technologies, the technology has independent of temperature
The expensive instruments such as circulating instrument (PCR), and high sensitivity, specificity is good, the advantages that reaction speed is fast.Therefore, as a kind of new
Emerging technology is used for field quick detection, animal embryo sex identification and the gene of clinical disease diagnosis, popular bacterium or virus
The fields such as chip development.In field of virus detection, relied on LAMP technology to develop at present includes second for quick diagnosis detection
Method (the Hong et of a variety of popular viruses including liver hepatitis viruse, influenza virus, sars coronavirus, herpes simplex virus
al.,2004；Enomoto et al.,2005；Kaneko et al.,2005；).In Bacteria Detection field, the technology is also extensive
Applied to Mycobacterium tuberculosis, Escherichia coli, streptococcus pneumonia, Shigella dysenteriae quick detection (Iwamoto et al.,
2003).Carried out based on the difference of distinguished sequence on Y chromosome, Hirayama etc. (2004) using LAMP technology to ox early stage
The quick detection of embryo gender, LAMP technology a brand-new field is applied to.
Since PCR and LAMP inventions, since the specific amplification for realizing nucleic acid, it has been widely used, still
Due to highly sensitive, high specific and high efficiency, false positive results are caused to occur.The reason for causing false positive the failure of an experiment is a lot：
1. sample room cross pollution：Sample contamination mainly has the container for collecting sample to be contaminated, or during sample placement, due to poorly sealed
Overflow in outside container, or be stained with sample outside container and cause mutual cross pollution；Sample nucleic acid template in extraction process, due to
Inhaling the pollution of sample rifle causes to pollute between sample；2. the pollution of reagent：Mainly due to during preparation of reagents, due to sample loading gun,
Container, distilled water and other solution are by the nucleic acid-templated pollutions of PCR.3. amplified production pollutes：This be in amplified reaction it is main most
Common pollution problem.The PCR primer pollution of denier, so that it may form false positive.4. Aerosol Pollution：In air and liquid side
It can form aerosol during friction, reaction tube is more tempestuously shaken in operation, when uncapping, when inhaling sample and pollution sample injection gun
Can all aerosol be formed and pollute by inhaling sample repeatedly.5. the pollution of cloned plasmids in laboratory：Because cloned plasmids are in unit volume
Interior content is at a relatively high, is needed in addition in purge process with more apparatus and reagent, and the plasmid in living cells, due to work
The simplicity of the growth and breeding of cell and there is very strong vitality, its possibility of pollution is also very big.In molecular biology experiment
Room and some control laboratories that positive control is made of cloned plasmids, this problem are relatively common.
Especially as the growing of LAMP technology, two are hindered the bottleneck problem of its application in basic unit also gradual
Highlight；It is the Aerosol that LAMP sensitivity is brought first, due to extension of the LAMP method for target sequence
Sensitive and amplification amount is big, to tackle this problem, typically requires " strict in actual application at present for LAMP technology
Subregion, standard operation ", i.e. experiment is divided into by " sample treatment area, solution preparation area, template addition according to LAMP operating processes
Area, detection zone ", and being eventually adding in operation in strict accordance with template, first negative, the rear positive, from low to high dense
Degree is operated.Requirement for such stringent, it is difficult to accomplish during actual basic unit's use, and then causes the technology
It is difficult to promote in basic unit's Site Detection.Next to that the result evidence visualization of Site Detection, in order that it is very clear with result,
There is more easily visual detecting system.
It can thus be seen that when detecting the harsh scene of environmental requirement or field carries out nucleic acid molecules quick detection, such as
What reasonably avoids the Aerosol Pollution in detection process, while and can solves the Site Detection knot in nucleic acid molecules sample detection
The visualization problem of fruit evidence, can turn into so that nucleic acid molecules detect soon preferably serves one that unit detects in basic unit
It can't steer clear of, the problem of also can not escape.
The content of the invention
A technical problem to be solved by this invention is to provide one kind for the present situation of prior art and be avoided that and detected
The closed experimental system setup of the Rapid identification nucleic acid amplification product of Aerosol Pollution in journey.
Another technical problem to be solved by this invention be for prior art present situation provide it is a kind of using above-mentioned
The closed experimental system setup of Rapid identification nucleic acid amplification product is in non-diagnostic purpose ring mediated isothermal amplification and the lateral layer of nucleic acid
Application in analysis flowing test strips combination quick detection target nucleic acid amplification product method.
