CN2649597Y - PCR amplification tube for multi-step reaction - Google Patents
PCR amplification tube for multi-step reaction Download PDFInfo
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- CN2649597Y CN2649597Y CN 200320110590 CN200320110590U CN2649597Y CN 2649597 Y CN2649597 Y CN 2649597Y CN 200320110590 CN200320110590 CN 200320110590 CN 200320110590 U CN200320110590 U CN 200320110590U CN 2649597 Y CN2649597 Y CN 2649597Y
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- amplification
- pipe
- solution pool
- pcr
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Abstract
The utility model discloses a PCR augmenter used for multi step reactions, which includes an amplification pipe assembly. The amplification pipe assembly is composed of pipe covers and amplification pipes. The amplification pipe assembly is equipped with a solution pool opening upward and a solution flow passage. The solution pool lies inside of the amplification pipe. The utility model positions the liquid storage tank inside of the reaction tube, and the reaction tube itself can form a relatively sealed space, and therefore the gene amplification and the post amplification operation can be finished in the relatively sealed space. The multi-step reaction of the gene amplification and the related gene detection can be completed in the same PCR amplification pipe in the sealed space.
Description
One, technical field
The utility model relates to a kind of pcr amplification device that can be used for finishing polystep reaction, is used for gene amplification and genes involved thereof and detects.
Two, background technology
The utility model relates to a kind of pcr amplification pipe (PCR is a technology well known to those skilled in the art) that is used for gene amplification and directly detects, and refers in particular to a kind of flushing-free pcr amplification device that can directly detect amplified production.
At present, in molecular biology research and medical clinic applications process, polymerase chain reaction (PCR) technology is indispensable.PCR normally carries out in the tube chamber of a sealing, and it can amplify detected target gene tens thousand of to millions of times.But, after the gene amplification, generally need detect amplified production with methods such as electrophoresis.Because PCR method sensitivity is high, its amplified production inevitably can enter in the air in testing process.When testing, these skyborne products that float may be amplified out again next time, and Here it is, and PCR detects the reason that obtains so-called " false positive " easily.For this reason, some biotech companies have developed enclosed gene amplification and detection technique in the world.As fluorescent quantitative PCR technique.But this Technology Need has very complicated dynamic optical proofing unit, and detecting instrument is very complicated, is difficult to promote in China.Simultaneously, the each test of this technology can only detect the information of a gene, can not satisfy the requirement of bioinformation epoch people's needs to the detection of a large amount of gene informations.
Biochip technology provides strong instrument for people detect a large amount of gene informations simultaneously.Gene chip is fixed in many different nucleic acid probes (single stranded oligonucleotide) on the slide, the amplified production mark fluorescent of gene in the sample, and hybridizes with the nucleic acid probe on the chip, detects by the fluorescent scanning instrument.Though what gene chip obtained contains much information, the most gene chip is mainly used in laboratory studyes such as the mRNA detection of expression application of cell at present.In important pathogenic micro-organism context of detection, seldom there are sophisticated application example and product really up to the mark to come out so far.The operating process such as processing, amplification label, hybridization, detection that mainly are sample with regard to its reason all divide to come to be carried out separately, thereby make whole testing process complex operation, complexity, and therefore increased the chance of polluting, reduced gained result's reliability.Although gene amplification technology and chip detecting method are integrated at present, but be characterized in that whole gene amplification process carries out in a micro reaction pool, thereby, need be to the cooling that heats up repeatedly of the same position of device, this can't dynamically follow the tracks of and real-time quantitative analysis the result.For this reason, some investigators propose a gene chip and combine with micro-fluidic technologies both at home and abroad, realize the integrated of all operations process.The applicant also studies this, and has proposed gene amplification microarray probe circulating detection type biological chip etc.Yet, because this chip preparation difficulty, and need the special reactor of development supporting with it, so its cost is very high.Simultaneously, gene chip requires the amplified production mark fluorescent of target gene, detects the hybridization state of nucleic acid probe by fluorescent signal.This just requires will mix fluorescence in the sample preparation process, and the cost that this has not only improved sample preparation has increased difficulty and uncertain factor.And, in this process,, influence the reliability that detects because of the labeling effciency problem.Simultaneously, because the complicacy of entire operation process improves,, be unfavorable for integrated detection as the cleaning repeatedly of the non-specific probe of condition control and hybridization back of sample preparation process etc.Therefore, can to carry out the biochip technology cheaply that non-marked detects to detected gene order be one of key that realizes biochip a large amount of practical applications in fields such as medical science and life science in development.
