CN102534011B - Totally-enclosed fast fluorescence detection method and device for target nucleic acid amplification products - Google Patents
Totally-enclosed fast fluorescence detection method and device for target nucleic acid amplification products Download PDFInfo
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Abstract
The invention relates to a totally-enclosed fast fluorescence detection method and a device for target nucleic acid amplification products. The method is characterized in that when an amplification reaction is over, a reaction tube is placed in a closed device under the condition that a cover is not opened, the wall of the reaction tube is broken in the closed device so as to enable amplificationproducts in the reaction tube to react with detection liquid arranged in the closed device in advance, then fluorescence detection is performed, and the result can be read. The device comprises a PCR(Polymerase Chain Reaction) tube, a centrifuge tube and an ejector plug; the PCR tube can be placed in the centrifuge tube; the ejector plug is provided with a puncture needle capable of puncturing the PCR tube; and the centrifuge tube is provided with a tube cap capable of being closed. With the method and the device, fluorescence detection can be performed for target nucleic acid amplification products quickly, and the target nucleic acid amplification products can be prevented from being contaminated so as to avoid false positive result. The invention can be applied to clinical infectious pathogen detection, food pathogen detection as well as species identification and the like in the genic level in the field of agriculture, industry, customs, animal husbandry and the like.
Description
Technical field
The proofing unit of the totally-enclosed target nucleic acid amplified production of the present invention, more particularly, relate to the method for utilizing fluorescent probe technique rapid detection target nucleic acid amplified production in totally-enclosed proofing unit, the invention still further relates to test kit and the purposes in detecting infectious disease pathogens thereof of utilizing this totally enclosed device for fast detecting rapid detection target nucleic acid amplified production.
Prior art
The history in existing more than 100 year of nucleic acids research, late 1960s, the beginning of the seventies, people are devoted to study the gene in vitro isolation technique, and nineteen eighty-three U.S. scientist Kary Mullis drives to travel at the interstate highway that wriggles, and has been pregnant with the blank of PCR.Through the effort in 2 years, confirm the conception of PCR experimentally, and applied for first patent of relevant PCR in 1985, delivered first piece of PCR academic paper at the science magazine.From then on round pcr has obtained the generally approval of life science circle.Therefore Kary Mullis has also obtained Nobel chemistry Prize.
Round pcr itself is very simple, and can satisfy the different requirements that the biologist operates DNA to greatest extent.Round pcr is development with surprising rapidity again, and the speed that is penetrated into each biology subscience and clinical each section also makes other biotechnology hope and give birth to and sigh.PCR has important application value in many scientific domains such as molecular biology, medical research, life science, biotechnology, genetic engineering, medical diagnosis on disease, medical jurisprudence, archeology, and huge pushing effect is played in the development of modern molecular biology techniques such as the gene clone of having set up, dna sequence analysis.PCR can provide the very believable Accurate Analysis of genetic stew, than responsive 10,000 to 1,000,000 times of any prior art.Someone predicts that 21 century is biological century, and PCR is most important technology of biological century so.
The concrete application of PCR has following big aspect:
1, PCR can be used for the early diagnosis of tumour and the mensuration of oncotherapy effect
2, PCR can be used for the mensuration of pathogeny microorganism, as: HAV, HBV, HCV, HIV, pathogenic bacterium, virus etc.
3, the diagnosis of heredopathia.
4, the diagnosis of the diagnosis of presenile dementia and treatment and Parkinson disease.
5, legal medical expert's identification and analysis, as: paternity test, crime scene analyzing specimen.
The PCR product is generally 10
13Copy/ml gets 0.1ml and is 10
9Copy, and the 1mg human gene group DNA just contains 1.4 * 10
6Therefore the single copy gene fragment of copy makes pcr amplification reaction have powerful amplification ability, has improved the susceptibility that detects.Have powerful amplification efficiency just because of the PCR reaction, also cause its shortcoming of easily polluting, the pollution of denier just can cause false positive results.In the PCR reaction, mainly there are three source of pollution: 1. the crossed contamination between sample; 2. the pollution of laboratory cloned plasmids; 3.PCR the pollution of amplified production.Wherein main with the 3rd source of pollution, just can recognize the seriousness of PCR product pollution by a following example.In the reaction tubes of 100ml, can produce 10
12A copy molecule, if with these molecules a standard Olympics swimming pool (after the dilution, the 0.1ml diluent contains 400 amplifier molecules that copy in 50m * 25m * 2m), considerably beyond pcr amplification reaction detect the limit of several copies.Therefore, when PCR detects micro-infectant, must note the problem of product residual contamination, especially should be like this to clinical labororatory.So how to eliminate a great problem that the PCR false positive results has then become researchers to solve.