Another technical problem to be solved by this invention be for prior art present situation provide it is a kind of using above-mentioned
The closed experimental system setup of Rapid identification nucleic acid amplification product non-diagnostic purpose prevent nucleic acid amplification product pollution, avoid gas
Application in colloidal sol pollution method.
A technical problem to be solved by this invention uses the closed experiment of above-mentioned Rapid identification nucleic acid amplification product
Identification of Species, gene of the system and device in testing food pathogenic and agricultural, industry, customs, animal husbandry on gene level are dashed forward
The application become in the non-diagnostic mesh purposes in detection.
Technical scheme is used by the present invention solves above-mentioned technical problem：The Rapid identification nucleic acid amplification product it is closed
Experimental system setup, it is characterised in that：Including be arranged successively from top to bottom reaction tube, sampling sample mixing device and reaction indication box,
Wherein：
The reaction tube, there is sealable lid；
The sampling sample mixing device, including pipe sleeve and the upper lid that the pipe sleeve opening can be sealed, and have in the middle part of the pipe sleeve
The cut substrate of the reaction tube can be cut, it is described in the state of the reaction lid upper tube cap is placed into sampling sample mixing device
Reaction tube can be gently against in the cut substrate, and is being linked closely on the sampling sample mixing device in the state of lid, the reaction tube energy edge
The inner tubal wall for the sampling sample mixing device moves down 4~5 millimeters, finally enables the cut substrate in the sampling sample mixing device to draw
Break the bottom of the reaction tube；
The reaction indication box, its middle part has the sharp protrusion thing that can puncture the sampling sample mixing device, described sharp convex
The outer peripheral face for playing thing is provided with least one breach, and the tail end of the reaction indication box has can accommodate lateral chromatography test strips
Cavity volume, and be fitted with bonnet in the distal opening of the cavity volume.
Preferably, the cutting angle of the cut substrate is between 30 °~60 °.
The end of the reaction indication box has internal thread, and accordingly, the end of the sampling sample mixing device is then with outer
Screw thread, and the end of the sampling sample mixing device is also fitted with sealing ring.External thread bolt connection in sampling sample mixing device end refers in reaction
In the state of showing box end internal thread, sampling sample mixing device can be fixed in reaction indication box, and sealing ring can be utilized to sample
The sealing of sample mixing device is fixed in reaction indication box.
Reaction tube is further better achieved to be fixed in sampling sample mixing device, the pipe sleeve of the sampling sample mixing device inwardly prolongs
Stretching has the annular bead that can support the reaction tube.
The invention discloses a kind of closed experimental system setup using above-mentioned Rapid identification nucleic acid amplification product non-
Diagnostic purpose ring mediated isothermal amplification and nucleic acid lateral f chromatographic flow test strips combination quick detection target nucleic acid amplification product method
In application.
The present invention also provides a kind of closed experimental system setup using above-mentioned Rapid identification nucleic acid amplification product non-
Diagnostic purpose prevents nucleic acid amplification product pollution, avoids application in Aerosol Pollution method.
The invention also discloses a kind of closed experimental system setup using above-mentioned Rapid identification nucleic acid amplification product to exist
In Identification of Species on gene level of testing food pathogenic and agricultural, industry, customs, animal husbandry, detection in Gene Mutation
Application in non-diagnostic mesh purposes.
Compared with prior art, the invention has the advantages that：After amplified reaction terminates in the above method, reaction is not opened
The lid of pipe, it can avoid forming serious Aerosol Pollution in target nucleic acid amplification product release air, amplified production is at this
Fluoroscopic examination can be carried out using the nucleic acid lateral f chromatographic flow test strips reacted in indication box in completely closed device, its is excellent
Point is that only needs mark biotin and FITC probes on a primer, can be within 5-15min minutes after nucleic acid amplification reaction
Nucleic acid amplification product detection is completed, and product specificities can be differentiated, sensitivity is pg levels, and specific gene can be shown after hybridization
Result is shown, there is visualization directly perceived, quick advantage and convenient；The device of sealing is not opened after detection, can be all useless by it
Abandon in safe place；Whole process is carried out in closed environment, can effectively prevent target nucleic amplifier cross pollution, avoids false sun
Property；Nucleic acid amplification quickly can be analyzed and detected in medical and health, food security and routine experimentation using the device of the present invention
Product, judges whether object to be checked contains false positive, such as personation foodstuff traceability, food-borne pathogens detection, and food transfer
Genetic test, palm off the detection of specific meats product etc. type, therefore highly promotion and application at the scene.
