CN2615141Y - Non-flushing PCR amplificating tube capable of directly detecting gene - Google Patents

Abstract

The utility model discloses a flushing-free PCR augmentation pipe which can detect gene comprising a, wherein the reactor pipe is composed of a pipe cap and a pipe body. A molecule beacon is fixed in the reactor pipe which is used in PCR. The domain where the molecule beacon is fixed in the PCR reactor pipe is equipped with a transparent window. The molecule beacon is fixed inside of the transparent window. The technical project of which the molecule beacon is introduced into the utility model can reduce operation of gene amplification, crossbreed and detecting unitization. Fluorescence can not need to be filtered when target gene increases thereby cutting the cost and improving the reliability of result.

Description

But the flushing-free pcr amplification pipe of direct gene detection

One, technical field

The utility model relates to a kind of pcr amplification pipe (the pcr amplification pipe 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 pipe that can directly detect amplified production.

Two, technical background

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 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 hybridize, detect by the fluorescent scanning instrument with the nucleic acid probe on the chip.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, there are not 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 are sample with regard to its major cause all divide to come to be carried out separately, cause prior art to exist the defective of complex operation, complexity thus, 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 and since the entire operation process complicacy improve, as the cleaning repeatedly of the non-specific probe of condition control and hybridization back of sample preparation process etc., be unfavorable for integrated detection.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

But technical problem the utility model need not to mix fluorescence when providing a kind of operation that can make gene amplification, hybridization and detect integrated working method to be able to easy, target gene amplification, cost is minimized and can make the flushing-free pcr amplification pipe of the direct gene detection that gained result's reliability is improved.

But technical scheme the utility model is a kind of flushing-free pcr amplification pipe of direct gene detection, comprise reaction tubes and this reaction tubes by the pipe cover 11 and body 12 form, at reaction tubes 1 internal fixing that is used for PCR molecular beacon 2 is arranged.1. beneficial effect can constitute the space of a relative closure owing to the utility model is arranged at reaction tubes reaction tubes self with molecular beacon, make gene amplification, hybridization and detect incorporate working method and can finish in the space of above-mentioned relative closure as follows: the gene extract to be amplified that A) will prepare, amplification liquid (comprising corresponding primer and enzyme etc.) add in the reaction tubes body, and block is held on the 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 the amplification after the solution stream molecular beacon of falling fixed zone, with the molecular beacon effect on the pipe internal surface, control certain temperature and make it carry out specific hybridization; D), read the fluorescent signal of molecular beacon on the reaction tubes, thereby obtain the hybridization information of gene by optical detecting method.And this method is just because of the utility model has been arranged, could pass through some simple operations, realize gene amplification, hybridization, detecting operation continuously, and need not in the amplification procedure of target gene to add marker such as fluorophor, avoid behind pcr amplification, opening the amplification pipe, to clean the target gene of non-specific hybridization, and add operation such as hybridization solution, not only simplified operation steps, the more important thing is the detection false positive of having avoided pcr amplification product fully and make this method can adopt existing equipment or existing installation improved a little after equipment, reduced cost.Molecular beacon is different with common nucleic acid probe (single stranded nucleic acid probe).Molecular beacon is made up of 4 parts such as the cane district of fluorophor, fluorescent quenching group and double-strandednucleic acid, single-stranded loop forming core acid districts.Wherein, the zone discerned for molecular beacon and detected target gene hybridization, single-stranded loop forming core acid district.When molecular beacon with after target gene combines, the stem stalk of double-strandednucleic acid is opened, thereby the distance between fluorescence group and the fluorescent quenching group is changed, and causes the change of the fluorescence radiation character of molecular beacon.And the dna single chain probe of common nucleic acid be by with the target gene hybridization that is labeled fluorescence molecule, realize detecting.The general direct target gene that in solution, detects of molecular beacon, but this can only detect a kind of target gene.The utility model detects target gene with immobilized molecular beacon method, not only can detect a plurality of genes simultaneously, and can need not target gene mixed special manipulation such as fluorescence, also need not the non-specific adsorption of hybridizing on the chip of back is cleaned, to reduce fluorescence background.The utility model is directly fixed on molecular beacon in the pcr amplification pipe exactly, realizes that in PRC amplification pipe gene amplification and hybridization detect integrated.Fluorophor and quenching of fluorescence group in the molecular beacon, when hybridization detects, be used for not needing to carry out fluorescent mark with the nucleic acid fragment of molecular beacon hybridization, after the hybridization, nucleic acid fragment combines with molecular beacon, fluorophor and quenching of fluorescence group that molecular beacon inside is had separate in the space, the signal of its fluorophor can be detected, simultaneously, fluorophor that the molecular beacon of not hybridizing has and quenching of fluorescence group spatially keep slight distance, and fluorescent signal can not be detected, so do not need to wash, reduce operation steps, reduced the difficulty that detects.To sum up, that the operation that the utility model can make gene amplification, hybridization and detect integrated working method is able to is easy, cost is minimized and gained result's reliability is improved, and can realize flushing-free.

