CN116478800A - Integrated anti-pollution detection tube and nucleic acid detection method based on same - Google Patents
Integrated anti-pollution detection tube and nucleic acid detection method based on same Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 153
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 115
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- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 115
- 230000003321 amplification Effects 0.000 claims abstract description 117
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 117
- 239000012188 paraffin wax Substances 0.000 claims abstract description 70
- 238000005336 cracking Methods 0.000 claims abstract description 64
- 238000007789 sealing Methods 0.000 claims abstract description 43
- 238000005119 centrifugation Methods 0.000 claims abstract description 28
- 238000003745 diagnosis Methods 0.000 claims abstract description 25
- 229940031182 nanoparticles iron oxide Drugs 0.000 claims abstract description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 57
- 108091033409 CRISPR Proteins 0.000 claims description 51
- 238000010354 CRISPR gene editing Methods 0.000 claims description 47
- 238000006243 chemical reaction Methods 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 23
- 230000009089 cytolysis Effects 0.000 claims description 21
- 238000003776 cleavage reaction Methods 0.000 claims description 18
- 230000008569 process Effects 0.000 claims description 13
- 230000001678 irradiating effect Effects 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 9
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N iron oxide Inorganic materials [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 claims description 8
- NDLPOXTZKUMGOV-UHFFFAOYSA-N oxo(oxoferriooxy)iron hydrate Chemical compound O.O=[Fe]O[Fe]=O NDLPOXTZKUMGOV-UHFFFAOYSA-N 0.000 claims description 8
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- 238000001746 injection moulding Methods 0.000 description 4
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- 238000011109 contamination Methods 0.000 description 2
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- 101100385364 Listeria seeligeri serovar 1/2b (strain ATCC 35967 / DSM 20751 / CCM 3970 / CIP 100100 / NCTC 11856 / SLCC 3954 / 1120) cas13 gene Proteins 0.000 description 1
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 1
- 241000907316 Zika virus Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 210000005006 adaptive immune system Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention discloses an integrated anti-pollution detection tube and a detection method based on the same, and belongs to the technical field of nucleic acid molecule diagnosis. The integrated anti-pollution detection tube comprises an amplification tube, a detection tube is sleeved outside the amplification tube, a cracking tube is arranged inside the amplification tube, and a tube cap is arranged at the top of the detection tube; the cracking tube comprises cracking tube sealing paraffin arranged at the bottom, and the amplification tube comprises amplification tube sealing paraffin arranged at the bottom; the paraffin wax is a mixed paraffin wax doped with iron oxide nanoparticles having a diameter of 10 nm. The nucleic acid detection method based on the integrated 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 laser lamp irradiation 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 integrated anti-pollution nucleic acid detection tube and a CRISPR molecular diagnosis detection method based on the same.
Background
CRISPR, collectively known as "regularly clustered spaced short palindromic repeats" (clusterides), is a widely occurring adaptive immune system in archaebacteria and bacteria. 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 doudna team, the university of california, berkeley, inc. 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, CRISPR nucleic acid detection techniques have problems in that it is generally necessary to cleave and amplify a sample nucleic acid prior to performing a CRISPR nucleic acid detection reaction, and then to perform the detection after adding the sample nucleic acid to a CRISPR reaction system. In the process, operations such as uncovering of the detection tube, transferring of the reagent, closing of the detection tube and the like are needed, so that not only is laboratory aerosol pollution easily caused, but also high false positive problem of subsequent results is caused, and cross contamination of laboratories and related equipment is easily caused, a great deal of time is needed to be consumed to finish a series of operations, and waste of manpower and material resources is caused. It is therefore highly desirable to find a convenient and practical device or method that allows for integrated sample-in-and-out operation of sample nucleic acids without decreasing sensitivity, and that is simple and pollution-free.
Disclosure of Invention
In order to realize CRISPR-based integrated, simple and pollution-free nucleic acid detection, the invention aims to provide an integrated pollution-prevention detection tube and a molecular diagnosis detection method based on the same. The invention aims to solve the problems that false positive or sensitivity is reduced and the integration can not be realized to obtain a detection result because of operations such as uncovering, transferring and closing of sample nucleic acid and the like in the process of detecting nucleic acid based on CRISPR technology; the integrated 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 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 integrated anti-pollution detection tube, which comprises an amplification tube, wherein the outside of the amplification tube is sleeved with a detection tube, the inside of the amplification tube is provided with a cracking tube, and the top of the detection tube is provided with a tube cap; wherein, the schizolysis pipe includes the bottom setting and plays sealed effect's paraffin, and the amplification pipe includes the bottom setting and plays sealed effect's paraffin.
