CN113621682A - Biological sample processing method, collection container and kit - Google Patents
Biological sample processing method, collection container and kit Download PDFInfo
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- CN113621682A CN113621682A CN202010377054.5A CN202010377054A CN113621682A CN 113621682 A CN113621682 A CN 113621682A CN 202010377054 A CN202010377054 A CN 202010377054A CN 113621682 A CN113621682 A CN 113621682A
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- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/305—Fixative compositions
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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
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Abstract
The invention discloses a biological sample processing method, an acquisition container and a kit, wherein the biological sample processing method comprises the following steps: (1) immobilizing the biological sample with an immobilization reagent, the immobilization reagent being adapted to inactivate pathogens in the biological sample and the immobilization reagent being adapted to form cross-linked products with proteins or nucleic acid molecules in the biological sample; and (2) subjecting the immobilized product obtained in step (1) to a decrosslinking treatment using a decrosslinking reagent to decrosslink the crosslinked product to obtain a nucleic acid molecule or protein suitable for detection. According to the biological sample processing method provided by the embodiment of the invention, the collected biological sample can be fixed by adopting the fixing reagent, and when the reagent carrying aldehyde groups is adopted, the sample can be effectively fixed, so that pathogens such as viruses or bacteria in the sample can not have infectivity or toxicity any more, and the operation is safe.
Description
Technical Field
The invention relates to the technical field of collection containers for collecting samples, in particular to a biological sample processing method, a collection container and a kit.
Background
The collection container in the related art includes a containing cavity for storing a sample and a liquid storage cavity for storing a reagent, and the sample needs to be fixed by adding the reagent after being added into the containing cavity. The reagent needs to be sealed in the liquid storage groove in advance, namely in the process of packaging the reagent, the reagent is firstly added into the liquid storage groove, and then the liquid storage groove is sealed by a sealing film so as to form a sealed liquid storage cavity. Therefore, the liquid storage cavity cannot be subjected to tightness detection before the reagent is packaged, and the problem of reagent leakage or impurity mixing is easy to occur.
In addition, since some biological samples involve toxic or infectious materials such as viruses, bacteria and other pathogens, the collection of these biological samples often raises operational safety issues.
Therefore, there is still a need for an improved collection container for use in biological testing.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a biological sample processing method and a collection container applicable to the method, and the collection container has the advantages of good sealing effect, reliable work and the like.
In one aspect of the invention, the invention features a method of processing a biological sample, the method including, according to an embodiment of the invention: (1) immobilizing a biological sample with an immobilization reagent, the immobilization reagent being adapted to inactivate pathogens in the biological sample and the immobilization reagent being adapted to form cross-linked products with proteins or nucleic acid molecules in the biological sample; and (2) subjecting the immobilized product obtained in step (1) to a decrosslinking treatment using a decrosslinking reagent to decrosslink the crosslinked product so as to obtain the nucleic acid molecule or protein suitable for the detection. Therefore, by adopting the method, the collected biological sample can be fixed by adopting the fixing reagent, and when the reagent carrying aldehyde group is adopted, the sample can be effectively fixed, so that pathogens such as viruses or bacteria in the sample can not have infectivity or toxicity any more, and the operation is safe. In addition, the human body can be effectively prevented from contacting the immobilized reagent, so that the injury or negative effect of the immobilized reagent on the human body is avoided.
According to some embodiments of the invention, the immobilized reagent comprises a reagent carrying an aldehyde group.
According to some embodiments of the invention, the reagent bearing aldehyde groups comprises at least one of formaldehyde, glutaraldehyde, and paraformaldehyde.
According to some embodiments of the invention, the immobilization reagent comprises at least one selected from the group consisting of:
(a) a solution containing calcium carbonate and formalin;
(b) formalin solution with pH value of 7.0-7.5;
(c) a paraformaldehyde solution with a pH of 7.0-7.5;
(d) a solution containing glutaraldehyde and formaldehyde;
(e) a solution containing formaldehyde, mercuric chloride and sodium acetate;
(f) a solution containing acetic acid and formaldehyde;
(g) solution containing picric acid, acetic acid and formaldehyde
(h) A solution containing picric acid and formaldehyde; and
(i) a solution comprising periodate, lysine and paraformaldehyde.