Pollute the false positive that causes in order to solve pcr amplification product, many scholars try every means to solve this problem.
The isolation of workspace before and after the PCR reaction: comprise preparation and the preparation of reagent; Dna profiling is extracted in the processing of clinical samples; The amplification of template; The detection of amplified production and calibrating etc.Isolate PCR reaction front and back workspace, and in the PCR operation, seal covering, can improve the specificity of extension increasing sequence.Think at present PCR reaction work is divided into: reagent preparation area, template preparation district, PCR reaction zone, product detection zone four parts; The workspace environment should be adhered to frequent disinfection by ultraviolet light, and this is the main measure that prevents that PCR from polluting.
Use PCR prevention contamination reagent box: the dUTP (UNG) that PCR pollution prevention test kit is modified for employing replaces the dTTP in the PCR reactive system.The amplification after product can be degraded by specificity under uridylic glycosyl enzyme (UNG) effect before next PCR reaction, and this enzyme is to natural nucleic acid-templated having no effect, therefore previous amplified production pollutes the PCR system, can not become the template of next pcr amplification, pollute the dUTP amplified production thereby can fundamentally remove PCR.Equally have template, hybridization, clone's character with n DNA, just effect influences to some extent to restriction enzyme.Also available dU replaces dT in the primer in addition, thereby makes UNG can block the primer amplification of pollution at the very start.Use PCR prevention contamination reagent box to note: annealing temperature preferably is higher than 5 ℃ in the PCR reaction, because UNG just has activity below 5 ℃, if annealing temperature is lower than 5 ℃, and the new synthetic dUDNA of the DNA degradable of remnants after the sex change.Common 95 ℃ can not complete inactivation UNG; Amplified production preferably detects immediately.
RS-PCR method (RNA-specific PCR) is also referred to as strand-specific RT-PCR, mainly refer to be used for the specific PCR method of RNA template, and this method can obviously reduce false positive and not influence the susceptibility of PCR.Its key is to design primer, and 3 of reverse transcriptase primer ' end (A district) has about 20 Nucleotide and to be the specificity of template sequence not mutually, and 20 Nucleotide of 5 ' end (C district) are for adding modified base.Behind the mRNA reverse transcription, through ultracentrifugation cDNA and unnecessary primer are separated, using with second primer (C) again is the synthetic second chain cDNA of template with the first chain cDNA, with the B district of reverse transcriptase primer and the primer C tailing cDNA that increases, and DNA or the plasmid DNA of pollution just can not be amplified in the later PCR circulation.
Fluorescence quantitative PCR method: also claim fluorescent PCR technology (fluoresence PCR, F-PCR), be at first to be succeeded in developing by the U.S. PE company nineteen ninety-five, it has merged the susceptibility of PCR, the specificity of DNA hybridization and the advantage of spectroscopic techniques accurate quantification, computer is followed the tracks of synchronously, and datamation is handled, and the variation in the direct detection PCR process is to obtain quantitative results, do not need to do PCR aftertreatment or detection, stopped pipe operation fully.Probe mark is except using TET and FAM, and also available HEX, JOE are as report fluorescence, and the quenching group of 3 ' end is used TAMRA always.When probe was kept perfectly, the fluorescence of fluorescence report group was suppressed the group cancellation by fluorescence, and after probe was cut off, the fluorescence report group just sent report fluorescence, and the quantity of intensity of fluorescence and PCR product is proportional.The fluoroscopic examination instrument sees through wavelength and the length variations that the PCR tube wall can directly detect fluorescent signal.Though fluorescence quantitative PCR method detects target nucleic acid and can play totally-enclosed, automatization and detect, because the instrument costliness, the strict and operator of operation field must take appointment with certificate, and detect the cost height, have limited the widespread use of detection of nucleic acids.