Preferably, the amplification tube comprises a first hollow cylinder upper body, a first conical middle body and a first hollow cylinder lower body which are fixedly connected, an opening is formed in the closing side of the first hollow cylinder lower body, and paraffin is arranged in the opening for sealing; the detection 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 tube comprises a third hollow cylinder and a third conical lower body, and paraffin is arranged at the bottom of the third conical lower body for sealing.
Preferably, the materials of the lysis tube, the amplification tube, the detection tube and the tube cap are virgin polypropylene.
Preferably, the lysis tube, the amplification tube, the detection tube and the tube cap are connected in sequence through clearance fit.
Preferably, the lysis tube, the amplification tube, the detection tube and the tube cap are connected in sequence through threaded fit.
Preferably, the cracking tube sealing paraffin and the amplification tube sealing paraffin are paraffin doped with 10 nm-diameter ferric oxide nano particles.
Further preferably, the cracking tube sealing paraffin and the amplification tube sealing paraffin can be processed in advance according to the required size, and then the sealing of the cracking tube and the amplification tube is realized through interference fit; or placing the opening bottoms of the cracking tube and the amplification tube in paraffin in a molten state, and sealing the cracking tube and the amplification tube after the paraffin is cooled.
The invention discloses a CRISPR molecular diagnosis detection method based on the anti-pollution detection tube, which comprises the following steps:
1) After the nucleic acid amplification reagent dry powder is placed in an amplification tube, fixing a cracking tube in the middle of the amplification tube; freeze-drying the CRISPR reagent and placing the CRISPR reagent in a detection tube, and fixing an amplification tube on the middle upper part of the detection tube; placing a nucleic acid cracking reagent in a cracking tube, and 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 tube of the reagent tube to be detected obtained in the step 1); covering a tube cap, and performing a nucleic acid cleavage reaction procedure of the sample nucleic acid to be detected in a constant temperature environment;
3) After the nucleic acid cracking reaction is finished, a laser lamp with the wavelength of 1.5w 806 nm is used for irradiating a cracking tube to seal the paraffin (5), the paraffin is melted, and the process of nucleic acid amplification reaction is carried out through one-stage forward and reverse centrifugation and uniform mixing;
4) After the nucleic acid amplification reaction is finished, a laser lamp with the wavelength of 1.5w 806 nm is used for irradiating the sealed paraffin (6) of the amplification tube, paraffin is melted, and the two-stage positive and negative centrifugation mixing is carried out for carrying out a nucleic acid detection reaction;
5) Finally, the detection test tube is irradiated by a UV lamp or a blue light gel cutting instrument to obtain the detection result of CRISPR molecular diagnosis.
Preferably, in the step 2), the constant temperature environment for the nucleic acid cleavage reaction is 38-40 ℃ and the time is 15-20 min; in the step 3), the temperature of the nucleic acid amplification reaction is 38-40 ℃ and the time is 12-18 min; in the step 4), the temperature of the nucleic acid detection reaction is 38-40 ℃ and the time is 12-18 min; the rotational speed of one-stage forward and reverse centrifugation in the step 3) is 600-6000 rpm, the time of one-stage forward rotation centrifugation is 3-5 s, the time of reverse rotation centrifugation is 3-5 s, and the forward rotation and the reverse rotation are respectively alternated 3-5 times; in the step 4), the rotating speed of the two-stage forward and reverse centrifugation is 600-6000 rpm, the time of the two-stage forward rotation centrifugation is 3-5 s, the time of the reverse rotation centrifugation is 3-5 s, and the forward rotation and the reverse rotation are respectively alternated for 3-5 times; the irradiation time of a laser lamp with the wavelength of 1.5w 806 nm in the step 3) is 1-2 s; in the step 4), the irradiation time of a laser lamp with the wavelength of 1.5w 806 nm is 5-7 s.