According to some embodiments of the invention, the de-crosslinking reagent comprises at least one of citric acid, citrate, EDTA and Tris.
According to some embodiments of the invention, the decrosslinking treatment is performed at a temperature above room temperature.
According to some embodiments of the invention, the de-crosslinking treatment is performed at a temperature of 70 to 100 degrees celsius.
According to some embodiments of the invention, the nucleic acid molecule comprises at least one of DNA and RNA.
According to some embodiments of the invention, the nucleic acid sample suitable for the detection contains RNA molecules of no more than 300nt in length.
According to some embodiments of the invention, the pathogen comprises a bacterium and a virus.
According to some embodiments of the invention, the virus comprises an RNA virus.
According to some embodiments of the invention, the RNA virus comprises at least one of a hiv virus, a hepatitis c virus, a encephalitis b virus, an influenza virus, a rhinovirus, a poliovirus, a coxsackie virus, a dengue virus, a rotavirus, a tobacco mosaic virus, a coronavirus, a marburg virus, an ebola virus.
According to some embodiments of the invention, the coronavirus comprises at least one of a SARS virus, a MERS virus, and 2019-nCoV.
According to some embodiments of the invention, the biological sample comprises at least one of an alveolar or bladder or stomach or intestine or uterus or nasal lavage fluid, a suspension containing cells or bacteria or fungi or viruses obtained by rinsing in a liquid after collection of a sample by puncturing or brushing or scraping, a cell or bacteria or fungi or virus sample cultured in a liquid, blood, body fluid, interstitial fluid, tears, stool, sputum, urine, saliva, semen, nasal discharge, pleural fluid, ascites, prostatic fluid, hair, skin, epithelial tissue.
According to some embodiments of the invention, after step (1), the immobilized treated product is stored at room temperature before step (2).
According to some embodiments of the invention, after step (2), further comprising: isolating the nucleic acid molecule from the nucleic acid sample.
According to some embodiments of the invention, after step (2), further comprising: the nucleic acid molecules are detected by in situ hybridization using a probe.
Further, according to a second aspect of the present invention, to achieve the above object, there is provided a collecting container according to an embodiment of the present invention, including: a container body defining a first reservoir chamber therein having an opening; the cover body is movably arranged at the opening, a second liquid storage cavity is defined in the cover body, the cover body is provided with a liquid injection port and a liquid outlet which are communicated with the second liquid storage cavity, and a pierceable film covers the liquid outlet; the puncture part is arranged on the container body or the cover body and used for puncturing the pierceable film so as to allow the first liquid storage cavity to be communicated with the second liquid storage cavity.
The collecting container provided by the embodiment of the invention has the advantages of good sealing effect, reliability in operation and the like.
In addition, the collecting container according to the above embodiment of the present invention may further have the following additional technical features:
according to some embodiments of the invention, the pierceable membrane is integrally formed with at least a portion of the cap.
According to some embodiments of the invention, the collection container further comprises a stopper for closing the pouring opening.
According to some embodiments of the invention, the pouring outlet is located on a side surface of the lid body facing away from the opening.
According to some embodiments of the present invention, the liquid outlet is located on a side surface of the cover body facing the opening, and a projection of the liquid outlet in a plane of the opening is located inside the opening.
According to some embodiments of the invention, the cover comprises: the second liquid storage cavity is formed in the cover body; the extension portion is arranged on the cover body, a liquid passing channel is arranged in the extension portion, one end of the liquid passing channel is communicated with the second liquid storage cavity, the other end of the liquid passing channel forms the liquid outlet, and the pierceable membrane is arranged in the liquid passing channel.
According to some embodiments of the invention, the wall surface of the second reservoir chamber has a guide surface extending circumferentially around the liquid passage channel, the guide surface extending obliquely downward in a direction approaching the liquid passage channel.
According to some embodiments of the invention, the piercing part is provided on the cover body, and at least a part of the piercing part is fitted in the liquid passing channel.
According to some embodiments of the invention, the piercing part is provided with at least one flow guide groove at a position close to the extension part, and the flow guide groove extends along the length direction of the piercing part.
According to some embodiments of the invention, the extension is disposed on a side of the cap body facing the opening, and the extension is adapted to extend from the opening into the first reservoir.