2. because the utility model can make working method carry out multipass amplification, hybridization and detection,, the utility model detects and the acquisition quantitative result so can making this method realize dynamically following the tracks of.

3. the utility model adopts the connection of " molecular beacon is connected on the transparent window by chemical active radical ", has in conjunction with firmly, be not easy to break away from, and the advantage that can repeatedly use repeatedly, the test sample that needs simultaneously is few, the density height of molecular probe.

4. the utility model technical measures of " molecular beacon being fixed in the high-molecular gel medium, being fixed on the transparent window " have improved the density of fixed member beacon, have strengthened hybridization signal, make gained result's reliability be able to further raising.

5. the utility model method of " transparent window being divided into several regions; assembling has the nucleotide sequence molecular beacon of similar and different base arrangement mode on different zones respectively ", can in a PCR test, hybridize detection, can carry out high-throughout detection several even tens thousand of gene segments.

Four, description of drawings

Fig. 1 is a structural representation of the present utility model.

Fig. 2 is the direct chemical annexation figure of the utility model molecular beacon.

Five, specific embodiments

But the flushing-free pcr amplification pipe of 1 one kinds of direct gene detection of embodiment, comprise reaction tubes and this reaction tubes by the pipe cover 11 and body 12 form, at reaction tubes 1 internal fixing that is used for PCR molecular beacon 2 is arranged, on the zone of PCR reaction tubes internal fixing molecular beacon, transparent window 3 is arranged, molecular beacon 2 is transparent window 3 inboards fixedly, on transparent window 3, be provided with its group's layer of chemically reactive, molecular beacon 2 is located on the chemical group layer, and this chemical group layer can adopt amino layer, the aldehyde radical layer, the cyano group layer, present embodiments such as sulfydryl layer can take following concrete way that molecular beacon is fastened on the transparent window by chemical nsg layer:

On surfaces such as clear plastic window such as polystyrene, glass by plasma body activate, uv-radiation activates and method such as chemokinesis, on the transparent window surface, connect chemical active radical as amino, aldehyde radical, cyano group, sulfydryl etc., utilize the chemical group reaction on above-mentioned chemical active radical and the molecular beacon again, molecular beacon is connected on the transparent window.

2. the modifying method of clear plastic window, glass surface is had a variety of, as the pentanedial decoration method), polylysine modifies method, sulfydryl modification method, polyose modification method, BSA-NHS and modifies method, hydrogel modification method etc.,

The chemical treatment of transparent glass window: put into the solution of forming by 1/3 hydrogen peroxide (30%) and 2/3 sulfuric acid (18M), soak 1 hour (21).Again with deionized-distilled water flushing 3 times; Putting into deionized-distilled water boiled 10 minutes; Dry under argon gas stream, preserve standby in the drying place.

Aminosilane processing: the solution 10 minutes that pretreated substrate is immersed 95% acetone that contains 1%3-aminopropyltriethoxysilane (aminosilane), after the taking-up, clean with acetone and deionized water rinsing, after under 120 ℃ dry 45 minutes, place dry place to preserve.Slide behind the silanization is put into the PBS solution that contains 3%～4% glutaraldehyde, and soaking at room temperature 2 hours is taken out, and cleans, and dries up with nitrogen, places 4 ℃ of preservations standby.

BSA-NHS modifies preparation: clear plastic window, the slide of silanization are put into and are contained 1.76g N-N '-disuccinimidyl carbonate, 1.2ml N-N '-diisopropylethylamine, in the solution A of 68.8ml N-N '-dimethylformamide (DMF), room temperature reaction took out after 3 hours, immersion contains the PBS solution room temperature of 1%BSA placed 12 hours, took out then and put into solution A, and room temperature continues reaction 3 hours, take out, clean dry up standby.