Further preferably, in the step 2), the constant temperature environment of the acid cleavage reaction is 39 ℃ and the time is 15min; in the step 3), the temperature of the nucleic acid amplification reaction is 39 ℃ and the time is 12min; in the step 4), the temperature of the nucleic acid detection reaction is 39 ℃ and the time is 15min; in the step 3), the rotating speed of one-stage positive and negative centrifugation is 1000rpm, the positive and negative centrifugation is performed three times, and the time of each positive and negative centrifugation is 4s; in the step 4), the rotating speed of the two-stage positive and negative centrifugation is 1000rpm, the positive and negative centrifugation is performed three times, and the time of each positive and negative centrifugation is 4s; the irradiation time of the laser lamp with the wavelength of 1.5w 806 nm in the step 3) is 2s; the laser lamp irradiation time of 1.5w 806 nm in step 4) was 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 a cracking tube, an amplification tube and a detection tube are sequentially sleeved from inside to outside, three detection stages required in CRISPR molecular diagnosis can be divided into three independent detection spaces, the cracking tube and the amplification tube are sealed by paraffin doped with iron oxide nanoparticles with different concentrations, the cracking reagent and the amplification reagent are stored separately, and then the paraffin doped with the iron oxide nanoparticles with different concentrations is melted in different irradiation time by irradiation of a 808nm laser lamp, so that batch mixing of the reagents from inside to outside is realized, and the staged implementation of the three detection stages is satisfied. 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 amplification tube, schizolysis pipe and the detecting tube that have hollow cylinder structure and class toper structure to constitute respectively, utilize geometry's cooperation, 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 adopting clearance fit connection or thread fit connection, convenient assembly between pipes can be realized; through interference fit or screw thread fit connection of the pipe cap and the detection pipe, the tightness of the system in centrifugal treatment is ensured, and laboratory aerosol pollution in the nucleic acid detection process is effectively prevented.
Further, the cracking tube and the amplification tube are sealed by using paraffin doped with ferric oxide nano particles with different concentrations, so that the three processes of nucleic acid cracking, nucleic acid amplification and CRISPR detection can be separated, and meanwhile, the paraffin doped with the ferric oxide nano particles can be melted in a time-sharing manner under the irradiation of a 808nm laser lamp, so that the effective connection of the three processes of nucleic acid cracking, nucleic acid amplification and CRISPR detection is realized.
The invention also discloses a CRISPR molecular diagnosis detection method based on the integrated anti-pollution detection tube, when the CRISPR molecular diagnosis is carried out by adopting the integrated anti-pollution detection tube, 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 808nm laser lamp irradiation 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 also 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 integrated 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-inactivation environment, and based on the integrated anti-pollution detection tube, the CRISPR molecular diagnosis detection reagent can be pre-stored, and when the nucleic acid detection is needed, the nucleic acid sample to be detected is only needed to be added into the test tube (the reagent pre-storage is realized), so that the household nucleic acid detection can be realized.
Drawings
FIG. 1 is a schematic structural view of an integrated 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 structure of the detection tube of the present invention;
FIG. 4 is a schematic view of an amplification tube of the present invention;
FIG. 5 is a schematic view of a pyrolysis tube of the present invention;
FIG. 6 is a schematic illustration of the reagent addition assembly of the integrated anti-contamination test tube of the present invention; wherein, (a) is a schematic diagram of adding a nucleic acid amplification reagent to an amplification tube; (b) is a schematic diagram of the cooperation of the lysis tube and the amplification tube; (c) schematic representation of the addition of CRISPR reagent to the detection tube; (d) is a schematic diagram of the cooperation of the amplification tube and the detection tube; (e) adding nucleic acid lysate to the lysis tube; (f) An integrated anti-pollution detection tube schematic diagram added with various reagents;
FIG. 7 is a schematic diagram of a process for CRISPR molecular diagnosis using an integrated anti-pollution detection tube according to the present invention; wherein, figure (a) is a schematic diagram after the laser lamp of 808nm irradiates the cracking tube to seal paraffin; FIG. b is a schematic view of the amplification tube after the sealing paraffin wax is irradiated by a 808nm laser lamp;
wherein: 1-pipe cap, 2-detecting pipe, 3-cracking pipe, 4-amplifying pipe, 5-cracking pipe sealing paraffin, 6-amplifying pipe sealing paraffin.
Description of the embodiments
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 invention provides an integrated anti-pollution detection tube, which sequentially comprises a tube cap 1, a detection tube 2, a cracking tube 3, a cracking tube sealing paraffin 5, an amplification tube 4 and an amplification tube sealing paraffin 6 from top to bottom.