According to further embodiments of the present invention, the cap body includes: the base is arranged at the opening and is connected with the container main body, and the base is provided with a sampling port; the installation shell is arranged on the base in a turnover mode so as to open and close the sampling port, the installation shell limits the second liquid storage cavity, and the extending portion is arranged on the installation shell and extends into the base.
According to some embodiments of the invention, the collection container further comprises a filter element disposed within the base and below the liquid outlet.
According to some embodiments of the invention, the cap is threadably connected to the container body.
According to some embodiments of the invention, the collection container further comprises: and the pressing part is arranged on the cover body and is connected with the puncture part, and the pressing part drives the puncture part to puncture the puncture membrane after being pressed down.
According to some embodiments of the invention, the pressing part comprises: the pressing body is arranged on the surface of one side of the cover body, which is far away from the opening, and the pressing body is connected with the puncture part; a deformable portion located outside the pressing body and surrounding the pressing body, the deformable portion being connected between the cover and the pressing body.
According to some embodiments of the invention, the deformable portion forms a pleated structure.
According to some embodiments of the invention, the pressing portion, the puncturing portion and at least a portion of the cover are integrally formed.
According to some embodiments of the invention, a fixed reagent is disposed in the second reservoir chamber, the fixed reagent comprising a reagent carrying an aldehyde group. Therefore, in the using process, the fixing reagent can be used for fixing the collected biological sample, and when the reagent carrying aldehyde groups is adopted, the sample can be effectively fixed, so that pathogens such as viruses or bacteria in the sample can not have infectivity or toxicity any more, and the operation is safe. In addition, through adopting this collection container, can avoid human contact fixed reagent effectively to avoid fixed reagent to the injury or the negative effects of human body. According to an embodiment of the invention, the pathogens include bacteria and viruses. According to an embodiment of the invention, the virus comprises an RNA virus. According to an embodiment of the invention, the RNA virus comprises at least one of a hiv virus, a hepatitis c virus, a encephalitis b virus, an influenza virus, a rhinovirus, a poliovirus, a coxsackie virus, a dengue virus, a rotavirus, a tobacco mosaic virus, a coronavirus, a marburg virus, an ebola virus. According to an embodiment of the invention, the coronavirus comprises at least one of a SARS virus, a MERS virus and 2019-nCoV.
According to some embodiments of the invention, the reagent bearing aldehyde groups comprises at least one selected from the group consisting of formaldehyde, glutaraldehyde and paraformaldehyde. Thus, the nucleic acid or protein can be protected by effectively causing a crosslinking reaction between the nucleic acid or protein and the fixing agent using the fixing agent, and can be stored for a long period of time, for example, at room temperature for at least one month. These cross-linked products can subsequently be subjected to a de-cross-linking treatment by a de-cross-linking reagent in order to detect the corresponding nucleic acids or proteins.
It should be noted that, according to the embodiments of the present invention, the biological sample that can be used is preferably a liquid biological sample, including but not limited to lavage fluid of alveoli, bladder, stomach, intestine, uterus or nasal cavity, suspension containing cells or bacteria or fungi or viruses obtained by washing in liquid after collecting a sample by puncturing or brushing or scraping, a cell or bacteria or fungi or viruses sample cultured in liquid, blood, body fluid, tissue fluid, tears, feces, sputum, urine, saliva, semen, nasal discharge, pleural fluid, ascites, prostatic fluid, etc.
In a third aspect of the invention, the invention proposes a kit comprising, according to an embodiment of the invention: the collection container as described above, and a fixed reagent disposed in the second reservoir chamber of the collection container. As mentioned above, the kit can be used for biological sample collection and immobilization treatment, so that the immobilized reagent can perform immobilization treatment on the collected biological sample in the using process, and when the reagent carrying aldehyde group is used, the sample can be effectively immobilized, so that pathogens such as viruses or bacteria in the sample can not have infectivity or toxicity any more, and the operation is safe. In addition, through adopting this collection container, can avoid human contact fixed reagent effectively to avoid fixed reagent to the injury or the negative effects of human body. It should be noted that the features and advantages described above in relation to the collection container apply equally to the kit and are not described in further detail here.