Full closed target nucleic amplifier fast testing device: its principle is after amplified reaction is finished, do not open the pipe lid of reaction tubes fully, form pollution in the air to avoid target nucleic amplifier to be discharged into, under covered situation, be placed in reaction tubes in the device of closure, target nucleic amplifier is transferred on the test strip by reaction tubes in the environment of physical property sealing, numerical value by fluoroscopic examination, sentence read result, do not open the device of sealing after the detection, it all can be discarded in the safe place.
Pollution in target nucleic acid amplification operating process between the Chang Kejian sample, pollution may by atmospheric pollution or aerosol, cause false positive reaction from the known or unknown positive material of following in the negative sample disposal process; Yet what see is the product of target nucleic acid amplification last time more, becoming the template of next target nucleic acid reaction, mainly is after finishing by the target nucleic acid amplified reaction, must open the pipe lid of PCR reaction tubes, the sucking-off amplified production carries out the traditional detection method that gel electrophoresis or other must open type and detects.The amplification extremely sample target sequence of trace is polluted, and can increase up to a million times at short notice, judges thereby influence detected result.The instrument that can antipollutionly exempt to uncap or device has such or such shortcoming at present: as the isolation method of workspace before and after the PCR reaction, can not guarantee to prevent completely to pollute, if and experiment operator carries the PCR product on one's body, the effect that so this method does not have yet accidentally.PCR prevention contamination reagent box method is used the antipollution prerequisite of this system, be that whole PCR system must adopt dUTP to replace dTTP from the beginning, and the price of the anti-pollution reagent of UNG-dUTP system also makes the use of this reagent be restricted.The RS-PCR method requires very high to design of primers, be not suitable for the operator of basic unit equally.Fluorescence quantitative PCR method is so because expensive, the operational requirement of quantitative real time PCR Instrument is high is not suitable for the operator of basic unit and uses.Though full closed target nucleic amplifier fast testing device is simple and convenient.But sealing effectiveness is bad, and is careless slightly, can cause the leakage of PCR product equally and pollutes.Moreover cost is too high, so be difficult to promote the use of.If can have a kind of not only simple and convenient not only fast but also the low method of cost will huge meaning be arranged to field of biology.
Summary of the invention
In order to solve target nucleic acid amplified production pollution problems better, and in conjunction with above all can prevent the relative merits of target nucleic acid amplified production pollution method, the applicant's combined with fluorescent probe technique, the rapid fluorescence of developing total closed type rapid detection target nucleic acid amplified production detects the device of thing, is called total closed type target nucleic acid amplified production rapid fluorescence proofing unit.
The invention provides a kind of total closed type target nucleic acid amplified production rapid fluorescence detection method, be after amplified reaction finishes, under covered situation, be placed in reaction tubes in the device of stopping property, in this stopping property device reaction tubes by broken wall, make the detection liquid reaction of presetting in the interior amplified production of reaction tubes and the closed unit, carry out fluoroscopic examination, sentence read result then.
After amplified reaction finishes in the aforesaid method, do not open the pipe lid of reaction tubes, can avoid forming in the target nucleic acid amplified production release air serious aerosol and pollute; Amplified production can carry out fluoroscopic examination in this airtight fully device; Do not open the device of sealing after the detection, it all can be discarded in the safe place.Whole process is carried out in closed environment, can prevent the target nucleic amplifier crossed contamination effectively, avoids false positive.
The invention provides a kind of product with PCR, constant-temperature amplification or the amplification of other method and use the total closed type target nucleic acid amplified production rapid fluorescence proofing unit of fluoroscopic examination.It comprises PCR pipe, thimble plug, centrifuge tube, and described PCR pipe can place centrifuge tube, and described thimble plug has the puncture needle that can puncture the PCR pipe, and centrifuge tube has sealable pipe lid.
Described thimble plug comprises cock body and puncture needle, and in order to allow the liquid in the PCR pipe enter centrifuge tube smoothly, there is outstanding pin ridge in the puncture needle outside, and cock body is provided with engraved structure, and ridge can be 1 or many.Also has liquid-leaking nozzle on the puncture needle.
Said apparatus also comprises the scissors pincers that cooperate this device.Can be inverted in the 1.5ml centrifuge tube with the 0.2ml PCR pipe after guaranteeing to shear, the top of PCR pipe and centrifugal body coincide, thereby guarantee that the PCR pipe enters centrifuge tube and can not put askew.