In the present invention, the contamination prevention detecting tube includes a cap 1, the structure of which is shown in fig. 2. The pipe cap 1 has a good sealing effect on the detection pipe 2, so that the sample nucleic acid to be detected is prevented from volatilizing outside the integrated anti-pollution detection pipe, and aerosol pollution is avoided.
Wherein, the material of the pipe cap 1 is preferably polyolefin material; the cap 1 is preferably manufactured by injection moulding.
In the invention, the anti-pollution detection tube also comprises a detection tube 2, wherein the detection tube 2 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. 3 for its structure. In the present invention, the detection tube 2 is used to store a CRISPR reagent, and after the nucleic acid amplification reaction is completed, the detection tube 2 serves as a container in which the CRISPR nucleic acid detection reaction occurs.
Wherein, the material of the detection tube 2 is preferably a virgin polypropylene material; the test tube 2 is preferably manufactured by injection molding.
In the invention, the anti-pollution detection tube further comprises an amplification tube 4, the amplification tube 4 comprises a first hollow cylinder upper body, a first conical middle body and a first hollow cylinder lower body which are sequentially connected from top to bottom, an opening is arranged on the closing-in side of the first hollow cylinder lower body, and the opening on the closing-in side of the first hollow cylinder lower body is sealed through an amplification tube paraffin 6, and the structure is shown in fig. 4. In the present invention, the amplification tube 4 functions as a container in which a nucleic acid amplification reaction occurs and a reservoir for a nucleic acid amplification reagent.
Wherein, the material of the amplification tube 4 is preferably a virgin polypropylene material; the amplification tube 3 is preferably manufactured by an injection molding process.
In the invention, the amplification tube 4 and the detection tube 2 are assembled through clearance fit; the amplification tube 4 is clamped inside the detection tube 2, and the detection tube 2 plays a role in fixedly supporting the amplification tube 4.
In the invention, the integrated anti-pollution detection tube further comprises a cracking tube 3, the structure of which is shown in fig. 5, and it can be seen that the cracking tube 3 comprises a third hollow cylindrical upper body and a third conical lower body, and the bottom of the third conical lower body is sealed by using cracking tube sealing paraffin 5. In the present invention, the cleavage tube 3 functions as a container in which a nucleic acid cleavage reaction occurs and a reservoir for a nucleic acid cleavage reagent.
In the invention, the outer diameter of the cracking tube 3 is matched with the inner diameter of the amplification tube 4 and generates clearance fit; the cracking tube 3 is clamped inside the amplification tube 4, after the assembly is completed, the height of the cracking tube 3 is higher than that of the upper part of the first hollow cylinder of the amplification tube 4, the assembly of the whole integrated anti-pollution detection tube is facilitated, and the amplification tube 4 plays a role in fixedly supporting the cracking tube 3.
Wherein, the cracking tube 3 is preferably made of virgin polypropylene; the pyrolysis tube 3 is preferably manufactured by an injection molding process.
In the invention, the cracking tube sealing paraffin 5 and the amplifying tube sealing paraffin 6 can bear a certain volume of liquid under the condition of no strong external force interference, the volume is far more than the volume required by experiments, and the cracking tube sealing paraffin 5 and the amplifying tube sealing paraffin 6 can be melted in different time due to different concentrations of doped ferric oxide nano particles under the irradiation of 808nm laser lamps.
In particular, in a specific embodiment of the present invention, the lysis tube sealing paraffin 5 and the amplification tube sealing paraffin 6 are preferably paraffin doped with iron oxide nanoparticles having a diameter of 10 nm. In the invention, the amplification tube sealing paraffin 6 is later than the cracking tube sealing paraffin 5 to melt, and the higher the concentration of doped ferric oxide, the more energy absorbed in unit time is, and the more easily the melting point of the paraffin is reached, so the concentration of the doped ferric oxide nano particles of the amplification tube sealing paraffin 6 is lower than the concentration of the doped ferric oxide nano particles of the cracking tube sealing paraffin 5.
