CN113150965B - Anti-pollution detection tube and CRISPR molecular diagnosis detection method based on same - Google Patents
Anti-pollution detection tube and CRISPR molecular diagnosis detection method based on same Download PDFInfo
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- CN113150965B CN113150965B CN202110432678.7A CN202110432678A CN113150965B CN 113150965 B CN113150965 B CN 113150965B CN 202110432678 A CN202110432678 A CN 202110432678A CN 113150965 B CN113150965 B CN 113150965B
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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
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an anti-pollution detection tube and a CRISPR molecular diagnosis detection method based on the same, and belongs to the technical field of nucleic acid molecular diagnosis. The anti-pollution detection tube comprises an amplification inner tube, a detection outer tube is sleeved outside the amplification inner tube, a cracking inner tube is arranged inside the amplification inner tube, and a tube cap is arranged at the top of the amplification inner tube; wherein, the cracking inner tube comprises a cracking inner tube sealing film arranged at the bottom, and the amplifying inner tube comprises an amplifying inner tube sealing film arranged at the bottom; the pipe cap comprises a pipe cap body, a pressing rebound part is arranged on a pipe cap column body, a pipe needle is arranged in the pipe cap body, and the pipe needle is fixedly connected with the pressing rebound part. The CRISPR molecular diagnosis detection method based on the anti-pollution detection tube reduces the operations of uncovering, transferring sample nucleic acid, closing the cover and the like, and simplifies the operation steps and improves the detection efficiency by simple pressing and centrifugation steps; meanwhile, the problems of laboratory aerosol pollution and the like caused by operations such as uncapping, sample nucleic acid transferring and the like are avoided.
Description
Technical Field
The invention belongs to the technical field of nucleic acid molecular diagnosis, and relates to an anti-pollution detection tube and a CRISPR molecular diagnosis detection method based on the same.
Background
The rapid, accurate, sensitive and quantitative detection of specific nucleic acid sequences plays an increasingly important role in the fields of diagnosis of human infectious diseases, food safety, determination of pathogens, global biosafety, tracking of biological pollution in environmental analysis, environmental quality monitoring and the like. The epidemic of the novel coronavirus brings great threat to the life safety of people. The cause of rapid outbreaks of novel coronaviruses lacks effective detection means; at present, the standard detection method uniformly approved in the world is PCR (polymerase chain reaction) detection, but the method has certain defects, the detection period is long, and about two to three hours are needed for completing one-time nucleic acid detection; in addition, PCR detection requires specialized technicians, large instrumentation, and these conditions limit the applicability of the method. Besides the traditional standard PCR detection, there are also the main nucleic acid amplification detection technologies such as RPA (recombinase polymerase amplification) technology, LAMP (loop mediated isothermal amplification) technology, RCA (rolling circle amplification) technology, CPA (cross primer amplification) technology, PSR (polymerase spiral reaction) technology and the like, but these methods have certain advantages and disadvantages, such as the RPA technology, which has high response speed, exponentially amplified but lower RPA sensitivity, and can not meet the clinical detection requirements; for example, the LAMP technology has higher sensitivity and specificity, but the primer design is complex. Therefore, there is a lack of a simple and effective nucleic acid detection method, which shortens the detection time, improves the detection sensitivity, and suppresses the propagation speed of viruses.
CRISPR, collectively known as "regularly clustered, short palindromic repeats" (Clustered regularly interspaced short palindromic repeats), is an adaptive immune system that is widely found in archaebacteria and bacteria. CRISPR systems were first discovered in the genome of escherichia coli at the beginning of the 90 s of the 20 th century. In 2013, the gene site-directed editing technology CRISPR/Cas9 was discovered by researchers and successfully applied, and since this CRISPR technology began to be the most popular gene editing tool. In 2017, the professor Zhang Feng, the university of harvard, a top scholars in the field of gene editing, applied CRISPR technology to the field of nucleic acid detection, which technology has been used to detect nucleic acid molecules of various pathogenic organisms (e.g., zika virus, dengue virus, mycobacterium tuberculosis, etc.) in its co-effort with the Jennifer dounna team of the university of california, berkeley. Thereafter, various nucleic acid detection platforms based on CRISPR enzymes such as Cas12, cas13, cas14, etc. have also been developed. Compared with the traditional nucleic acid detection method, the CRISPR technology has great advantages in the aspects of detection cost, efficiency, portability, specificity, simplicity and the like, has better biocompatibility, can be combined with other technologies, is simpler and more convenient and sensitive to detect nucleic acid, and is also known as a novel nucleic acid detection technology of the next generation. However, the CRISPR technology has some problems, in order to improve the detection sensitivity, it is generally necessary to amplify the nucleic acid before performing the CRISPR nucleic acid detection reaction, and then add a CRISPR reaction system to the amplified nucleic acid to detect the target nucleic acid. The operations of uncovering, transferring and closing the reagent after amplification are needed, so that the laboratory aerosol pollution is easily caused, the subsequent result is high in false positive problem, the cross pollution of the laboratory and related equipment is easily caused, and a great amount of time is needed to finish a series of operations, so that the waste of manpower and material resources is caused. Therefore, it is urgently required to find a convenient and feasible device or method to solve the problems of laboratory aerosol pollution and the like caused by uncapping, sample nucleic acid transferring, closing and the like in the process of amplifying nucleic acid to detecting nucleic acid, which are faced by the CRISPR technology.