According to an embodiment of the present invention, the kit may further include: a de-crosslinking reagent comprising at least one of citric acid, citrate, EDTA, and Tris. Thus, these crosslinked products can be subjected to a decrosslinking treatment by a decrosslinking reagent to detect the corresponding nucleic acids or proteins.
According to an embodiment of the present invention, further comprising: a detection reagent adapted to perform at least one of in situ hybridization, immunohistochemistry, ELISA, western blot, nucleic acid extraction, PCR reaction, nucleic acid sequencing.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
fig. 1 is a cross-sectional view of a collection container according to some embodiments of the present invention, wherein the piercing portion is in an initial state without piercing the pierceable membrane.
Fig. 2 is an enlarged view at D in fig. 1.
Fig. 3 is a cross-sectional view of a collection container according to some embodiments of the present invention, wherein the piercing portion is in a state to pierce the pierceable membrane.
Fig. 4 is an enlarged view at E in fig. 3.
Fig. 5 is a cross-sectional view of a collection container according to further embodiments of the present invention, wherein the puncturing portion is in an initial state without puncturing the pierceable membrane.
Fig. 6 is an enlarged view at F in fig. 5.
Fig. 7 is a cross-sectional view of a collection container according to further embodiments of the present invention, wherein the puncturing portion is in a state of puncturing the pierceable membrane.
Fig. 8 is an enlarged view at G in fig. 7.
Fig. 9 is a partial cross-sectional view of a cover of a collection container according to further embodiments of the present invention.
Fig. 10 is a schematic view of a piercing portion of a collection container according to further embodiments of the present invention.
Fig. 11 is a schematic view of a piercing portion of a collection container according to further embodiments of the present invention.
FIG. 12 is a schematic diagram showing a process of performing a crosslinking immobilization reaction and a decrosslinking reaction on a nucleic acid and a protein according to one embodiment of the present invention.
FIG. 13 is a schematic diagram of the results of human urine specimen collection, immobilization and in situ hybridization assays using a collection container according to one embodiment of the invention.
Fig. 14 is a graph illustrating the results of the fixation and immunohistochemical detection of a human mesenchymal stem cell culture sample using a collection container according to another embodiment of the present invention.
FIG. 15 is a schematic representation of the results of collecting, fixing and PCR testing of a human bronchial brush sample using a collection container according to another embodiment of the present invention.
Reference numerals: the collection container 1, the container main body 100, the first reservoir 101, the opening 102, the lid 200, the second reservoir 201, the liquid injection port 202, the liquid outlet 203, the pierceable membrane 210, the lid body 220, the base 221, the sampling port 2211, the mounting shell 222, the extension portion 230, the liquid passing channel 231, the guide surface 240, the piercing portion 300, the guide groove 310, the stopper 400, the filter 500, the pressing portion 600, the pressing body 610, and the deformable portion 620.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
In one aspect of the invention, the invention features a method of processing a biological sample, the method including, according to an embodiment of the invention: (1) immobilizing a biological sample with an immobilization reagent, the immobilization reagent being adapted to inactivate pathogens in the biological sample and the immobilization reagent being adapted to form cross-linked products with proteins or nucleic acid molecules in the biological sample; and (2) subjecting the immobilized product obtained in step (1) to a decrosslinking treatment using a decrosslinking reagent to decrosslink the crosslinked product so as to obtain the nucleic acid molecule or protein suitable for the detection. Therefore, by adopting the method, the collected biological sample can be fixed by adopting the fixing reagent, and when the reagent carrying aldehyde group is adopted, the sample can be effectively fixed, so that pathogens such as viruses or bacteria in the sample can not have infectivity or toxicity any more, and the operation is safe. In addition, the human body can be effectively prevented from contacting the immobilized reagent, so that the injury or negative effect of the immobilized reagent on the human body is avoided.