Three, technology contents
Technical problem: the utility model provides a kind of pcr amplification pipe that is used for polystep reaction, and polystep reaction is finished in same pcr amplification pipe under closed state.
Technical scheme: the utility model is a kind of pcr amplification device that is used for polystep reaction, comprises amplification pipe assembly, and amplification pipe assembly is made up of pipe lid and amplification pipe, is provided with solution pool and the solution runner that opening makes progress on amplification pipe assembly, and solution pool is positioned at the amplification pipe.
Technique effect: 1. liquid storing pool is arranged in the reaction tubes owing to the utility model, reaction tubes self can constitute the space of a relative closure, gene amplification and amplification back operation can be finished in the space of above-mentioned relative closure: the gene extract to be amplified that A) will prepare, amplification liquid (comprising corresponding primer and enzyme etc.) add in the reaction tubes body, in liquid storing pool, add gene amplification afterreaction system again, sealing PCR reaction tubes; B) reaction tubes that sealing is got well is positioned over and increases in the corresponding gene-amplificative instrament; Gene-amplificative instrament can use various popular instruments, also can be amplified hybridization and detection integrated instrument by improved special use; C) will manage in after the amplification solution stream after, make two kinds of liquid mixing, will mix back liquid again and firmly be thrown to bottom the PCR reaction tubes; D) after solution mixes, carry out next step gene amplification reaction again.Therefore, the utility model can make polystep reactions such as gene amplification or genes involved detection finish in same pcr amplification pipe under closed state.
2. because the utility model can make the RT-PCR (reverse transcription gene amplification) of two step method realize in single PCR pipe.Reaction reagent in the PCR pipe carries out transcriptive process,reversed so that produce dna segment in the solution, for another example before described method carry out the reverse transcription system and mix with solution in the liquid storing pool, carry out PCR again and react.In closed system, carry out the RT-PCR of two step method, the reaction yield height is arranged, be difficult for producing advantages such as pollution, sensitivity height.
3. the utility model adopts and stores two kinds of reaction systems in the closed system, can carry out two step gene amplification reactions under the state that is not subjected to external interference, as can significantly reduce the operation steps of experiment in the nest-type PRC reaction, improves the repeatability and the accuracy of experiment.
4. the utility model adopts and stores two kinds of reaction systems in the closed system, wherein a kind of is the gene amplification mixed system, and stores hybridization buffer in liquid storing pool, can be after the PCR reaction finishes, mix with hybridization buffer, nucleic acid probe last and that be fixed on the PCR inside pipe wall is hybridized.Be amplified the result to detect gene,, can confirm as and before gene amplification, have detected gene if clear and definite hybridization signal is arranged.
Four, description of drawings
Fig. 1 is the structural representation of the utility model embodiment 1.
Fig. 2 is the structural representation of the utility model embodiment 2.
Fig. 3 is the structural representation of the utility model embodiment 3.
Fig. 4 is the structural representation of the utility model embodiment 4.
Wherein 1: pipe lid, 2: regular-PCR pipe, 3: fluid passage, 4: solution pool
Five, specific embodiments
Embodiment 1
A kind of pcr amplification device that can be used for finishing polystep reaction, using method is:
1, the preparation of chip
According to each target-gene sequence that will detect, design an oligonucleotide probe of 20-50 base of the effective hybridization of minus strand (promptly identical) with it with normal chain, amino of one end mark, by the point sample method these probes are fixed to the pipe lid inner face of handling according to certain array, preparation pipe cover core sheet.