In 2017, the RPA reagent for nucleic acid amplification and the CRISPR reagent for nucleic acid detection are mixed by a professor team [ Gootenberg, J.S. et al Nucleic acid detection with CRISPR-Cas13a/C2C2 [ J ]. Science 356, 438-442 (2017) ] of the university of Harvard university of Bode institute Zhang Feng of the same gene editing field, so that the two reactions are simultaneously carried out in the same test tube, thereby realizing the detection while amplification. Thus, the nucleic acid amplification and the nucleic acid detection are combined into one step, aerosol pollution caused by operations such as uncovering, transferring sample nucleic acid and the like is avoided, the efficiency of the nucleic acid detection is improved, but the detection sensitivity is reduced by the method for detecting the nucleic acid while amplifying. In 2019 Huihui Liu, yongming Wang [ Huihui Liu, yongming Wang et al Cas12aVDet A CRISPR/Cas12a-Based Platform for Rapid and Visual Nucleic Acid Detection [ J ]. American Chemical Society 91, 12156-12161 (2019) ] et al propose a CRISPR/Cas12a-based method called "Cas12aVDet" for rapid nucleic acid detection, avoiding aerosol pollution caused by uncapping operation, reducing experimental operation and improving detection efficiency. The method is to integrate Recombinase Polymerase Amplification (RPA) and a CRISPR reagent in a single reaction system, but add the CRISPR reagent to the inner wall of a tube which is uniquely designed, and after the RPA reaction is finished, add the CRISPR reagent to an RPA reaction solution by centrifugation to realize nucleic acid detection. Thus, detection can be completed within 30min, and the problems of aerosol pollution and the like caused by operations such as uncapping, sample nucleic acid transferring and the like are avoided, but the method is difficult to operate in the actual use process, has the problem that CRISPR reagent and RPA solution are mixed in advance, and reduces sensitivity. Mengyao Zhang, chengzhi Liu et al [ Mengyao Zhang, chengzhi Liu et al Selective endpoint visualized detection of Vibrio parahaemolyticus with CRISPR/Cas12a assisted PCR using thermal cycler for on-site application [ J ]. Science Direct 214, 1873-3573 (2020) ] developed a detection platform for detecting Vibrio parahaemolyticus by means of CRISPR and PCR techniques. In the method, CRISPR reagent is added into a test tube cover in advance, then the PCR reagent cover is closed on a micro thermal cycler to carry out PCR reaction, and the CRISPR reagent is adsorbed on the test tube cover by the adsorption force between liquid drops, so that the two reagents are separated. In this process, to avoid deactivation of the enzyme, the lid of the thermocycler would be opened, leaving the entire reaction partially on the thermocycler and partially exposed to air. After amplification is completed, the CRISPR reagent adsorbed on the test tube cover is thrown to the bottom of the test tube by centrifugation, mixed with the PCR reagent and used for nucleic acid detection by replacing dTTP in a PCR system with dUTP, so that operations such as cover opening and sample nucleic acid transfer are avoided.
In addition, in the above-mentioned prior art CRISPR molecular diagnostic detection method, the nucleic acid sample to be detected still needs to be cracked outside the test tube, and then added into the detection test tube to perform subsequent diagnostic detection, that is, the prior art method cannot realize one-step operation of sample in-out and out-out, thus reducing the detection efficiency of nucleic acid detection. Therefore, the prior art still cannot realize one-tube and simple pollution-free operation of sample inlet and sample outlet under the conditions of no false positive and no sensitivity reduction.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide an anti-pollution detection tube and a CRISPR molecular diagnosis detection method based on the anti-pollution detection tube. The invention aims to solve the problems that false positive or sensitivity is reduced and one-tube detection result cannot be obtained due to operations such as uncovering, transferring and closing of sample nucleic acid in a nucleic acid detection process based on a CRISPR technology; the anti-pollution detection tube provided by the invention can ensure that the detection system has higher robustness, reduces experimental operation, prevents pollution and improves the efficiency of nucleic acid detection on the premise of not reducing sensitivity and causing higher false positive in the nucleic acid detection based on the CRISPR technology.
In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:
the invention discloses an anti-pollution detection tube, which comprises an amplification inner tube, wherein a detection outer tube is sleeved outside the amplification inner tube, a cracking inner tube is arranged inside the amplification inner tube, and a tube cap is arranged at the top of the amplification inner tube; wherein, the cracking inner tube comprises a cracking inner tube sealing film arranged at the bottom, and the amplifying inner tube comprises an amplifying inner tube sealing film arranged at the bottom; the pipe cap comprises a pipe cap body, a pressing rebound part is arranged on a pipe cap column body, a pipe needle is arranged in the pipe cap body, and the pipe needle is fixedly connected with the pressing rebound part.
Preferably, the cap cylinder is provided with an outer eave structure for clamping and limiting, and the pressing rebound part is arranged at the upper part of the cap cylinder; the amplification inner tube comprises a first hollow cylinder upper part and a first conical lower part which are fixedly connected, an opening is arranged on the closing side of the first conical lower part, and a sealing film of the amplification inner tube is arranged on the opening for sealing; the detection outer tube comprises a second hollow cylindrical upper body and a second type of conical lower body with the bottom closed, which are fixedly connected; the cracking inner tube comprises a third hollow cylinder, and the sealing film of the cracking inner tube is arranged at the bottom of the third hollow cylinder for sealing.
Preferably, the pressing rebound part is a polyolefin elastic sleeve, and the tail part of the tubular needle is fixedly connected with the inner side surface of the bottom of the polyolefin elastic sleeve.
Preferably, the inner tube for cracking, the inner tube for amplifying, the outer tube for detecting and the tube cap are connected through interference fit in sequence.
Preferably, the inner tube for cracking, the inner tube for amplifying, the outer tube for detecting and the tube cap are connected in sequence through threaded fit.
Preferably, the lysis inner tube sealing film and the amplification inner tube sealing film are both polyolefin films.
Further preferably, the thickness of the film of the inner tube sealing film is 50-100 mu m, and the thickness of the film of the inner tube sealing film is 50-100 mu m.