The size of thimble plug cock body is when the thimble plug is positioned in the centrifuge tube, and its position makes the outstanding centrifugal mouth of pipe 3-10mm of the afterbody of the PCR pipe that is inverted into.The mode that punctures the PCR pipe is to utilize centrifuge tube pipe cap seal to close in the process of centrifuge tube, and pipe lid extruding PCR pipe pipe tail produces pressure, promotes the PCR pipe and moves down, and PCR pipe pipe lid is punctured by thimble; The PCR pipe that thimble needle point sharpness can be guaranteed just to have inserted does not puncture the PCR pipe, has and does not puncture PCR pipe top before the mouth of pipe contact at centrifuge tube pipe drowning, only contacts totally enclosed environment of formation with the mouth of pipe at centrifuge tube pipe lid, and the thimble needle point punctures PCR and manages.In the design of the ridge of wire surface the PCR pipe is punctured between back center and pipe lid and produces the slit, under centrifugal action, the interior liquid of PCR pipe can flow out from the PCR pipe of original sealing; The funnel-like of thimble plug body part design is worn sky can make the liquid that flows out from the PCR pipe enter the reaction of centrifuge tube bottom by cock body, and in the PCR pipe, cock body all do not have liquid residue.Hollow out around the thimble plug cock body can make the centrifugal on the tube wall enter the pipe end.The body of thimble plug cock body is long to be 2-6mm, and the reaction liquid in the centrifuge tube all is suitable for below 0.5ml.
The invention also discloses a kind of method of rapid fluorescence detection target nucleic acid amplified production, be to use above-mentioned total closed type target nucleic acid amplified production rapid fluorescence proofing unit to detect the amplification of nucleic acid sequences thing.
The invention also discloses a kind of nucleic acid amplification product crossed contamination that prevents, avoid false-positive method, is to make
With above-mentioned total closed type target nucleic acid amplified production rapid fluorescence proofing unit amplification of nucleic acid sequences thing.
The invention also discloses the purposes in the kind evaluation on clinical infectious disease pathogens detection, testing food pathogenic and gene level of above-mentioned total closed type target nucleic acid amplified production rapid fluorescence detection method or device.
The present invention will utilize fluorescent probe technique, detect amplified production.According to optimization experiment scheme of the present invention, in aforesaid method, comprise the target nucleic acid reaction solution in the described enclosed appts (centrifuge tube) and detected liquid, and can in the environment of sealing, fully mix and reaction, just need not open and directly to detect fluorescence, thereby the article that may pollute in the anti-locking apparatus leak because of unexpected.
According to more preferably experimental program of the present invention, in aforesaid method, if the pipe of PCR pipe is stamped protuberance, can cover into round with shearing the PCR pipe with the scissors embedding, but do not destroy the stopping property of PCR pipe, can make the PCR pipe be inverted smoothly in the centrifuge tube again, the pipe of the PCR after shearing pipe lid is pressed close to the centrifuge tube tube wall as far as possible simultaneously, play the effect to a relative positioning of PCR pipe, make the mid-way of the corresponding PCR pipe of thimble pipe lid.
Total closed type target nucleic acid amplified production rapid fluorescence proofing unit of the present invention is compared with conventional target nucleic acid detection technique, mainly contains following advantage:
(1) total closed type detects.Whole process all need not be uncapped, and seals, and has well avoided the pollution of the target nucleic acid amplified production that causes because uncap:
A) the PCR reaction tubes finishes all to seal to being inverted centrifuge tube from nucleic acid amplification;
B) the PCR pipe is punctured when centrifuge tube pipe lid closes enclosed space of formation fully, the cut maximum that puncture produces when covering to pipe;
C) since the amount of liquid of PCR pipe less than 100 μ l, even the PCR pipe is inverted its liquid of back because the capillary siphoning phenomenon is stayed the top, only at the bottom of thimble punctures PCR pipe pipe lid fully and just can enter the centrifuge tube pipe by centrifugal its;
(2) rapid fluorescence detects.Owing to add fluorescent probe and the restriction enzyme that specific recognition target nucleic acid reaction product is arranged in advance in the centrifuge tube bottom, target nucleic acid product amplified production enters the centrifuge tube bottom through centrifugal one and namely begins reaction with its mixing: probe A and probe B identify target nucleic acid sequence simultaneously, the three forms a special construction, in case form this special construction, just can identify by being limited property restriction endonuclease, restriction enzyme is marked with between fluorophor FAM and quenching group Q at probe A and cuts, FAM separates with the Q group and comes off, the Q group is cancellation FAM again, FAM sends fluorescence, be attached to the new cutting of generation on the target sequence again with stylish probe A, fluorescent signal constantly is exaggerated, and fluorescent signal can reach the strongest about 10 minutes;
(3) high specificity.Owing to need two adjacent probe identify target sequence simultaneously, forms a special space structure after, restriction enzyme just begins cutting and generation fluorescent signal (see figure 4);
(4) highly sensitive.This method the first step target nucleic acid amplification has generally acknowledged high sensitivity, and the second step probe in detecting also is a method for amplifying signal under the effect of Restriction Enzyme simultaneously;
(5) simple to operate, quick.Whole testing process only needs about ten to 15 minutes time;
(6) detecting need only a small fluorescent detector.