Brief description of the drawings
The structural representation of an angle in Fig. 1 embodiment of the present invention 1；
The structural representation of another angle in Fig. 2 embodiment of the present invention 1；
Fig. 3 Fig. 2 A-A is to diagrammatic cross-section；
Reaction tube is not inserted into the structural representation of sampling sample mixing device in Fig. 4 embodiment of the present invention 1；
The diagrammatic cross-section of reaction tube in Fig. 5 embodiment of the present invention 1；
The structural representation of sample mixing device is sampled in Fig. 6 embodiment of the present invention 1；
The diagrammatic cross-section of sample mixing device is sampled in Fig. 7 embodiment of the present invention 1；
The diagrammatic cross-section of cut substrate in Fig. 8 embodiment of the present invention 1；
The structural representation of sharp protrusion thing in Fig. 9 embodiment of the present invention 1；
Figure 10 is Fig. 9 top view；
The structural representation of indication box is reacted in Figure 11 embodiment of the present invention 1；
Figure 12 is Figure 11 left view；
Figure 13 is Figure 12 B-B direction diagrammatic cross-section；
Figure 14 is the result figure of embodiment 3；
Figure 15 is the result figure of embodiment 4.
Embodiment
Below by way of drawings and Examples are combined, the invention will be further described.
Embodiment 1
The closed experimental system setup of this Rapid identification nucleic acid amplification product includes the reaction tube being arranged successively from top to bottom
1st, sample mixing device 2 and reaction indication box 3 are sampled, wherein：
Reaction tube 1, there is sealable lid 11；
Sample mixing device 2, including the upper lid 22 that pipe sleeve 21 and energy sealed tube 21 are open are sampled, and the middle part of pipe sleeve 21 has and can cut
The cutting angle of broken reaction tube 1 is 30 ° of cut substrate 4, and the pipe sleeve 21 for sampling sample mixing device 2 extends internally can support reaction tube
1 annular bead 211, cover lid 11 in reaction tube 1 and be placed into sampling sample mixing device 2 in the state of annular bead 211, reaction
Pipe 1 can be gently against in cut substrate 4, and is being linked closely on sampling sample mixing device 2 in the state of lid 22, and reaction tube 1 can be along sampling sample mixing
The inner tubal wall of device 2 moves down 4 millimeters, the final bottom for enabling the cut substrate 4 sampled in sample mixing device 2 to scratch reaction tube 1；
Indication box 3 is reacted, is made up of lower end rectangular pipe and upper end pipe and be in the structure of strip cast, is had in the middle part of it
The sharp protrusion thing 5 of sampling sample mixing device 2 can be punctured, the outer peripheral face of sharp protrusion thing 5 is evenly arranged with multiple breach 51, and reacts
The tail end of the lower end rectangular pipe of indication box 3 has the cavity volume 31 that can accommodate lateral chromatography test strips, and in the distal opening of cavity volume 31
It is fitted with bonnet 32；And the upper end for reacting indication box 3 has internal thread 33, accordingly, the end of sampling sample mixing device 2 then has
There is external screw thread 23, and the end of the sampling sample mixing device 2 is also fitted with sealing ring, the spiral shell of external screw thread 23 in sampling sample mixing device 2 end
It is connected in the state of the reaction end internal thread 33 of indication box 3, and sealing ring can be utilized to sample the sealing of sample mixing device 2 and be fixed on reaction
In indication box 3.
In experimentation is carried out, operating procedure it is as follows：
(1) closed sample adding device is first passed through to be loaded, it is also possible to pipettor by nucleic acid and enzyme and other necessary reagents,
Add in the reaction tube 1 for having inserted probe and carry out amplification experiment, after the completion of amplification experiment, reaction tube 1 is inserted into sampling sample mixing
In device 2, reaction tube 1 is gently leaned against in the beveled edge of the sharp cut substrate 4 in sampling sample mixing device 2, sample mixing is sampled by fastening
The upper lid 22 of device 2 makes inner tubal wall of the reaction tube 1 along sampling sample mixing device 2 move down 4 millimeters so that in sampling sample mixing device 2
Sharp cut substrate 4 can scratch the bottoms of reaction tube 1, the bottom-open of reaction tube 1,
(2) reaction tube 1 will have been inserted again and upper cover of lock 22 fastens closed sampling sample mixing device 2 and is put into centrifuge, by
The nucleic acid amplification product reaction solution that amplified reaction is completed in reaction tube 1, centrifugation are extremely sampled the front end of sample mixing device 2 by centrifugal force,
Complete previous work.