The invention discloses a CRISPR molecular diagnosis detection method based on the anti-pollution detection tube, which comprises the following steps:
1) Freeze-drying the CRISPR reagent, placing the CRISPR reagent in a detection outer tube, and fixing an amplification inner tube in the middle upper part of the detection outer tube; after the nucleic acid amplification reagent dry powder is placed in the amplification inner tube, fixing the cracking inner tube in the middle of the amplification inner tube; placing a nucleic acid cracking reagent in the cracking inner tube, and then covering a tube cap; obtaining a reagent tube to be detected;
2) Opening a tube cap, and adding the sample nucleic acid to be detected into the cracking inner tube of the reagent tube to be detected obtained in the step 1); covering a tube cap, and performing a nucleic acid cleavage reaction of the sample nucleic acid to be detected in a constant temperature environment;
3) After the nucleic acid cleavage reaction is finished, pressing the tube cap, enabling the tube needle to pierce the inner tube sealing film, and performing a one-stage centrifugation to perform a nucleic acid amplification reaction;
4) After the nucleic acid amplification reaction is finished, pressing the tube cap, enabling the tube needle to pierce the inner tube sealing film, and performing two-stage centrifugation to perform a nucleic acid detection reaction;
5) Finally, the detection result of CRISPR molecular diagnosis is obtained through UV lamp irradiation.
Preferably, in the step 2), the constant temperature environment for the nucleic acid cleavage reaction is 37-40 ℃ and the time is 10-20 min; in the step 3), the temperature of the nucleic acid amplification reaction is 37-40 ℃ and the time is 10-20 min; in the step 4), the temperature of the nucleic acid detection reaction is 37-40 ℃ and the time is 15-20 min; in the step 3), the rotating speed of one-stage centrifugation is 600-6000 rpm, and the time of one-stage centrifugation is 5-20 s; in the step 4), the rotating speed of the two-stage centrifugation is 600-6000 rpm, and the time of the two-stage centrifugation is 5-20 s; in the step 3) and in the step 4), the pressing time of the pipe cap (1) is 2-7 s.
Further preferably, in the step 2), the constant temperature environment of the acid cleavage reaction is 39 ℃ and the time is 10min; in the step 3), the temperature of the nucleic acid amplification reaction is 39 ℃ and the time is 20min; in the step 4), the temperature of the nucleic acid detection reaction is 39 ℃ and the time is 18min; in the step 3), the rotating speed of the one-stage centrifugation is 3000rpm, and the time of the one-stage centrifugation is 10s; in the step 4), the rotating speed of the two-stage centrifugation is 3000rpm, and the time of the two-stage centrifugation is 10s; in step 3) and in step 4), the pressing time of the cap (1) is 5s.
Preferably, the reagent tube to be detected obtained in the step 1) is directly stored and used for direct detection and taking of CRISPR molecular diagnosis.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses an anti-pollution detection tube, which is characterized in that three detection stages required in CRISPR molecular diagnosis can be divided into three detection spaces through a cracking inner tube, an amplification inner tube and a detection outer tube which are sequentially sleeved from inside to outside, and the three detection stages can be carried out in stages by designing a tube cap to be provided with a structure with a pressing rebound part and a tube needle and installing the tube cap on the top of the amplification inner tube. Therefore, the anti-pollution detection tube has reasonable structural design, forms an independent separated and totally-enclosed detection tube structure, avoids the problems of laboratory aerosol pollution and the like caused by operations such as uncapping, transferring sample nucleic acid, closing the cover and the like, simultaneously realizes one-tube operation of sample inlet and sample outlet, simplifies operation steps in the nucleic acid detection process, and enhances the applicability of nucleic acid detection.
Further, through adopting each of the amplification inner tube and the detection outer tube that have hollow cylinder structure and class toper structure constitution respectively, utilize the cooperation of geometry, can make it in the built-in assembly process, leave the filling space that satisfies the test reagent, guarantee to detect going on smoothly.
Further, by taking the polyolefin elastic sleeve as the pressing rebound part, the three processes of nucleic acid splitting, nucleic acid amplification and CRISPR detection can be seamlessly connected in one tube, so that experimental operation steps are simplified, and the problems of laboratory aerosol pollution and the like caused by operations such as uncapping, sample nucleic acid transferring and closing are avoided.
Further, by adopting interference fit connection or screw fit connection, close fit between pipes can be realized, and system tightness in centrifugal treatment is ensured, so that laboratory aerosol pollution in the nucleic acid detection process is effectively prevented.
Further, by adopting the polyolefin film as the inner tube sealing film for cracking and the inner tube sealing film for amplifying, the three processes of nucleic acid cracking, nucleic acid amplification and CRISPR detection can be separated, and meanwhile, the sealing film can be pierced by a tube needle, so that the effective connection of the three processes of nucleic acid cracking, nucleic acid amplification and CRISPR detection is realized.
Further, the film thickness of schizolysis inner tube sealing film more preferably is 70 mu m, and the film thickness of the sealed film of inflation inner tube more preferably is 90 mu m, not only can guarantee that corresponding sealing film can bear the reagent of corresponding volume, but also can be easy by the tubular needle puncture, links up three process orderly.
The invention also discloses a CRISPR molecular diagnosis detection method based on the anti-pollution detection tube, when the anti-pollution detection tube is adopted to carry out CRISPR molecular diagnosis, the operations of opening the cover, transferring sample nucleic acid at each stage, closing the cover and the like are reduced, and sample inlet and outlet can be realized through simple pressing and centrifugation steps, so that the operation steps are simplified, and the efficiency of nucleic acid detection is improved; meanwhile, the problems of laboratory aerosol pollution and the like caused by operations such as uncapping, sample nucleic acid transferring and the like are avoided, and an important difficult problem in the CRISPR detection technology and even the whole molecular diagnosis technology is solved. Meanwhile, the method can be used for direct detection and taking of CRISPR molecular diagnosis, and improves universality and detection efficiency of nucleic acid detection.
Furthermore, ideal experimental results can be obtained by adjusting experimental parameters when the anti-pollution detection tube is used for detecting nucleic acid, and the detection efficiency is improved.
Furthermore, the reagent tube to be detected obtained in the step 1) is directly stored in a refrigerator or other non-inactivating environment, and based on the anti-pollution detection tube, the pre-storage of CRISPR molecular diagnosis detection reagent can be realized, and when nucleic acid detection is needed, only the nucleic acid sample to be detected is needed to be added into the test tube (reagent pre-storage is realized), so that the detection of household nucleic acid can be realized.