Because this device can make the detection of target nucleic acid amplified production finish under the air-tight state fully, greatly reduce the pollution of uncapping and causing because of electrophoresis detection in the nucleic acid detection method of tradition amplification back.This method is simple to operate, and fast, it is low to detect cost, the reliability height.This closed target nucleic acid amplified production rapid fluorescence proofing unit can increase the accuracy that the result judges in each field use, shorten detection time, and the overall cost in each field is reduced.This device because simple to operate, quick, cost is low and remarkable to preventing the effect that target nucleic amplifier pollutes, and concerning the technician of field of biology work, very big help is arranged.
Because being extensive use of of nucleic acid amplification detection technique is so this device of the present invention can be applied to the field that all need use nucleic acid amplification detection technique method to detect.Identify and the application of aspect such as detection in Gene Mutation as the kind of fields such as clinical infectious disease pathogens detection, testing food pathogenic and the animal husbandry of agro-industry customs on gene level.As:
1. total closed type target nucleic acid amplified production rapid fluorescence proofing unit of the present invention can be applicable to the antenatal diagnosis of inherited disease.Use the fetus amnion cell, amniotic fluid or even female blood can check sex of fetus, this is necessary in related genetic diseases diagnosis with sex chromosome.For inherited disease occurred frequently, as thalassemia, sickle-cell anaemia, deficiency of coagulation factors, DMD etc. in clinical application for many years, for contribution has been done in prenatal and postnatal care, for the disease that genetic predisposition is arranged especially geriatric disease, as the part in diabetes, hyperlipidemia even the tumour.
2. total closed type target nucleic acid amplified production rapid fluorescence proofing unit of the present invention can be applicable to the detection of infective pathogen body.The gene of external source invasion, in case illustrate its part nucleotide sequence, just can design primer or probe, detect with PCR, PT-PCR or hybridizing method, its scope comprises all microorganisms such as bacterium, virus, protozoon and parasite, mould, rickettsia, chlamydozoan and mycoplasma, the sensitivity of the feature detection of PCR diagnosis and specificity are all far above current immunological method, required time has also reached clinical requirement, this is for the virus that is difficult to cultivate (hepatitis B), and is particularly suitable bacterium (as tuberculosis, anerobe) and the protozoon (as treponema pallidum) etc.
3. total closed type target nucleic acid amplified production rapid fluorescence proofing unit of the present invention can be applicable to detection and the diagnosis of oncogene.Though the research major part to oncogene also belongs to basic stage, but canceration is that this brass tacks that is caused by genovariation is unquestionable, so oncogene, antioncogene are gone into the research of anti-metastasis gene, leaving the diagnostic means of molecular level can't carry out, applicable example has quantitative (the comprising slow grain and anxious grain) of leukemia residual cell clinically, the inactivation of antioncogene such as P53 and Rb in the lung cancer, the activation of neurotic knurl N-myc gene and expression.Observe particular cancer gene and the implantation of anti-metastasis gene and antisense oligonucleotide has all become the gene therapy in modern age to the blocking-up of strongly expressed oncogene the starting point by in situ hybridization.
4. total closed type target nucleic acid amplified production rapid fluorescence proofing unit of the present invention can be applicable to dna fingerprint, individual recognition, parent discriminating and forensic.This problem of attracting attention for the public security organs, procuratorial organs and people's court part obtains legal acceptance in some country.Myohaemoglobin moonlet gene, the polypeptide of beta-globin gene, ApoB gene etc. and the difference of multiplicity all are applied to identifying, its sensitivity has reached a hair, a cell, a sperm and has obtained the personal feature collection of illustrative plates, and this field has also been developed into marrow or organ transplantation and joined in the research of type and animal kind system.