2, the design of primers of gene-amplification
According to each target-gene sequence that will detect, design the amplimer about a pair of 20 bases at two ends, therein one with normal chain complementary primer 5 ' end fluorescence molecule of mark (as FAM, CY3, CY5 etc.).
3, gene-amplification
In common PCR amplification pipe, add PCR or RT-PCR reaction system, primer and the DNA that handles well or the RNA template that mixes in advance.Fill in solution pool then, add the hybridization buffer of 30ul at solution pool.Cover the pipe lid for preparing at last.On the pcr amplification instrument, carry out amplified reaction according to certain program.
4, hybridization detects
After pcr amplification finished, the pipe that will increase was inverted and is firmly made PCR reaction solution and hybridization solution to mix and gets rid of to pipe lid end, places 37 ℃ of hybridization 1 hour.
5, chip detection
To manage the cover core sheet places the fluorescent scanning microscopically to carry out the results of hybridization detection.
Embodiment 2
A kind of pcr amplification pipe that is used for polystep reaction comprises that reaction tubes and this reaction tubes be made up of the liquid storing pool of the storage liquid in PCR pipe and the pipe.Be respectively applied for the specific primer (referring to the open primer of WHO) that detects the relevant coronavirus of severe acute respiratory syndrome, in the PCR pipe, add the reverse transcription system, in liquid storing pool, add the gene amplification system simultaneously, and add an amount of SYBGREEN.The gene amplification condition of using WHO to recommend, after being provided with on the PCR instrument, carry out reverse transcription after, the PCR pipe be inverted make two kinds of solution mix mutually, carry out pcr amplification again.Before and after the amplification, can be in direct viewing under the ultraviolet lamp, if fluorescence, then gene is amplified, and contains the relevant coronavirus of detected severe acute respiratory syndrome in the tested systems.
A kind of pcr amplification device that can be used for finishing polystep reaction, using method is:
1, the preparation of chip
According to each target-gene sequence that will detect, design an oligonucleotide probe of 20-50 base of the effective hybridization of minus strand (promptly identical) with it with normal chain, with Beacon designer2.1 software this probe design is become molecular beacon, by the point sample method these probes are fixed to the pipe lid inner face of handling according to certain array then, preparation pipe cover core sheet.
2, the design of primers of gene-amplification
According to each target-gene sequence that will detect, design the amplimer about a pair of 20 bases at two ends.
3, the amplification of PCR, hybridization and detection
The amplification of PCR, hybridization and detection are with scheme 1
Embodiment 4
A kind of pcr amplification device that is used for polystep reaction, comprise amplification pipe assembly, amplification pipe assembly by pipe cover 1 and amplification manage 2 and form, on amplification pipe assembly, be provided with solution pool and the solution runner 3 that opening makes progress, solution pool is positioned at amplification pipe 2, solution pool hangs to be located to manage and covers on 1, and the outer bottom of solution pool is a ramped bottom surface.
Embodiment 5
A kind of pcr amplification device that is used for polystep reaction, comprise amplification pipe assembly, amplification pipe assembly by pipe cover 1 and amplification manage 2 and form, on amplification pipe assembly, be provided with solution pool and the solution runner 3 that opening makes progress, solution pool is positioned at amplification pipe 2, and solution pool is annular solution 4, and solution runner 3 is positioned at the internal perisporium of annular solution pool 41, solution pool is located in the amplification pipe 2, and the outer wall of solution pool matches with the amplification inside pipe wall.
Embodiment 6
Solution pool lacks shape solution pool 42 for circle, its cross section lacks shape for circle, solution runner 3 is the gap between scarce shape solution pool 42 of circle and amplification pipe 2 inwalls, the cross section that circle lacks shape solution pool 42 is that central angle is greater than the scarce shape of 180 ° circle, and be located in the amplification pipe 2, circle lacks the outer wall of shape solution pool 42 and the inwall of amplification pipe 2 matches, and the end face that circle lacks shape solution pool 42 is the scarp.