Drawings
FIG. 1 is a schematic view of the structure of an anti-pollution test tube according to the present invention;
FIG. 2 is a schematic view of the cap structure of the present invention;
FIG. 3 is a schematic view of the stylet of the present invention;
FIG. 4 is a schematic illustration of the cap and stylet of the present invention attached together by adhesive;
FIG. 5 is a schematic view of an amplification inner tube and its amplification inner tube sealing film according to the present invention;
FIG. 6 is a schematic view of a cracking inner tube and its sealing film according to the present invention;
FIG. 7 is a schematic view of a detection outer tube of the present invention;
FIG. 8 is a schematic illustration of the reagent addition assembly of the anti-fouling detection tube according to the present invention; wherein, (a) is a schematic diagram of the addition of a lyophilized CRISPR reagent to the detection outer tube; (b) is a schematic diagram of the cooperation of the amplification inner tube and the detection outer tube; (c) Schematic drawing of adding nucleic acid amplification reagent to the amplification inner tube; (d) is a schematic diagram of the cooperation of the inner tube for cleavage and the inner tube for amplification; (e) adding nucleic acid lysate to the inner tube; (f) A schematic diagram is assembled for the whole anti-pollution detection tube;
FIG. 9 is a schematic diagram of a CRISPR molecular diagnostic process using an anti-fouling detection tube according to the invention; wherein, (a) is a schematic diagram of pressing the tube cap to puncture the sealing film of the cracking inner tube; (b) is a schematic diagram of the mixing of the amplification inner tubes; (c) Schematic drawing for puncturing and amplifying the inner tube sealing film for pressing the tube cap; (d) is a schematic diagram for detecting the mixing of the outer tube;
wherein: 1-pipe cap, 2-pipe needle, 3-amplification inner pipe, 4-cracking inner pipe, 5-detection outer pipe, 6-cracking inner pipe sealing film and 7-amplification inner pipe sealing film.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the attached drawing figures:
referring to fig. 1, the present invention provides an anti-contamination detection tube comprising, from top to bottom, a cap 1, a stylet 2, an amplification inner tube 3, an amplification inner tube sealing film 7, a lysis inner tube 4, a lysis inner tube sealing film 6, and a detection outer tube 5.
In the present invention, the contamination prevention detecting tube includes a cap 1, the structure of which is shown in FIG. 2; comprising a stylet 2, the structure of which is seen in figure 3. The pipe cap 1 comprises a pipe cap column body and a pressing rebound part, an outer eave structure is arranged on the pipe cap column body, the pipe cap 1 is clamped and limited on the anti-pollution detection pipe when the outer eave structure is used for being assembled integrally, a hole for penetrating the pipe needle 2 is formed in the middle of the pipe cap 1, the pipe needle 2 penetrates through the hole, and the tail of the pipe needle 2 is connected with the pressing rebound part.
Wherein, the cooperation theory of pipe cap 1 and stylet 2 is as follows:
the effect of pipe cap 1 is under the effect that external force pressed, can drive pipe needle 2 downwards and pierce through schizolysis inner tube sealing film 6 and amplification inner tube sealing film 7 in proper order, after the external force disappears, because of the elasticity that its pressure resilience portion self had, state before can resume, drive pipe needle 2 and keep away from schizolysis inner tube sealing film 6 and amplification inner tube sealing film 7, accomplish cracked nucleic acid sample and accomplish the nucleic acid sample of amplification and will be able to enter into respectively the amplification inner tube 3 and detect outer tube 5 along sealing film (schizolysis inner tube sealing film 6 and amplification inner tube sealing film 7) damage department, participate in the reaction of next step, pipe cap 1 plays a good sealing effect to the amplification inner tube 3 simultaneously, guarantee that the sample nucleic acid that waits to detect can not volatilize outside the anti-pollution detection pipe, avoid producing the aerosol and pollute.
Wherein, the material of the pipe cap 1 is preferably polyolefin material; the cap 1 is preferably manufactured by injection moulding. The material of the stylet 2 is preferably a photosensitive resin composition material; the stylet 2 is preferably manufactured using additive manufacturing techniques.
Specifically, in a specific embodiment of the present invention, the pressing rebound portion in the cap 1 is a polyolefin elastic sleeve. The tail of the tubular needle 2 is adhered to the bottom of the polyolefin elastic sleeve through strong glue from the inside of the polyolefin elastic sleeve. The connection schematic of the tube cap 1 and the tube needle 2 is shown in fig. 4.
In the invention, the anti-pollution detection tube further comprises an amplification inner tube 3, the amplification inner tube 3 comprises a first hollow cylinder upper part and a first conical lower part which are sequentially connected from top to bottom, an opening is arranged on the closing side of the first conical lower part, and the opening on the closing side of the first conical lower part is sealed through an amplification inner tube sealing film 7, and the structure is shown in fig. 5. In the invention, the anti-pollution detection tube further comprises a detection outer tube 5, wherein the detection outer tube 5 comprises a second hollow cylindrical upper body and a second type conical lower body, wherein the second hollow cylindrical upper body and the second type conical lower body are sequentially communicated from top to bottom; the maximum inner diameter of the second conical lower body is smaller than or equal to the inner diameter of the second hollow cylinder upper body. See fig. 7 for its structure. In the present invention, the detection outer tube 5 is used to store a CRISPR reagent, and after the nucleic acid amplification reaction is completed, the detection outer tube 5 serves as a container in which a CRISPR nucleic acid detection reaction occurs. In the invention, the bottom of the amplification inner tube 3 is sealed by using the amplification inner tube sealing film 7 in advance, the amplification inner tube 3 serves as a container for nucleic acid amplification reaction and a reservoir for nucleic acid amplification reagent, and also serves as a tube cover of the detection outer tube 5, so that the detection outer tube 5 is sealed, and the amplified nucleic acid sample is prevented from volatilizing from the detection outer tube 5 to the outside of the anti-pollution detection tube, and aerosol pollution is avoided. In the invention, the amplification inner tube 3 and the detection outer tube 5 are connected through interference fit; the amplification inner tube 3 is clamped inside the detection outer tube 5, the height of the amplification inner tube 3 is higher than the height of the upper body of the second hollow cylinder of the detection outer tube 5, good sealing of the whole anti-pollution detection tube is guaranteed, the cracking inner tube 4 is nested inside the amplification inner tube 3, and a one-pipe use operation method for sample inlet and sample outlet is achieved. The detection outer tube 5 plays a role in fixedly supporting the amplification inner tube 3.