5. total closed type target nucleic acid amplified production rapid fluorescence proofing unit of the present invention can be applicable to the animal and plant quarantine.Sensitive, special, quick diagnosis detection method is the entrance guard of China import-export ports, whether the personnel at the inspection discrepancy gateway of a country, animal and plant (breeding stock, seed) etc. carry deadly infectious disease (AIDS, animal virus, plant virus etc.) pathogenic agent, food, feed etc. whether be with Salmonellas etc. all need the gene diagnosis means with these germs refuse outside the gateway of a country, be the necessary guarantee that improves china's overall national strength.
6. total closed type target nucleic acid amplified production rapid fluorescence proofing unit of the present invention can be applicable to the application in the high-tech biomedical sector.In genetically modified animals and plants, check the existence of implanting gene.
Description of drawings
Fig. 1 is the structure of totally-enclosed target nucleic amplifier fast testing device of the present invention.
Fig. 2 is the structure of proofing unit thimble plug of the present invention.
Fig. 3 is the operation steps of proofing unit of the present invention.
Fig. 4 is that totally-enclosed target nucleic amplifier fast testing device of the present invention detects principle.
Fig. 5 is that totally-enclosed target nucleic amplifier fast testing device of the present invention detects staphylococcus aureus gene.
Fig. 6 totally-enclosed target nucleic amplifier fast testing device of the present invention detects the streptococcus aureus drug resistant gene.
Fig. 7 totally-enclosed target nucleic amplifier fast testing device of the present invention detects the chlamydia trachomatis gene
Embodiment
Can specify technical scheme of the present invention below in conjunction with drawings and Examples, but not constitute the restriction to protection scope of the present invention.
As Fig. 1, a kind of totally-enclosed target nucleic amplifier fast testing device, it comprises PCR reaction tubes 11, thimble plug 12 and centrifuge tube 13; The nucleic acid amplification reaction that carries out in PCR pipe 11 finishes, nucleic acid amplification reaction liquid 14 is arranged in the PCR reaction tubes 11, make pipe 11 be inverted into centrifuge tube 13, centrifuge tube 13 presets thimble plug 12 and detects liquid 16 (containing probe A, the probe B and the restriction enzyme that detect nucleic acid amplification reaction liquid), utilize centrifuge tube pipe cap seal to close in the process of centrifuge tube, pipe lid extruding PCR pipe pipe tail produces pressure, promotes PCR pipe 11 and moves down, and PCR pipe pipe covers 15 and punctured by thimble; Thimble needle point sharpness can guarantee that the PCR pipe of just having inserted can not be punctured, namely have at centrifuge tube pipe drowning and do not puncture the PCR pipe with thimble before the mouth of pipe contacts and cover 15, only contact with the mouth of pipe at centrifuge tube pipe lid and form a totally enclosed environment, the thimble needle point punctures PC R pipe.In the design of the ridge of wire surface the PCR pipe is punctured between back center and pipe lid and produces the slit, under centrifugal action, the interior liquid 14 of PCR pipe can flow out from the PCR pipe of original sealing; The funnel-like of thimble plug body part design is worn sky and can be made the liquid that flows out from the PCR pipe enter the centrifuge tube bottom by cock body to detect liquid 16 and mix and react with presetting, and in PCR manages, cock body all do not have liquid residue.Hollow out around the thimble plug cock body can make the centrifugal on the tube wall enter the pipe end.
As Fig. 2, the thimble plug comprises puncture needle 21, the fixing cock body 23 of puncture needle, and puncture needle and cock body all have the leakage design.Particularly, the thimble plug comprises pin 21, ridge projection 22, cock body curved surfaces 23, pin plug leakage mortise 24, cock body leakage mortise 25.
The size of the needle point of thimble plug design can guarantee that it can puncture not frangibility of PCR pipe pipe lid and self, can guarantee that around the ridge projection 22 of thimble wearing out pipe lid back needle point at puncture needle 21 produces the slit between covering with pipe, can flow out the PCR liquid in pipe.It is not residual that the design of the curved surfaces of cock body curved surfaces 23 can make the liquid in the PCR pipe enter the cock body surface liquid.Funnel-like aperture mortise 25 can guarantee that the PCR liquid in pipe comes out by centre hole and can pass completely through thimble plug cock body when centrifugal in the middle of puncture needle body hollow out mortise 24, the cock body, does not have liquid residue.