(3) in the state of the external screw thread 23 of sampling sample mixing device 2 end is screwed onto the reaction end internal thread 33 of indication box 3,
And sealing ring can be utilized to sample the sealing of sample mixing device 2 and be fixed in reaction indication box 3.Meanwhile and using reacting in indication box 3
Sharp protrusion thing 5 is punctured sample mixing device 2 is sampled, and sampling sample mixing device 2 is broken, and reaction solution lateral chromatography in indication box 3 is reacted tries
Under the siphonage of paper slip (LFD), moved to lateral chromatography test strips direction.
(4) because LFD lateral chromatography test strips have C (nature controlling line), T (detection line), when only C lines develop the color, for feminine gender
Reaction, it is the positive when C, T two-lines react, when not developing the color for two, reaction failure.
(5) the closed experimental system setup of this Rapid identification nucleic acid amplification product is not opened after detecting, because of sealing gasket and close
Seal all using silica gel material, reacts the acrylonitritrile-styrene resin AS materials of indication box 3, sampling sample mixing device 2 and reaction
Pipe 1 is made using medical grade polyvinylchloride, last gamma rays sterilizing, its good airproof performance, and nontoxic, you can by its whole
Discard in safe place.
Embodiment 2
The closed experimental system setup of this Rapid identification nucleic acid amplification product includes the reaction tube being arranged successively from top to bottom
1st, sample mixing device 2 and reaction indication box 3 are sampled, wherein：
Reaction tube 1, there is sealable lid 11；
Sample mixing device 2, including the upper lid 22 that pipe sleeve 21 and energy sealed tube 21 are open are sampled, and the middle part of pipe sleeve 21 has and can cut
The cutting angle of broken reaction tube 1 is 45 ° of cut substrate 4, and the pipe sleeve 21 for sampling sample mixing device 2 extends internally can support reaction tube
1 annular bead 211, cover lid 11 in reaction tube 1 and be placed into sampling sample mixing device 2 in the state of annular bead 211, reaction
Pipe 1 can be gently against in cut substrate 4, and is being linked closely on sampling sample mixing device 2 in the state of lid 22, and reaction tube 1 can be along sampling sample mixing
The inner tubal wall of device 2 moves down 4 millimeters, the final bottom for enabling the cut substrate 4 sampled in sample mixing device 2 to scratch reaction tube 1；
Indication box 3 is reacted, is made up of lower end rectangular pipe and upper end pipe and be in the structure of strip cast, is had in the middle part of it
The sharp protrusion thing 5 of sampling sample mixing device 2 can be punctured, the outer peripheral face of sharp protrusion thing 5 is evenly arranged with multiple breach 51, and reacts
The tail end of the lower end rectangular pipe of indication box 3 has the cavity volume 31 that can accommodate lateral chromatography test strips, and in the distal opening of cavity volume 31
It is fitted with bonnet 32；And the upper end for reacting indication box 3 has internal thread 33, accordingly, the end of sampling sample mixing device 2 then has
There is external screw thread 23, and the end of the sampling sample mixing device 2 is also fitted with sealing ring, the spiral shell of external screw thread 23 in sampling sample mixing device 2 end
It is connected in the state of the reaction end internal thread 33 of indication box 3, and sealing ring can be utilized to sample the sealing of sample mixing device 2 and be fixed on reaction
In indication box 3.
In experimentation is carried out, operating procedure it is as follows：
(1) closed sample adding device is first passed through to be loaded, it is also possible to pipettor by nucleic acid and enzyme and other necessary reagents,
Add in the reaction tube 1 for having inserted probe and carry out amplification experiment, after the completion of amplification experiment, reaction tube 1 is inserted into sampling sample mixing
In device 2, reaction tube 1 is gently leaned against in the beveled edge of the sharp cut substrate 4 in sampling sample mixing device 2, sample mixing is sampled by fastening
The upper lid 22 of device 2 makes inner tubal wall of the reaction tube 1 along sampling sample mixing device 2 move down 4 millimeters so that in sampling sample mixing device 2
Sharp cut substrate 4 can scratch the bottoms of reaction tube 1, the bottom-open of reaction tube 1,
(2) reaction tube 1 will have been inserted again and upper cover of lock 22 fastens closed sampling sample mixing device 2 and is put into centrifuge, by
The nucleic acid amplification product reaction solution that amplified reaction is completed in reaction tube 1, centrifugation are extremely sampled the front end of sample mixing device 2 by centrifugal force,
Complete previous work.