Wherein, the material of the amplification inner tube 3 is preferably polymethyl methacrylate material; the amplification inner tube 3 is preferably manufactured by injection molding.
Wherein, the detection outer tube 5 is preferably made of polypropylene; the detecting outer tube 5 is preferably manufactured by injection molding.
In the present invention, the anti-pollution test tube further comprises a lysis inner tube 4, the structure of which is shown in fig. 6, and it is known that the lysis inner tube 4 comprises a third hollow cylinder, the bottom of which is sealed by a lysis inner tube sealing film 6, and the lysis inner tube 4 functions as a container for the nucleic acid lysis reaction and a reservoir for the nucleic acid lysis reagent. In the invention, the outer diameter of the cracking inner tube 4 is matched with the inner diameter of the amplification inner tube 3 and generates interference fit; the inner tube 4 is clamped inside the inner tube 3, the height of the inner tube 4 is lower than that of the upper part of the first hollow cylinder of the inner tube 3, and the sealing of the whole anti-pollution detection tube is facilitated (because when the height of the inner tube 4 is higher than that of the inner tube 3, the sealing of the inner tube 4 is required to be realized on the basis of sealing the inner tube 3, thus the overall sealing performance is adversely affected, and the possibility of aerosol pollution in a laboratory is increased), and the inner tube 3 plays a role of fixedly supporting the inner tube 4.
Wherein, the material of the cracking inner tube 4 is preferably polymethyl methacrylate material; the cracking inner tube is preferably manufactured by an injection molding process.
In the present invention, the inner tube sealing membrane 6 and the inner tube sealing membrane 7 can carry a volume of liquid which is far more than the volume required for the experiment without strong external force interference, and the inner tube sealing membrane 6 and the inner tube sealing membrane 7 can be pierced by the tube needle 2.
Specifically, in the embodiment of the present invention, the materials of the cracking inner tube sealing film 6 and the amplification inner tube sealing film 7 are preferably polyolefin materials; the sealing film is preferably manufactured by adopting a calendaring process. The thickness of the amplified inner tube sealing film 7 is thicker than that of the cleaved inner tube sealing film 6, the amplified inner tube sealing film 7 is required to bear the volumes of the amplified reagent and the cleaved reagent, and the cleaved inner tube sealing film 6 only needs to bear the volume of the cleaved reagent, so that the thickness of the amplified inner tube sealing film 7 is thicker than that of the cleaved inner tube sealing film 6 according to the difference of the volumes of the born liquids. Wherein, the film thickness of schizolysis inner tube seal film 6 is 50~100 mu m, and the film thickness of the sealed film of inflation inner tube 7 is 50~100 mu m.
The invention also provides a detection method for CRISPR molecular diagnosis by using the anti-pollution detection tube, which comprises the following steps: after freeze-drying CRISPR reagent is added into the detection outer tube 5, the amplification inner tube 3 is fixed in the detection outer tube 5; after adding a nucleic acid amplification reagent to the amplification inner tube 3, fixing the cleavage inner tube 4 in the amplification inner tube 3; after adding a nucleic acid cracking reagent into the cracking inner tube 4, covering the amplification inner tube 3 with a tube cap 1 with a tube needle 2, and storing in a refrigerator at minus 20 ℃; when CRISPR molecular diagnosis is needed, only opening a pipe cap 1 and adding a sample to be detected into the cracking inner pipe 4, then covering the pipe cap 1, putting the whole anti-pollution detection pipe into a needed constant temperature environment, and carrying out nucleic acid cracking reaction of sample nucleic acid to be detected; after the nucleic acid cleavage reaction is completed, the cap 1 is pressed by hand to pierce the inner tube sealing film 6 by the stylet 2, and the nucleic acid amplification reaction is performed by one-stage centrifugation; after the nucleic acid amplification reaction is completed, pressing the cap 1 by hand to pierce the inner tube sealing film 7 with the stylet 2, and performing two-stage centrifugation to perform a nucleic acid detection reaction; finally, the detection result of CRISPR molecular diagnosis is obtained through UV lamp irradiation.
The rotational speeds of the first-stage centrifugation and the second-stage centrifugation are preferably 600-6000 rpm, more preferably 3000rpm, and the time of the first-stage centrifugation and the second-stage centrifugation is preferably 5-20 s, more preferably 10s; the two-stage centrifugation is performed by transferring all the nucleic acid sample subjected to cleavage remaining in the cleavage inner tube 4 to the amplification inner tube 3, uniformly mixing the sample nucleic acid with the nucleic acid detection reagent, transferring all the liquid remaining in the amplification inner tube to the detection outer tube as much as possible, and uniformly mixing the amplified nucleic acid sample with the CRISPR reagent.