The invention also provides a detection method for CRISPR molecular diagnosis by using the anti-pollution detection tube, which comprises the following steps: after adding a nucleic acid amplification reagent or dry powder to the amplification tube 4, fixing the lysis tube 3 in the amplification tube 4; after adding CRISPR reagent or dry powder to the detection tube 2, fixing the amplification tube 4 in the detection tube 2; after adding a nucleic acid cracking reagent into the cracking tube 4, covering the detection tube 2 with a tube cover 1, 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 pipe 3, 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, a 808nm laser lamp is used for irradiating the cleavage tube sealing paraffin 5, and a procedure of performing one-stage forward and reverse rotation centrifugation and performing nucleic acid amplification reaction is performed; after the nucleic acid amplification reaction is finished, irradiating the amplification tube sealing paraffin 6 by using a 808nm laser lamp, performing two-stage forward and reverse rotation centrifugation, and performing a nucleic acid detection reaction; finally, the detection result of CRISPR molecular diagnosis is obtained by irradiation through a UV lamp or by being placed on a blue light gel cutting instrument.
Specifically, in the embodiment of the present invention, after the CRISPR reagent is added to the detection tube 2, the amplification tube 4 is immobilized in the detection tube 2. 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 10 microliters; the CRISPR reagent is preferably added to the bottom of the detection tube 2; a schematic illustration of the addition of CRISPR reagent to the detection tube 2 is shown in fig. 6 (c); a schematic diagram of the detection tube 2 after the amplification tube 4 is immobilized is shown in FIG. 6 (d). In the present invention, after adding the nucleic acid amplification reagent dry powder to the amplification tube 4, the lysis tube 3 is fixed to the amplification tube 4. 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 10. Mu.l. In the present invention, the nucleic acid amplification reagent is preferably added to the bottom of the amplification tube 4. In the present invention, a schematic diagram of adding a nucleic acid amplification reagent dry powder to the amplification tube 4 is shown in FIG. 6 (a); a schematic diagram after fixing the lysis tube 3 in the amplification tube 4 is shown in FIG. 6 (b). In the present invention, a nucleic acid cleavage reagent is added to the cleavage tube 3, and the tube cap 1 is covered. In the present invention, the volume of the nucleic acid cleavage reagent is preferably 10. Mu.l. In the present invention, the nucleic acid cleavage reagent is added to the bottom of the cleavage tube 3. In the present invention, a schematic diagram of the addition of a nucleic acid cleavage reagent to the cleavage tube 3 is shown in FIG. 6 (e).
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 detecting the CRISPR molecular diagnosis of nucleic acid, the CRISPR molecular diagnosis detection can be realized by simply irradiating the sample nucleic acid to be detected by using a 808nm laser lamp and centrifuging only 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 lysis tube 3, and the tube cap 1 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.
By the integrated anti-pollution detection tube and the CRISPR molecular diagnosis detection method based on the same, seamless connection of sample inlet to outlet can be realized, manual operation is reduced, time is saved, the efficiency of nucleic acid detection is improved, and laboratory aerosol pollution caused by operations such as uncapping, sample nucleic acid transferring and closing during the process of nucleic acid amplification to nucleic acid detection 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.
Examples
The structure of the anti-pollution detection tube and the schematic diagrams of each step provided in this embodiment are shown in fig. 6 and fig. 7, and the operation steps are as follows: firstly, adding a prepared RPA nucleic acid amplification reagent or dry powder into an amplification tube 4, then adding a cracking tube 3 into the amplification tube 4, wherein the cracking tube 3 is clamped in the amplification tube 4 and is fixed; then, the prepared CRISPR reagent or dry powder is added into the detection tube 2, and then the amplification tube 4 is added into the detection tube 2, so that the amplification tube 3 is clamped inside the detection outer tube 5 and is fixed; the prepared nucleic acid lysis reagent is added into the lysis tube 3, and then the tube cover 1 is covered, so that the nucleic acid lysis reagent can be immediately used, and can also be put into a refrigerator at minus 20 ℃ for sealing and storage. When CRISPR molecular diagnosis nucleic acid detection is needed, the integrated anti-pollution detection tube can be taken out, sample nucleic acid to be detected is added, then a tube cap 1 is covered, and then the reaction tube is put into a constant temperature centrifuge device at 39 ℃ for incubation for 15min; after incubation, irradiating the cracking tube with 808nm laser lamp to seal paraffin for 2s, after paraffin is melted, starting a centrifuge, and performing centrifugation treatment for the whole reaction tube at 1000rpm for 4s each time with positive and negative rotation time and 3 times each time, and transferring the cracked sample nucleic acid into the amplification tube 4 under the action of centrifugal force; then the integrated anti-pollution detection tube is further incubated in a constant temperature centrifuge device at 39 ℃ for 12min; after incubation, the cracking tube is irradiated by a 808nm laser lamp to seal paraffin for 5s, after paraffin is melted, a centrifuge is started, the whole reaction tube is subjected to centrifugation at 1000rpm for 4s each time in positive and negative rotation for 3 times alternately, and amplified sample nucleic acid is transferred into the detection tube 2 to be mixed with CRISPR reagent under the action of centrifugal force. And finally, incubating for 15min at the constant temperature of 39 ℃, and observing the experimental result under the irradiation of a UV lamp or a blue light gel cutting instrument.