Embodiment 7
A kind of pcr amplification device that is used for polystep reaction, comprising amplification pipe assembly, amplification pipe assembly by pipe cover 1 and amplification pipe 2 forms, be provided with solution pool and the solution runner 3 that opening makes progress increasing to manage on the assembly, solution pool is positioned at amplification pipe 2, and solution pool is fixed in the amplification pipe 2 by support 5.
Claims (9)
1, a kind of pcr amplification device that is used for polystep reaction, comprise amplification pipe assembly, amplification pipe assembly is made up of pipe lid (1) and amplification pipe (2), it is characterized in that being provided with solution pool and the solution runner (3) that opening makes progress on amplification pipe assembly, and solution pool is positioned at amplification pipe (2).
2, the pcr amplification device that is used for polystep reaction according to claim 1 is characterized in that solution pool hangs to be located on the pipe lid (1).
3, the pcr amplification device that is used for polystep reaction according to claim 1 and 2, the outer bottom that it is characterized in that solution pool is a ramped bottom surface.
4, the pcr amplification device that is used for polystep reaction according to claim 1 is characterized in that solution pool is annular solution pool (41), and solution runner (3) is positioned at the internal perisporium of annular solution pool (41).
5, the pcr amplification device that is used for polystep reaction according to claim 4 is characterized in that solution pool is located in the amplification pipe (2), and the outer wall of solution pool matches with the amplification inside pipe wall.
6, the pcr amplification device that is used for polystep reaction according to claim 1 is characterized in that solution pool is that circle lacks shape solution pool (42), and its cross section is that circle lacks shape, and solution runner (3) is the gap between the scarce shape solution pool (42) of circle and amplification pipe (2) inwall.
7, the pcr amplification device that is used for polystep reaction according to claim 6, the cross section that it is characterized in that the scarce shape solution pool (42) of circle is that central angle is greater than the scarce shape of 180 ° circle, and be located in the amplification pipe (2), circle lacks the outer wall of shape solution pool (42) and the inwall of amplification pipe (2) matches.
8, the pcr amplification device that is used for polystep reaction according to claim 7 is characterized in that the end face of the scarce shape solution pool (42) of circle is the scarp.
9, the pcr amplification device that is used for polystep reaction according to claim 1 is characterized in that solution pool is fixed in the amplification pipe (2) by support (5).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200320110590 CN2649597Y (en) | 2003-11-03 | 2003-11-03 | PCR amplification tube for multi-step reaction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200320110590 CN2649597Y (en) | 2003-11-03 | 2003-11-03 | PCR amplification tube for multi-step reaction |
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| Publication Number | Publication Date |
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| CN2649597Y true CN2649597Y (en) | 2004-10-20 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 200320110590 Expired - Lifetime CN2649597Y (en) | 2003-11-03 | 2003-11-03 | PCR amplification tube for multi-step reaction |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160431A (en) * | 2013-04-17 | 2013-06-19 | 东南大学 | Gene-detection integrally completed enclosed PCR (polymerase chain reaction) amplification tube |
| CN103194379A (en) * | 2013-04-17 | 2013-07-10 | 东南大学 | Closed type PCR (Polymerase Chain Reaction) amplification tube for integrated gene detection |
-
2003
- 2003-11-03 CN CN 200320110590 patent/CN2649597Y/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160431A (en) * | 2013-04-17 | 2013-06-19 | 东南大学 | Gene-detection integrally completed enclosed PCR (polymerase chain reaction) amplification tube |
| CN103194379A (en) * | 2013-04-17 | 2013-07-10 | 东南大学 | Closed type PCR (Polymerase Chain Reaction) amplification tube for integrated gene detection |
| CN103160431B (en) * | 2013-04-17 | 2014-07-23 | 东南大学 | Gene-detection integrally completed enclosed PCR (polymerase chain reaction) amplification tube |
| CN103194379B (en) * | 2013-04-17 | 2014-08-06 | 东南大学 | Closed type PCR (Polymerase Chain Reaction) amplification tube for integrated gene detection |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| AV01 | Patent right actively abandoned |
Effective date of abandoning: 20031103 |
|
| C25 | Abandonment of patent right or utility model to avoid double patenting |