(3) in the state of the external screw thread 23 of sampling sample mixing device 2 end is screwed onto the reaction end internal thread 33 of indication box 3,
And sealing ring can be utilized to sample the sealing of sample mixing device 2 and be fixed in reaction indication box 3.Meanwhile and using reacting in indication box 3
Sharp protrusion thing 5 is punctured sample mixing device 2 is sampled, and sampling sample mixing device 2 is broken, and reaction solution lateral chromatography in indication box 3 is reacted tries
Under the siphonage of paper slip (LFD), moved to lateral chromatography test strips direction.
(4) because LFD lateral chromatography test strips have C (nature controlling line), T (detection line), when only C lines develop the color, for feminine gender
Reaction, it is the positive when C, T two-lines react, when not developing the color for two, reaction failure.
(5) the closed experimental system setup of this Rapid identification nucleic acid amplification product is not opened after detecting, because of sealing gasket and close
Seal all using silica gel material, reacts the acrylonitritrile-styrene resin AS materials of indication box 3, sampling sample mixing device 2 and reaction
Pipe 1 is made using medical grade polyvinylchloride, last gamma rays sterilizing, its good airproof performance, and nontoxic, you can by its whole
Discard in safe place.
Embodiment 3
The closed experimental system setup of this Rapid identification nucleic acid amplification product includes the reaction tube being arranged successively from top to bottom
1st, sample mixing device 2 and reaction indication box 3 are sampled, wherein：
Reaction tube 1, there is sealable lid 11；
Sample mixing device 2, including the upper lid 22 that pipe sleeve 21 and energy sealed tube 21 are open are sampled, and the middle part of pipe sleeve 21 has and can cut
The cutting angle of broken reaction tube 1 is 60 ° of cut substrate 4, and the pipe sleeve 21 for sampling sample mixing device 2 extends internally can support reaction tube
1 annular bead 211, cover lid 11 in reaction tube 1 and be placed into sampling sample mixing device 2 in the state of annular bead 211, reaction
Pipe 1 can be gently against in cut substrate 4, and is being linked closely on sampling sample mixing device 2 in the state of lid 22, and reaction tube 1 can be along sampling sample mixing
The inner tubal wall of device 2 moves down 5 millimeters, the final bottom for enabling the cut substrate 4 sampled in sample mixing device 2 to scratch reaction tube 1；
Indication box 3 is reacted, is made up of lower end rectangular pipe and upper end pipe and be in the structure of strip cast, is had in the middle part of it
The sharp protrusion thing 5 of sampling sample mixing device 2 can be punctured, the outer peripheral face of sharp protrusion thing 5 is evenly arranged with multiple breach 51, and reacts
The tail end of the lower end rectangular pipe of indication box 3 has the cavity volume 31 that can accommodate lateral chromatography test strips, and in the distal opening of cavity volume 31
It is fitted with bonnet 32；And the upper end for reacting indication box 3 has internal thread 33, accordingly, the end of sampling sample mixing device 2 then has
There is external screw thread 23, and the end of the sampling sample mixing device 2 is also fitted with sealing ring, the spiral shell of external screw thread 23 in sampling sample mixing device 2 end
It is connected in the state of the reaction end internal thread 33 of indication box 3, and sealing ring can be utilized to sample the sealing of sample mixing device 2 and be fixed on reaction
In indication box 3.
In experimentation is carried out, operating procedure it is as follows：
(1) closed sample adding device is first passed through to be loaded, it is also possible to pipettor by nucleic acid and enzyme and other necessary reagents,
Add in the reaction tube 1 for having inserted probe and carry out amplification experiment, after the completion of amplification experiment, reaction tube 1 is inserted into sampling sample mixing
In device 2, reaction tube 1 is gently leaned against in the beveled edge of the sharp cut substrate 4 in sampling sample mixing device 2, sample mixing is sampled by fastening
The upper lid 22 of device 2 makes inner tubal wall of the reaction tube 1 along sampling sample mixing device 2 move down 5 millimeters so that in sampling sample mixing device 2
Sharp cut substrate 4 can scratch the bottoms of reaction tube 1, the bottom-open of reaction tube 1,
(2) reaction tube 1 will have been inserted again and upper cover of lock 22 fastens closed sampling sample mixing device 2 and is put into centrifuge, by
The nucleic acid amplification product reaction solution that amplified reaction is completed in reaction tube 1, centrifugation are extremely sampled the front end of sample mixing device 2 by centrifugal force,
Complete previous work.