Taking HBV as an example, the field workflow of the integrated anti-pollution consumable in this embodiment will be described.
1. As shown in FIG. 6 (a), a prepared HBV RPA nucleic acid amplification reaction reagent is added to the bottom of the amplification tube 4;
2. as shown in FIG. 6 (b), the lysis tube 3 is inserted into the amplification tube 4, and the lysis tube 3 is fixed;
3. as shown in fig. 6 (c), a prepared HBV CRISPR reagent is added to the bottom of the detection tube 2;
4. as shown in fig. 6 (d), the amplification tube 4 is inserted into the detection tube 2, and the amplification tube 4 is fixed;
5. as shown in FIG. 6 (e), a prepared nucleic acid lysate is added to the bottom of the lysis tube 4;
6. adding HBV sample nucleic acid to be detected to the bottom of the lysis tube 3;
7. as shown in fig. 6 (f), the tube cap 1 is covered, shaken and homogenized, centrifuged, and then the whole amplification tube is put into a thermostat for 15min to realize the cleavage of sample nucleic acid;
8. irradiating the cracking tube with 808nm laser lamp to seal paraffin for 2s, and melting paraffin;
9. as shown in fig. 7 (a), the forward and reverse transfer is carried out by centrifugation, the time of each forward and reverse rotation is 4s, the speed is 1000rpm, the forward and reverse transfer is carried out for 3 times, and the cracked sample nucleic acid is transferred into the amplification tube 4 and is uniformly mixed;
10. incubating for 12min in a constant temperature environment at 39 ℃ to realize amplification of sample nucleic acid;
11. irradiating the amplification tube with 808nm laser lamp to seal paraffin for 5s, and melting paraffin;
12. as shown in fig. 8 (b), the forward and reverse transfer is carried out by centrifugation, the forward and reverse transfer is carried out for 3 times at the speed of 1000rpm for 4s each time, and amplified sample nucleic acid is transferred into the detection tube 2 and uniformly mixed;
13. incubating for 15min in a constant temperature environment at 39 ℃, and then observing fluorescence, so that the detection of sample nucleic acid is conveniently and rapidly realized.
The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (9)
1. The integrated anti-pollution detection tube is characterized by comprising a detection tube (2), wherein an amplification tube (4) is sleeved in the detection tube (2), a cracking tube (3) is arranged in the amplification tube (4), and a tube cap (1) is arranged at the top of the detection tube (2);
wherein, the cracking tube (3) comprises cracking tube sealing paraffin (5) with the bottom part playing a role in sealing, and the amplifying tube (4) comprises amplifying tube sealing paraffin (6) with the bottom part playing a role in sealing;
the detection tube (2) and the tube cap (1) are connected through screw thread fit or interference fit.
2. The integrated anti-pollution detection tube according to claim 1, wherein the amplification tube (4) comprises a first hollow cylinder upper body, a first conical middle body and a first hollow cylinder lower body which are fixedly connected, an opening is arranged on the closing side of the first hollow cylinder lower body, and the amplification tube sealing paraffin (6) is arranged on the opening for sealing;
the detection tube (2) comprises a second hollow cylinder upper body and a second type of conical lower body with a closed bottom which are fixedly connected;
the cracking tube (3) comprises a third hollow cylinder upper body and a third conical lower body, an opening is formed in the closing side of the third conical lower body, and the cracking tube is sealed by paraffin (5) and is arranged in the opening for sealing.