Specifically, in the embodiment of the present invention, after the CRISPR reagent is added to the detection outer tube 5, the amplification inner tube 3 is fixed in the detection outer tube 5. The specific composition and concentration of the CRISPR reagent are not particularly limited, and the CRISPR reagent conventional in the art can be adopted; in the present invention, the volume of the CRISPR reagent is preferably 20 microliters for lyophilization preservation; the CRISPR reagent is preferably added to the bottom of the detection outer tube 5; a schematic of the addition of CRISPR reagent to the detection outer tube is shown in fig. 8 (a); a schematic diagram of the detection outer tube 5 after the amplification inner tube 3 is immobilized is shown in FIG. 8 (b). In the present invention, after adding a nucleic acid amplification reagent dry powder to the amplification inner tube 3, the cleavage inner tube 4 is fixed in the amplification inner tube 3. The specific composition and concentration of the nucleic acid amplification reagent are not particularly limited, and nucleic acid amplification reagents conventional in the art may be used. In the present invention, the volume of the nucleic acid amplification reagent is preferably 20. Mu.l. In the present invention, the nucleic acid amplification reagent is preferably added to the bottom of the amplification inner tube 3. In the present invention, a schematic diagram of adding a nucleic acid amplification reagent to the amplification inner tube 3 is shown in FIG. 8 (c); a schematic diagram after fixing the cleavage inner tube 4 in the amplification inner tube 3 is shown in FIG. 8 (d). In the present invention, a nucleic acid cleavage reagent is added to the cleavage inner tube 4, and the cap 1 of the amplification inner tube is covered. In the present invention, the volume of the nucleic acid cleavage reagent is preferably 20. Mu.l. In the present invention, the nucleic acid-cleaving reagent is added to the bottom of the cleavage inner tube 4. In the present invention, a schematic diagram of adding a nucleic acid cleavage reagent to the cleavage inner tube 4 is shown in FIG. 8 (e). In the present invention, a schematic view of the inner tube after capping the inner tube is shown in FIG. 8 (f).
Therefore, the anti-pollution detection tube can be stored in a refrigerator at the temperature of minus 20 ℃, and when the anti-pollution detection tube is used for nucleic acid detection CRISPR molecular diagnosis, the CRISPR molecular diagnosis detection can be realized by simply pressing and centrifuging the sample nucleic acid to be detected by taking out the anti-pollution detection tube and adding the sample nucleic acid to be detected.
Specifically, in the embodiment of the present invention, a sample nucleic acid to be detected is added to the amplification inner tube 3, and the cap 1 of the amplification inner tube 3 is covered. The invention is not particularly limited in the kind of the sample nucleic acid to be detected, and any kind of sample to be detected can be used. The sample nucleic acid to be detected has a volume of 5 microliters.
Specifically, in the embodiment of the present invention, after the cap 1 of the amplification inner tube 3 is covered, the nucleic acid cleavage reaction is first performed, after the nucleic acid cleavage reaction is completed, the cap 1 is pressed with force, the cap 1 drives the needle 2 to pierce the cleavage inner tube sealing film 6 of the cleavage inner tube 4, and then the force acting on the cap 1 is cancelled, and the nucleic acid amplification reaction is performed by one-stage centrifugation. In the invention, the procedure of the nucleic acid cleavage reaction is preferably 36-40 ℃, 10-20 min, more preferably 39 ℃,10min; the pressing time of the pipe cap 1 is 2-7 s, and more preferably 5s; the rotating speed of the one-stage centrifugation is preferably 600-6000 rpm, more preferably 3000rpm; the one-stage centrifugation time is preferably 5 to 20s, more preferably 10s. After the nucleic acid amplification reaction is completed, the cap 1 is pressed again with force, the cap 1 pierces the amplified inner tube sealing film 7 of the amplified inner tube 3 with the tube needle 2, and then the force acting on the cap 1 is canceled, and the nucleic acid detection reaction is performed by two-stage centrifugation. In the invention, the procedure of the nucleic acid amplification reaction is preferably 36-40 ℃, 10-20 min, more preferably 39 ℃,20min; the pressing time of the pipe cap 1 is 2-7 s, and more preferably 5s; the rotating speed of the two-stage centrifugation is preferably 600-6000 rpm, more preferably 3000rpm; the two-stage centrifugation time is preferably 5 to 20 seconds, more preferably 10 seconds. The procedure of the nucleic acid detection reaction is 36-40 ℃, 15-20 min, more preferably 39 ℃ and 18min. After the nucleic acid detection reaction is finished, the experimental result can be observed by using a UV lamp, so that the CRISPR molecular diagnosis detection method based on the anti-pollution detection tube is realized.
By the anti-pollution detection tube and the CRISPR molecular diagnosis detection method based on the same, seamless connection of sample nucleic acid to be detected in the process of detecting the sample nucleic acid can be realized, manual operation is reduced, time is saved, the efficiency of detecting the nucleic acid is improved, and laboratory aerosol pollution caused by operations such as uncovering, transferring sample nucleic acid and closing the cover in the process of amplifying the nucleic acid to detecting the nucleic acid is avoided.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The material and the preparation method of each part of the anti-pollution detection tube are provided in the embodiment.
1. The pipe cap 1 is made of high-quality polyolefin materials, the high-quality polyolefin materials are put into an injection molding machine, heated and melted by the injection molding machine, extruded into a mold cavity by a screw rod in a pressurizing way, and processed by cooling molding.
2. The material of the stylet 2 is selected from high-quality photosensitive resin composite materials, and is processed by using additive manufacturing technology.
3. The detection outer tube 5 is made of high-quality polypropylene materials, the high-quality polypropylene materials are put into an injection molding machine, heated and melted by the injection molding machine, pressed and extruded into a mold cavity by a screw rod, and processed by cooling molding.
4. The material of the amplification inner tube 3 is selected from high-quality PMMA material, the high-quality PMMA material is put into an injection molding machine, heated and melted by the injection molding machine, extruded into a mold cavity by a screw rod in a pressurizing manner, and processed by cooling molding.
5. The cracking inner tube 4 is made of high-quality PMMA material, the high-quality PMMA material is put into an injection molding machine, heated and melted by the injection molding machine, extruded into a mold cavity by a screw rod in a pressurizing manner, and processed by cooling molding.
6. The materials of the amplified inner tube sealing film 7 and the cracked inner tube sealing film 6 are selected from high-quality polyolefin materials, and the materials are manufactured and molded through a calendaring process.
7. Schematic diagrams of sealing the amplification inner tube 3 and the cleavage inner tube 4 with a sealing film are shown in FIGS. 5 and 6.