(3) in the state of the external screw thread 23 of sampling sample mixing device 2 end is screwed onto the reaction end internal thread 33 of indication box 3,
And sealing ring can be utilized to sample the sealing of sample mixing device 2 and be fixed in reaction indication box 3.Meanwhile and using reacting in indication box 3
Sharp protrusion thing 5 is punctured sample mixing device 2 is sampled, and sampling sample mixing device 2 is broken, and reaction solution lateral chromatography in indication box 3 is reacted tries
Under the siphonage of paper slip (LFD), moved to lateral chromatography test strips direction.
(4) because LFD lateral chromatography test strips have C (nature controlling line), T (detection line), when only C lines develop the color, for feminine gender
Reaction, it is the positive when C, T two-lines react, when not developing the color for two, reaction failure.
(5) the closed experimental system setup of this Rapid identification nucleic acid amplification product is not opened after detecting, because of sealing gasket and close
Seal all using silica gel material, reacts the acrylonitritrile-styrene resin AS materials of indication box 3, sampling sample mixing device 2 and reaction
Pipe 1 is made using medical grade polyvinylchloride, last gamma rays sterilizing, its good airproof performance, and nontoxic, you can by its whole
Discard in safe place.
The comma bacillus of embodiment 4, monokaryon Listeria, vibrio parahaemolytious, Shigella, Enterohemorrhagic E.coli (O157:
H7), the LAMP detections exemplified by Vibrio vulnificus, Yersinia ruckeri, vibrio alginolyticus, Vibrio harveyi, salmonella DNA sample
Laboratory：Biochip Beijing National ERC Ningbo branch center laboratory
Experimental standard product：Comma bacillus, monokaryon Listeria, vibrio parahaemolytious, Shigella, Enterohemorrhagic E.coli
(O157:H7), Vibrio vulnificus, Yersinia ruckeri, vibrio alginolyticus, Vibrio harveyi, the DNA sample of salmonella
Standard items source：Ningbo inspection and quarantining for import/export research institute
Probe synthesizes：For comma bacillus, monokaryon Listeria, vibrio parahaemolytious, Shigella, hemorrhagic large intestine bar
Bacterium, Vibrio vulnificus, Yersinia ruckeri, vibrio alginolyticus, Vibrio harveyi, the specific target gene of ten kinds of objects of salmonella,
Design is applied to the primer of LAMP amplifications, and probe of the fluorescein for follow-up LFD detections is marked on ring primer is added.
LFD lateral chromatography test paper：Using German GenLine HybriDetect test strip kits
Constant-temperature amplification and result detection：LAMP methods carry out target DNA amplification reaction, and kit has purchased from Rong Yan biotechnologies
Limit company.Carried out with experiment in the laboratory of biochip Beijing National ERC Ningbo branch center.Due to experiment
The various DNA extractions work in room is a lot, and laboratory Aerosol Pollution is serious, utilizes the present inventor's《One kind is used for multiple Mutiple Targets
The apparatus system (ZL201420076690.4) of the closed sample-adding of nucleic acid overall process》By ten groups of standard items (comma bacillus, monokaryon Li Si
Special bacterium, vibrio parahaemolytious, Shigella, Enterohemorrhagic E.coli (O157:H7), Vibrio vulnificus, Yersinia ruckeri, molten algae arc
Bacterium, Vibrio harveyi, salmonella) DNA sample, the buffer solution and the closed sample-adding of progress of enzyme, it is added to and is placed in advance in detection
In the reaction tube 1 of probe:
A, each primer concentration is in each reaction tube 1：
Each 1.6 μm of ol/L of FIP-bio and BIP,
Each 0.2 μm of ol/L of F3 and B3,
Each 0.8 μm of ol/L of LF-fitc and LB,
Total reaction system is 25 μ L.
B, by reaction tube 1,65 ° of lower 40~60min of constant-temperature incubation complete the expansion of LAMP target nucleic acids in constant-temperature incubation instrument
Increase.Then, by reaction tube 1, insert in the sampling sample mixing device 2 of preset hybridization buffer, and close the upper lid of sampling sample mixing device 2
22, the sampling sample mixing device 2 for having inserted reaction tube 1 is put into centrifuge, brief centrifugation 30 seconds, after will carry reaction tube 1
Sampling sample mixing device 2 by spiral way with reaction indication box 3 access and it is locking and closed after, utilize the point reacted in indication box 3
Sharp protrusion 5 is punctured sample mixing device 2 is sampled, and sampling sample mixing device 2 is broken, reaction solution lateral chromatography test paper in indication box 3 is reacted
Under the siphonage of bar (LFD), moved to lateral chromatography test strips direction, hybridization solution outflow is developed the color anti-by the siphon of LFD test strips
Should.