3. The integrated anti-pollution detection tube according to claim 1, wherein in order to facilitate the assembly of the detection tube (2), the lysis tube (3) and the amplification tube (4) and the facilitation of reagent filling, the top of the lysis tube (3) is higher than the top of the amplification tube (4) after the assembly of the lysis tube (3) and the amplification tube (4) is completed.
4. An integrated anti-pollution detection tube according to claim 1, characterized in that the lysis tube (3), the amplification tube (4) and the detection tube (2) are assembled by clearance fit in sequence.
5. The integrated anti-pollution detection tube according to claim 1, wherein the cracking tube sealing paraffin (5) and the amplification tube sealing paraffin (6) are paraffin doped with 10nm iron oxide nano particles; the concentration of the ferric oxide nano particles doped in the cracking tube sealing paraffin (5) is higher than that doped in the amplification tube sealing paraffin (6).
6. The integrated anti-pollution detection tube according to claim 5, wherein the paraffin (5) and the paraffin (6) can be processed in advance according to the required size, and then the sealing of the cracking tube (3) and the amplification tube (4) is realized through interference fit; or placing the bottoms of the openings of the cracking tube (3) and the amplifying tube (4) in paraffin in a molten state (the dimension in the height direction is 0.5 mm-0.8 mm in the molten state of the paraffin), and sealing the cracking tube (3) and the amplifying tube (4) after the paraffin is cooled.
7. A CRISPR molecular diagnostic detection method based on the anti-pollution detection tube according to any one of claims 1 to 6, characterized by comprising the steps of:
1) After a nucleic acid amplification reagent or reagent dry powder is placed in an amplification tube (4), fixing a cracking tube (3) in the middle of the amplification tube (4); placing CRISPR reagent or reagent dry powder in a detection tube (2), and fixing an amplification tube (4) on the middle upper part of the detection tube (2); placing a nucleic acid cracking reagent in a cracking tube (3), 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 tube (3) of the reagent tube to be detected, which is obtained in the step 1); covering a pipe cap (1), and completing a nucleic acid cleavage reaction procedure of the sample nucleic acid to be detected in a constant temperature environment;
3) After the nucleic acid cracking reaction is finished, a laser lamp with the wavelength of 1.5w 806 nm is used for irradiating a cracking tube to seal the paraffin (5), the paraffin is melted, and the process of nucleic acid amplification reaction is carried out through centrifugal mixing in the forward and reverse directions at one stage;
4) After the nucleic acid amplification reaction is finished, irradiating the sealed paraffin (6) of the amplification tube by using a laser lamp with the wavelength of 1.5w 806 nm, and carrying out a procedure of nucleic acid detection reaction by carrying out centrifugal mixing in the two-stage forward and reverse directions;
5) Finally, the detection test tube is irradiated by a UV lamp or a blue light gel cutting instrument to obtain the detection result of CRISPR molecular diagnosis.
8. The method for detecting CRISPR molecular diagnosis according to claim 1, wherein in the step 2), the constant temperature environment for the nucleic acid cleavage reaction is 38-40 ℃ for 15-20 min;
in the step 3), the temperature of the nucleic acid amplification reaction is 38-40 ℃ and the time is 12-18 min;
in the step 4), the temperature of the nucleic acid detection reaction is 38-40 ℃ and the time is 12-18 min;
in the step 3), the rotating speed of one-stage forward and reverse centrifugation is 600-6000 rpm, the time of one-stage forward and reverse centrifugation is 3-5 s, and the time of reverse centrifugation is 3-5 times respectively and alternately;
in the step 4), the rotating speed of the two-stage forward and reverse centrifugation is 600-6000 rpm, the time of the two-stage forward rotation centrifugation is 3-5 s, the time of the reverse rotation centrifugation is 3-5 s, and the forward rotation and the reverse rotation are respectively alternated for 3-5 times;
the irradiation time of a laser lamp with the wavelength of 1.5w 806 nm in the step 3) is 1-2 s;
in the step 4), the irradiation time of a laser lamp with the wavelength of 1.5w 806 nm is 5-7 s.
9. The method for detecting the molecular diagnosis of the CRISPR according to claim 8, wherein the reagent tube to be detected obtained in the step 1) is directly stored and used for direct detection and taking of the molecular diagnosis of the CRISPR.
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| CN202310523506.XA CN116478800A (en) | 2023-05-10 | 2023-05-10 | Integrated anti-pollution detection tube and nucleic acid detection method based on same |
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