Fig. 3 has illustrated the operation steps of proofing unit of the present invention.
In optimization experiment scheme of the present invention, described reaction tubes is the PCR reaction tubes, and pipe cover ultrathin design easily is punctured.Used centrifuge tube is the 1.5ml eppendorf centrifuge tube of standard.
A concrete implementation step is as follows:
A) after the target nucleic acid amplified reaction finishes, do not open the pipe lid of reaction tubes, be discharged into the serious aerosol pollution of formation in the air to avoid the target nucleic acid amplified production;
B) the PCR reaction tubes utilizes scissors pincers shearing pipe to cover into round and can be inverted in the centrifuge tube, and whole PCR pipe is the state of a sealing still;
C) by the thrust of centrifuge tube pipe lid to PCR pipe afterbody, inverted PCR pipe is pressed down, PCR pipe pipe lid is punctured by the thimble plug;
D) by centrifugal, the target nucleic acid reaction solution passes PCR pipe lid and the thimble consent that is punctured under centrifugal action, enter centrifuge tube;
E) owing to preset the fluorescent probe that detects the target nucleic acid reaction solution in the centrifuge tube, make amplified production in this airtight fully device, can carry out fluoroscopic examination;
F) numerical value by fluoroscopic examination, sentence read result;
G) do not open the device of sealing after detecting, because PCR pipe, centrifuge tube, thimble plug material are plastics, no metal device can all be discarded it in the safe place.
Following embodiment can specify technical scheme of the present invention, but does not constitute the restriction to protection scope of the present invention.
Embodiment 1: streptococcus aureus (ATCC25923) is that the target nucleic amplifier of example detects
1.PCR amplification:
2, detect
(1) the PCR pipe that will contain amplified material is placed in the pore of scissors pincers, and the scissors pliers grip that closes takes out the PCR pipe from pore;
(2) be inverted the PCR pipe, put into the centrifuge tube that is added with probe reaction liquid and thimble plug in advance;
(3) close centrifuge tube pipe lid and covering tightly;
(4) centrifugal 10 seconds rearmounted micro-fluorescence detectors;
Detect fluorescent value after (5) 10 minutes;
(6) the record result abandons centrifuge tube by the laboratory prescribed manner.
Embodiment 1 detected result is seen accompanying drawing 5.The fluorescence intensity of accompanying drawing 5 demonstration positive is reacted 10 minutes namely near 100, and negative sample does not have the fluorescence generation; Positive control fluorescent signal 10 minutes also about 80, shows that the result is effective.
Embodiment 2: methicillin-resistant staphylococcus aureus (ATCC33591) is that the target nucleic amplifier of example detects
1.PCR amplification:
2, detect
(1) the PCR pipe that will contain amplified material is placed in the pore of scissors pincers, and the scissors pliers grip that closes takes out the PCR pipe from pore;
(2) be inverted the PCR pipe, put into the centrifuge tube that is added with probe reaction liquid and thimble plug in advance;
(3) close centrifuge tube pipe lid and covering tightly;
(4) centrifugal 10 seconds rearmounted micro-fluorescence detectors;
Detect fluorescent value after (5) 10 minutes;
(6) the record result abandons centrifuge tube by the laboratory prescribed manner.
Embodiment 2 detected results are seen accompanying drawing 6.The fluorescence intensity of accompanying drawing 6 demonstration positive is reacted 10 minutes namely near 150, and negative sample does not have the fluorescence generation; The positive control fluorescent signal reached about 200 in 10 minutes, showed that the result is effective.
Embodiment 3: chlamydia trachomatis (ATCC VR1-19) is that the target nucleic amplifier of example detects
1, pcr amplification:
2, detect
(1) the PCR pipe that will contain amplified material is placed in the pore of scissors pincers, and the scissors pliers grip that closes takes out the PCR pipe from pore;
(2) be inverted the PCR pipe, put into the centrifuge tube that is added with probe reaction liquid and thimble plug in advance;
(3) close centrifuge tube pipe lid and covering tightly;
(4) centrifugal 10 seconds rearmounted micro-fluorescence detectors;
Detect fluorescent value after (5) 10 minutes;
(6) the record result abandons centrifuge tube by the laboratory prescribed manner.