C, result is as illustrated, NC is negative control；And swimming lane 1-10 correspond to respectively comma bacillus, monokaryon Listeria,
Vibrio parahaemolytious, Shigella, Enterohemorrhagic E.coli (O157:H7), Vibrio vulnificus, Yersinia ruckeri, vibrio alginolyticus, Ha Wei
Family name vibrios, salmonella, can be seen that from result figure, and doing ten kinds of LAMP experiments using the experimental system setup of the present invention finds no
Pollute and influence experimental result phenomenon, effect is ideal.
Target nucleic acid amplification analyte detection exemplified by the male's hair sample of embodiment 5
Laboratory：Biochip Beijing National ERC Ningbo branch center laboratory
Laboratory sample：Male's hair sample
Standard items source：Ningbo inspection and quarantining for import/export research institute
Probe synthesizes：Dalian treasured biotech firm
LFD lateral chromatography test paper：German GenLine HybriDetect test strip kits
By male's hair sample after digestion cracks, special DNA adsorption columns and the buffer solution of unique design are added
System, then by it is a series of quickly rinsing-centrifugation the step of, by the magazines such as salt, cell metabolite, albumen remove, finally use
Sterilized water collects pure extracting genome DNA.Draw the DNA sample gathered on a small quantity and be added to containing according to man
Property Y chromosome sry gene on particular sequence (DYZ1) design be applied to the specific primer of PCR amplifications, dNTP, enzyme ease up
In the reaction tube 1 of fliud flushing and the probe presented for follow-up LFD visualization results.
A, PCR reaction systems are 20 μ L：
The wherein μ L of DNA profiling 3, the μ L of sense primer 1, the μ L of 1 μ L, PCR reaction solution of anti-sense primer 15.Then by reaction tube 1
It is placed in PCR instrument, according to pre-degeneration：94℃5min；Denaturation：94 DEG C of 30s, annealing：55 DEG C of 30s, polymerization：72 DEG C of 60s, repeat
35 circulations；72 DEG C of extension 5min PCR amplification conditions complete the amplified reaction of target gene.
B, then, reaction tube 1 is inserted in the sampling sample mixing device 2 of preset hybridization buffer, and closes sampling sample mixing device
Lid 22 on 2, the sampling sample mixing device 2 for having inserted reaction tube 1 is put into centrifuge, brief centrifugation 30 seconds, after will carry it is anti-
Should pipe 1 sampling sample mixing device 2 by spiral way and reaction indication box 3 access and it is locking and closed after, using reacting in indication box 3
Sharp protrusion thing 5 punctured sample mixing device 2 is sampled, sampling sample mixing device 2 is broken, reaction solution lateral chromatography in indication box 3 is reacted
Under the siphonage of test strips (LFD), moved to lateral chromatography test strips direction, hybridization solution outflow is shown by LFD test strips siphons
Colour response.As a result show that the DNA sample comes from male's hair.
C, experimental result it is clear as illustrated, and whole detection process, efficient and convenient and whole pollution-free, effect
It is ideal.
Claims (6)
- A kind of 1. closed experimental system setup of Rapid identification nucleic acid amplification product, it is characterised in that：Including from top to bottom successively Reaction tube (1), sampling sample mixing device (2) and the reaction indication box (3) being arranged, wherein：The reaction tube (1), there is sealable lid (11)；The sampling sample mixing device (2), including pipe sleeve (21) and the upper lid (22) that the pipe sleeve (21) opening can be sealed, and the pipe There is the cut substrate (4) that can cut the reaction tube (1) in the middle part of set (21), lid (11) juxtaposition is covered in the reaction tube (1) In the state of entering to sample in sample mixing device (2), the reaction tube (1) can be gently against in the cut substrate (4), and in described take of linking closely Covered on sample sample mixing device (2) in the state of (22), the reaction tube (1) can be downward along the inner tubal wall of the sampling sample mixing device (2) It is mobile 4~5 millimeters, finally cause the cut substrate (4) in the sampling sample mixing device (2) that the bottom of the reaction tube (1) can be scratched；The reaction indication box (3), its middle part have the sharp protrusion thing (5) that can puncture the sampling sample mixing device (2), the point The outer peripheral face of sharp protrusion (5) is provided with least one breach (51), and the tail end of the reaction indication box (3) has and can accommodated The cavity volume (31) of lateral chromatography test strips, and it is fitted with bonnet (32), the cut substrate in the distal opening of the cavity volume (31) (4) cutting angle is between 30 °~60 °.