Agarose modify to be handled: agarose adds distilled water and is mixed with 1% agarose solution, mixes fully and boils 3 minutes.At the agarose solution that each sheet has all been toppled over 2 ml on the clear plastic window of silanization, wait to solidify the back in 37 ℃ of following dried overnight, preserve under the drying at room temperature condition.Use the NaIO of 20mM before using ₄Activated in water solution gets final product with distilled water is clean again.

Sulfydryl modification is handled: preparation contains the toluene solution of hydrosulphonyl silane 1%, and the clear plastic window that will handle is put into solution, spends the night under the room temperature.Take out slide, clean, dry up with trichloromethane, acetone and other organic solvent.

Polylysine is modified and is handled: the clear plastic window that cleans up is put into the PBS solution that contains 3% polylysine soaked 2 hours, clean, dry up, in 4 ℃ of preservations.

But the flushing-free pcr amplification pipe of 2 one kinds of direct gene detection of embodiment, comprise reaction tubes and this reaction tubes by the pipe cover 11 and body 12 form, at reaction tubes 1 internal fixing that is used for PCR some molecular beacons 2 are arranged, be respectively applied for the specific gene segment and the first type that detect the relevant coronavirus of severe acute respiratory syndrome, the specific gene segment of Influenza B virus, on the zone of PCR reaction tubes internal fixing molecular beacon, transparent window 3 is arranged, molecular beacon 2 is transparent window 3 inboards fixedly, present embodiment is divided into several regions to transparent window, on different zones, assemble nucleotide sequence molecular beacon respectively with similar and different base arrangement mode, present embodiment can also be fixed in molecular beacon 2 in the high-molecular gel medium, be fixed on the transparent window 3, above-mentioned high-molecular gel medium can be selected polyacrylamide for use again, agarose, polyvinyl alcohol etc.Test sample is after virolysis is handled, put into flushing-free pcr amplification pipe, the amplimer that adds reverse transcription PCR (RT-PCT) reagent and coronavirus and first type, Influenza B virus simultaneously behind the sealing amplification pipe, carries out carrying out on the PCR reaction unit amplified reaction.After amplification is finished, amplified production is introduced window region; If in test sample, have detected virus, then can detect fluorescent signal in corresponding molecular beacon FX.

But the flushing-free pcr amplification pipe of 3 one kinds of direct gene detection of embodiment, comprise reaction tubes and this reaction tubes by the pipe cover 11 and body 12 form, at reaction tubes 1 internal fixing that is used for PCR some molecular beacons 2 are arranged, be respectively applied for the specific gene segment and the first type that detect the relevant coronavirus of severe acute respiratory syndrome, the specific gene segment of Influenza B virus, on the zone of PCR reaction tubes internal fixing molecular beacon, transparent window 3 is arranged, in the present embodiment, on transparent window 3, be provided with transparent high-molecular gel layer 4 (as polyacrylamide, agarose, polyvinyl alcohol etc.), again molecular beacon 2 is fixed on the high-molecular gel medium layer.

Claims (5)

1. but the flushing-free pcr amplification pipe of a direct gene detection comprises that reaction tubes and this reaction tubes be made up of pipe lid (11) and body (12), and it is characterized in that has molecular beacon (2) being used for the reaction tubes of PCR (1) internal fixing.

2. but the flushing-free PCR reaction tubes of direct gene detection according to claim 1 is characterized in that transparent window (3) is arranged on the zone of PCR reaction tubes internal fixing molecular beacon, and molecular beacon (2) is transparent window (3) inboard fixedly.

3. but the flushing-free pcr amplification pipe of direct gene detection according to claim 1 and 2 is characterized in that being provided with its group's layer of chemically reactive on transparent window (3), and molecular beacon (2) is located on the chemical group layer.

4. but the flushing-free pcr amplification pipe of described direct gene detection according to claim 1 and 2, it is characterized in that on transparent window (3), being provided with transparent high-molecular gel layer (4), again molecular beacon (2) is fixed on the high-molecular gel medium layer (4).

5. but the flushing-free pcr amplification pipe of described direct gene detection according to claim 2, it is characterized in that transparent window is divided into several regions, on different zones, assemble nucleotide sequence molecular beacon respectively with similar and different base arrangement mode.

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