8. A schematic illustration of the bonding of the stylet 2 to the cap 1 by a strong glue is shown in fig. 4.
Example 2
The structure of the anti-pollution detection tube and the schematic diagram of each step provided in this embodiment are shown in fig. 8 and fig. 9, and the operation steps are as follows: the freeze-dried CRISPR reagent is first added to the detection outer tube 5, and then the amplification inner tube 3 is added to the detection outer tube 5. The amplification inner tube 3 is caught inside the detection outer tube 5, which will be fixed. The prepared RPA nucleic acid amplification reagent is added into the amplification inner tube 3, then the cleavage inner tube 4 is added into the amplification inner tube 3, and the cleavage inner tube 4 is blocked inside the amplification inner tube 3 and is fixed. The prepared nucleic acid lysis reagent is added into the lysis inner tube 4, and then the tube cap 1 is covered, so that the nucleic acid lysis inner tube can be immediately used, and can also be placed in a refrigerator at minus 20 ℃ for sealing and preservation. When CRISPR molecular diagnosis nucleic acid detection is needed, the anti-pollution detection tube can be taken out, sample nucleic acid to be detected is added, then the tube cap 1 is covered, then the reaction tube is put into a constant temperature centrifuge device at 39 ℃ for incubation for 10min, and after incubation is completed. The tube cap 1 is pressed by force, the tube needle 2 moves downwards under the action of the tube cap 1 to puncture the sealing film 6 of the cracking inner tube, the force is pressed for 5s, then the force is cancelled, the tube cap 1 is elastic, the tube needle 2 is far away from the sealing film 6 of the cracking inner tube, then the nucleic acid sample after the completion of the cracking can be transferred into the amplification inner tube 3, the centrifugal machine is started, the whole reaction tube is subjected to the centrifugal treatment of 3000rpm and 10s, and under the action of the centrifugal force, the cracked sample nucleic acid flows into the amplification inner tube 3 through the broken part of the sealing film 6 of the cracking inner tube. The sample nucleic acid entering the amplification inner tube 3 will activate the nucleic acid amplification reagent, and then the motor is used to forward and reverse the sample nucleic acid and the RPA nucleic acid amplification reagent to mix uniformly. The anti-contamination assay tube was then incubated in a thermostatic centrifuge device at 39℃for a further 20min, after which incubation was completed. The tube cap 1 is pressed by force, the tube needle 2 moves downwards under the action of the tube cap 1, the amplified inner tube sealing film 7 is pierced, the force is pressed for 5 seconds, then the force is cancelled, the tube needle 2 is driven to be far away from the amplified inner tube sealing film 7 due to the elasticity of the tube cap 1, then the nucleic acid sample after the amplification can be transferred into the detection outer tube 5, the centrifugal machine is started, 3000rpm and 10 seconds of centrifugal treatment are carried out on the whole reaction tube, and the amplified sample nucleic acid flows into the detection outer tube 5 through the damaged part of the amplified inner tube sealing film 7 under the action of the centrifugal force and is mixed with a CRISPR reagent. Finally, incubating for 18min at the constant temperature of 39 ℃, and observing the experimental result under the irradiation of a UV lamp.
Taking HBV as an example, the in-situ workflow of CRISPR anti-contamination consumables in this embodiment will be described.
1. As shown in fig. 8 (a), a prepared lyophilized CRISPR reagent was added to the bottom of the detection outer tube 5.
2. As shown in fig. 8 (b), the amplification inner tube 3 is fitted into the detection outer tube 5, and the amplification inner tube 3 is fixed.
3. As shown in FIG. 8 (c), the prepared RPA nucleic acid amplification reaction reagent was added to the bottom of the amplification inner tube 3.
4. As shown in FIG. 8 (d), the inner lysis tube 4 is inserted into the amplification inner tube 3, and the inner lysis tube 4 is fixed.
5. As shown in FIG. 8 (f), a prepared nucleic acid cleavage liquid was added to the bottom of the cleavage inner tube 4.
6. Sample nucleic acid to be amplified is added to the bottom of the lysis inner tube 4.
7. As shown in FIG. 8 (e), the whole amplification tube is put into a thermostat for 10min after the cap 1 is covered, shaken and centrifuged to cleave the sample nucleic acid.
8. As shown in fig. 9 (a), the cap 1 is pressed down with force, the cap 1 is compressed, the stylet 2 is moved downward, and then the inner tube sealing film 6 is ruptured to be pierced.
9. As shown in fig. 9 (b), when the cap 1 is released, the cap 1 returns to the previous state, and the stylet 2 is moved upward, away from the sealing film 6 of the cleavage inner tube, and returns to the previous state.
10. As shown in FIG. 9 (b), the cleaved sample nucleic acid will flow into the amplification tube 3 along the broken portion of the cleavage tube sealing film 6.
11. And uniformly mixing the sample nucleic acid and a nucleic acid amplification reagent, and incubating for 20min in a constant temperature environment at 39 ℃ to realize the amplification of the sample nucleic acid.
12. As shown in fig. 9 (c), the cap 1 is pressed down with force, the cap 1 is compressed, the stylet 2 is moved downward, and then the sealing film 7 is pierced.
13. As shown in fig. 9 (d), when the cap 1 is released, the cap 1 returns to the previous state, and the stylet 2 is moved upward, away from the inner tube sealing film 7, and returns to the previous state.
14. As shown in FIG. 9 (d), the cleaved sample nucleic acid will flow along the break of the sealing membrane 7 of the inner amplification tube into the outer detection tube, and the amplified nucleic acid solution will also have a good activation effect on the CRISPR reagent.
15. Shaking uniformly to mix sample nucleic acid and CRISPR reagent uniformly, then incubating for 18min in a constant temperature environment at 39 ℃, and then observing fluorescence, thereby realizing the detection of sample nucleic acid conveniently and rapidly.