- 2. the closed experimental system setup of Rapid identification nucleic acid amplification product according to claim 1, it is characterised in that：Institute The end for stating reaction indication box (3) has internal thread (33), and accordingly, the end of the sampling sample mixing device (2) is then with outer Screw thread (23), and the end of the sampling sample mixing device (2) is also fitted with sealing ring.
- 3. the closed experimental system setup of Rapid identification nucleic acid amplification product according to claim 1, it is characterised in that：Institute The pipe sleeve (21) for stating sampling sample mixing device (2) extends internally and has the annular bead (211) that can support the reaction tube (1).
- 4. a kind of usage right requires the closed reality of the Rapid identification nucleic acid amplification product described in 1~3 any one claim Check system device is in non-diagnostic purpose ring mediated isothermal amplification and nucleic acid lateral f chromatographic flow test strips combination quick detection target nucleus Application in sour amplified production method.
- 5. a kind of usage right requires the closed reality of the Rapid identification nucleic acid amplification product described in 1~3 any one claim Application of the check system device in non-diagnostic purpose prevents nucleic acid amplification product pollution, avoids Aerosol Pollution method.
- 6. a kind of usage right requires the closed reality of the Rapid identification nucleic acid amplification product described in 1~3 any one claim Identification of Species, gene of the check system device in testing food pathogenic and agricultural, industry, customs, animal husbandry on gene level The application in non-diagnostic mesh purposes in abrupt climatic change.
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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 |
| CN106497778B (en) * | 2016-11-04 | 2018-08-07 | 中国人民解放军军事医学科学院基础医学研究所 | A kind of device for the detection of constant temperature nucleic acid amplification quick visualization |
| CN108130270A (en) * | 2016-12-01 | 2018-06-08 | 李治国 | Instant genetic test packet |
| CN106906130A (en) * | 2017-04-19 | 2017-06-30 | 北京倍肯创新诊断技术研究院有限责任公司 | A kind of flat PCR reaction tubes of printing opacity high |
| CN107102000B (en) * | 2017-05-31 | 2024-03-08 | 苏州晶睿生物科技有限公司 | Nucleic acid detection device |
| CN107619775B (en) * | 2017-09-20 | 2021-03-26 | 宝瑞源生物技术(北京)有限公司 | Portable nucleic acid detection platform suitable for PCR chromatography |
| CN111548900A (en) * | 2019-02-12 | 2020-08-18 | 浙江大学 | Device and method for amplifying and detecting nucleic acid |
| CN211955507U (en) * | 2019-02-25 | 2020-11-17 | 上海快灵生物科技有限公司 | Biochemical reaction test paper strip pipe and kit |
| CN111424073B (en) * | 2020-04-30 | 2022-11-22 | 陕西科技大学 | Closed-tube nucleic acid amplification detection method and device |
| CN111647497A (en) * | 2020-05-14 | 2020-09-11 | 南京达伯可特生物科技有限公司 | Multiple closed nucleic acid amplification product rapid detection device |
| CN111733288B (en) * | 2020-06-22 | 2022-09-30 | 厦门大学 | Nucleic acid detection method and device and application in COVID-19 detection |
| CN113249196B (en) * | 2021-05-06 | 2024-07-26 | 北京谊安生物科技有限公司 | Heat expansion and cold contraction integrated reaction tube |
| CN116496878A (en) * | 2022-01-19 | 2023-07-28 | 江苏为真生物医药技术股份有限公司 | Sealing device applied to rapid detection and use method and application thereof |
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| CN1888902A (en) * | 2006-08-11 | 2007-01-03 | 杭州优思达生物技术有限公司 | Full closed target nucleic amplifier fast testing device |
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| US8911941B2 (en) * | 2011-04-14 | 2014-12-16 | Kenneth J. Michlitsch | Methods and apparatus for point-of-care nucleic acid amplification and detection |
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| CN1888902A (en) * | 2006-08-11 | 2007-01-03 | 杭州优思达生物技术有限公司 | Full closed target nucleic amplifier fast testing device |
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