Embodiment 2 detected results are seen accompanying drawing 7.The fluorescence intensity of accompanying drawing 7 demonstration positive is reacted 10 minutes namely near 80, and negative sample does not have the fluorescence generation; The positive control fluorescent signal reached about 100 in 10 minutes, showed that the result is effective.
More than closed nucleic acid amplification proofing unit detect staphylococcus, methicillin-resistant staphylococcus, trachoma doctor substance and detect all consistent with sequencing result.
Claims (7)
1. total closed type target nucleic acid amplified production rapid fluorescence proofing unit, comprise PCR pipe, centrifuge tube, it is characterized in that also comprising a thimble plug, described PCR pipe can place centrifuge tube, described thimble plug has the puncture needle that can puncture the PCR pipe, and the size of thimble plug cock body is when the thimble plug is positioned in the centrifuge tube, and its position makes the outstanding centrifugal mouth of pipe 3-10mm of the afterbody of the PCR pipe that is inverted into, cock body is provided with engraved structure, and centrifuge tube has sealable pipe lid.
2. total closed type target nucleic acid amplified production rapid fluorescence proofing unit according to claim 1 is characterized in that described thimble plug comprises cock body and puncture needle, and there is outstanding pin ridge in the puncture needle outside.
3. total closed type target nucleic acid amplified production rapid fluorescence proofing unit according to claim 2 is characterized in that on the described puncture needle liquid-leaking nozzle being arranged.
4. total closed type target nucleic acid amplified production rapid fluorescence proofing unit according to claim 1 is characterized in that also comprising the scissors pincers that cooperate this device.
5. the rapid fluorescence of a non-diagnostic purpose detects the method for target nucleic acid amplified production, is to use the total closed type target nucleic acid amplified production rapid fluorescence proofing unit of one of claim 1-4 to detect the amplification of nucleic acid sequences product.
6. a non-diagnostic purpose prevents the nucleic acid amplification product crossed contamination, avoids false-positive method that it comprises that right to use requires total closed type target nucleic acid amplified production rapid fluorescence proofing unit of one of 1-4 to detect the amplification of nucleic acid sequences product.
The total closed type target nucleic acid amplified production rapid fluorescence proofing unit of one of claim 1-4 identify in testing food pathogenic and agricultural, industry, customs, the animal husbandry kind on gene level, the non-diagnostic purpose purposes in the detection in Gene Mutation.
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| CN 201210014508 CN102534011B (en) | 2012-01-18 | 2012-01-18 | Totally-enclosed fast fluorescence detection method and device for target nucleic acid amplification products |
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| CN103160431B (en) * | 2013-04-17 | 2014-07-23 | 东南大学 | Gene-detection integrally completed enclosed PCR (polymerase chain reaction) amplification tube |
| CN105039149B (en) * | 2015-07-20 | 2017-11-17 | 宁波大学 | A kind of closed experimental system setup of Rapid identification nucleic acid amplification product and application |
| CN106978488A (en) * | 2017-04-02 | 2017-07-25 | 北京军秀咨询有限公司 | A kind of gene tester, detection kit and its application |
| CN108531377A (en) * | 2018-06-26 | 2018-09-14 | 广东省第二人民医院（广东省卫生应急医院） | It is a kind of can pre-filled reagent diaphragm type nucleic acid amplification airtight reactor tube |
| CN109735437B (en) * | 2019-01-28 | 2022-04-19 | 长春长光辰英生物科学仪器有限公司 | Vessel and method for collecting and processing cells after ejection sorting of cells |
| CN111548900A (en) * | 2019-02-12 | 2020-08-18 | 浙江大学 | Device and method for amplifying and detecting nucleic acid |
| CN111551703B (en) * | 2020-05-25 | 2020-12-25 | 安徽永生堂药业有限责任公司 | Efficient portable helicobacter pylori apparatus |
| CN112143780B (en) * | 2020-09-01 | 2022-04-08 | 浙江大学 | Method for confirming sample in gray zone after nucleic acid amplification |
| CN112877191A (en) * | 2021-02-22 | 2021-06-01 | 西安交通大学 | Anti-pollution consumable material and method for performing CRISPR molecular diagnosis by using same |
| WO2022213383A1 (en) * | 2021-04-09 | 2022-10-13 | 广州市达安医疗器械有限公司 | Closed nucleic acid test consumable and test method |
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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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