Claims (2)
1. An anti-pollution detection tube is characterized by comprising an amplification inner tube (3), wherein a detection outer tube (5) is sleeved outside the amplification inner tube (3), a cracking inner tube (4) is arranged inside the amplification inner tube (3), and a tube cap (1) is arranged at the top of the amplification inner tube (3);
wherein the cracking inner tube (4) comprises a cracking inner tube sealing film (6) arranged at the bottom, and the amplifying inner tube (3) comprises an amplifying inner tube sealing film (7) arranged at the bottom; the pipe cap (1) comprises a pipe cap body, a pressing rebound part is arranged on a pipe cap column body, a pipe needle (2) is arranged in the pipe cap body, and the pipe needle (2) is fixedly connected with the pressing rebound part;
the cracking inner tube (4), the amplification inner tube (3), the detection outer tube (5) and the tube cap (1) are connected in sequence through screw thread matching;
the pipe cap cylinder is provided with an outer eave structure for clamping and limiting, and the pressing rebound part is arranged at the upper part of the pipe cap cylinder;
the amplification inner tube (3) comprises a first hollow cylinder upper part and a first conical lower part which are fixedly connected, an opening is arranged at the closing side of the first conical lower part, and an amplification inner tube sealing film (7) is arranged at the opening to seal;
the detection outer tube (5) comprises a second hollow cylindrical upper body and a second type of conical lower body with the bottom closed, which are fixedly connected;
the cracking inner tube (4) comprises a third hollow cylinder, and a cracking inner tube sealing film (6) is arranged at the bottom of the third hollow cylinder for sealing;
the pressing rebound part is a polyolefin elastic sleeve, and the tail part of the tubular needle (2) is fixedly connected with the inner side surface of the bottom of the polyolefin elastic sleeve;
the cracking inner tube (4), the amplification inner tube (3), the detection outer tube (5) and the tube cap (1) are connected through interference fit in sequence;
the cracking inner tube sealing film (6) and the amplification inner tube sealing film (7) are polyolefin films;
the film thickness of the cracking inner pipe sealing film (6) is 50-100 mu m, and the film thickness of the amplification inner pipe sealing film (7) is 50-100 mu m;
the detection method of the anti-pollution detection tube comprises the following steps:
1) Freeze-drying a CRISPR reagent, placing the CRISPR reagent in a detection outer tube (5), and fixing an amplification inner tube (3) on the middle upper part of the detection outer tube (5); after the nucleic acid amplification reagent dry powder is placed in the amplification inner tube (3), fixing the cracking inner tube (4) in the middle of the amplification inner tube (3); placing a nucleic acid cracking reagent in the cracking inner tube (4), and then covering a tube cap (1); obtaining a reagent tube to be detected;
2) Opening a tube cap (1), and adding the sample nucleic acid to be detected into a cracking inner tube (4) of the reagent tube to be detected obtained in the step 1); covering the tube cap (1), and performing a nucleic acid cleavage reaction of the sample nucleic acid to be detected in a constant temperature environment;
3) After the nucleic acid cleavage reaction is finished, pressing the tube cap (1), enabling the tube needle (2) to pierce the cleavage inner tube sealing film (6), and performing a nucleic acid amplification reaction by one-stage centrifugation;
4) After the nucleic acid amplification reaction is finished, pressing the tube cap (1), enabling the tube needle (2) to pierce the inner tube sealing film (7), and performing two-stage centrifugation to perform a nucleic acid detection reaction;
5) Finally, a detection result of CRISPR molecular diagnosis is obtained through irradiation of a UV lamp;
the constant temperature environment of the nucleic acid cleavage reaction is 37-40 ℃ and the time is 10-20 min;
in the step 3), the temperature of the nucleic acid amplification reaction is 37-40 ℃ and the time is 10-20 min;
in the step 4), the temperature of the nucleic acid detection reaction is 37-40 ℃ and the time is 15-20 min;
in the step 3), the rotating speed of one-stage centrifugation is 600-6000 rpm, and the time of one-stage centrifugation is 5-20 s;
in the step 4), the rotating speed of the two-stage centrifugation is 600-6000 rpm, and the time of the two-stage centrifugation is 5-20 s;
in the step 3) and in the step 4), the pressing time of the pipe cap (1) is 2-7 s.
2. The anti-pollution detection tube according to claim 1, wherein the reagent tube to be detected obtained in the step 1) is directly stored and used for direct detection and access of CRISPR molecular diagnosis.
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| CN107964504B (en) * | 2017-10-28 | 2023-10-27 | 深圳职业技术学院 | Closed sample integrated detection device and detection method |
| CN113897277B (en) * | 2021-10-15 | 2023-12-01 | 圣湘生物科技股份有限公司 | Reaction apparatus and control method thereof |
| CN113980797A (en) * | 2021-10-29 | 2022-01-28 | 广东粤港澳大湾区国家纳米科技创新研究院 | Detection device and detection method for nucleic acid amplification product |
| CN115322884B (en) * | 2022-08-11 | 2023-07-18 | 上海金域医学检验所有限公司 | Nucleic acid detection device and method thereof |
| CN115678751B (en) * | 2022-12-30 | 2023-04-18 | 北京金诺美科技股份有限公司 | Sample tube |
| CN116855367A (en) * | 2023-09-04 | 2023-10-10 | 泰州蕾灵百奥生物科技有限公司 | Detection device and detection method for detecting multiple pathogenic genes |
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| JP3285227B2 (en) * | 1992-08-04 | 2002-05-27 | テルモ株式会社 | Syringe |
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| CN111548900A (en) * | 2019-02-12 | 2020-08-18 | 浙江大学 | Device and method for amplifying and detecting nucleic acid |
| CN110575810B (en) * | 2019-10-12 | 2024-02-23 | 安图实验仪器(郑州)有限公司 | Side-feeding reaction tube |
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| CN111621412A (en) * | 2020-06-17 | 2020-09-04 | 杭州奥盛仪器有限公司 | Reaction tube for two-step nucleic acid amplification and detection |
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| WO2008019603A1 (en) * | 2006-08-11 | 2008-02-21 | Ustar Biotechnologies (Hangzhou) Ltd. | A totally-enclosed device for quick detection of target nucleic acid amplification product |
| CN109706227A (en) * | 2019-01-24 | 2019-05-03 | 浙江大学 | A kind of quick visualization and the nucleic acid on-site inspection method that Aerosol Pollution can be prevented |
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