According to an embodiment of the present invention, the immobilized reagent contains a reagent carrying an aldehyde group. According to an embodiment of the invention, the reagent carrying aldehyde groups comprises at least one of formaldehyde, glutaraldehyde and paraformaldehyde. According to an embodiment of the invention, the immobilization reagent comprises at least one selected from the group consisting of: (a) a solution containing calcium carbonate and formalin; (b) formalin solution with pH value of 7.0-7.5; (c) a paraformaldehyde solution with a pH of 7.0-7.5; (d) a solution containing glutaraldehyde and formaldehyde; (e) a solution containing formaldehyde, mercuric chloride and sodium acetate; (f) a solution containing acetic acid and formaldehyde; (g) a solution containing picric acid, acetic acid and formaldehyde; (h) a solution containing picric acid and formaldehyde; and (i) a solution containing periodate, lysine, and paraformaldehyde. Thus, the biological sample can be effectively immobilized to kill pathogens, and the resulting treated biological sample can be preserved at room temperature for a long period of time.
According to an embodiment of the invention, the decrosslinking reagent contains at least one of citric acid, citrate, EDTA and Tris. According to an embodiment of the invention, the decrosslinking treatment is carried out at a temperature higher than room temperature. According to the embodiment of the invention, the de-crosslinking treatment is carried out at the temperature of 70-100 ℃. It can be understood by those skilled in the art that the de-crosslinking treatment is to further improve the detection efficiency of the subsequent biological sample, and if the crosslinking reaction does not significantly affect the detection of the subsequent biological sample, the de-crosslinking treatment is not required.
According to an embodiment of the invention, the nucleic acid molecule comprises at least one of DNA and RNA. According to an embodiment of the invention, said nucleic acid sample suitable for said detection contains RNA molecules of length not more than 300 nt. According to an embodiment of the invention, the pathogens include bacteria and viruses. According to an embodiment of the invention, the virus comprises an RNA virus. According to an embodiment of the invention, the RNA virus comprises at least one of a hiv virus, a hepatitis c virus, a encephalitis b virus, an influenza virus, a rhinovirus, a poliovirus, a coxsackie virus, a dengue virus, a rotavirus, a tobacco mosaic virus, a coronavirus, a marburg virus, an ebola virus.
According to an embodiment of the invention, the coronavirus comprises at least one of a SARS virus, a MERS virus and 2019-nCoV. According to an embodiment of the present invention, the biological sample may include at least one of blood, body fluid, tissue fluid, tears, stool, sputum, urine, saliva, semen, nasal discharge, pleural fluid, ascites, prostatic fluid, hair, skin, epithelial tissue. The biological sample may further comprise at least one of alveolar lavage, bladder lavage, gastric lavage, intestinal lavage, uterine lavage, nasal lavage. The biological sample may also include a suspension containing cells, bacteria, fungi or viruses obtained by washing in a liquid after collection of the sample by puncturing, brushing or scraping. The biological sample may also include a cell, bacterial, fungal or viral sample cultured in a liquid. According to an embodiment of the present invention, after step (1), the immobilization-treated product is stored at room temperature before step (2). According to an embodiment of the present invention, after the step (2), further comprising: isolating the nucleic acid molecule from the nucleic acid sample. According to an embodiment of the present invention, after the step (2), further comprising: the nucleic acid molecules are detected by in situ hybridization using a probe.
For ease of understanding, a collection container that can be adapted for use in the method of processing a biological sample of the present invention is described in detail below, and a collection container 1 according to an embodiment of the present invention is described below with reference to the accompanying drawings.
As shown in fig. 1 to 11, a collection container 1 according to an embodiment of the present invention includes a container body 100, a cap body 200, and a puncturing part 300.
The container body 100 defines a first reservoir 101 having an opening 102 therein. The cover body 200 is movably arranged at the opening 102, a second liquid storage cavity 201 is defined in the cover body 200, the cover body 200 is provided with a liquid injection port 202 and a liquid outlet 203 which are communicated with the second liquid storage cavity 201, and the liquid outlet 203 is covered with a pierceable membrane 210. The puncturing part 300 is disposed on the container body 100 or the cap body 200, and the puncturing part 300 is used for puncturing the pierceable film 210 to allow the first reservoir 101 to communicate with the second reservoir 201.
According to the collection container 1 of the embodiment of the present invention, by providing the first reservoir 101, the second reservoir 201 and the piercing part 300, one solution, for example, a collected sample, can be added to the first reservoir 101, and another solution, for example, a fixing solution for fixing a sample component, can be added to the second reservoir 201. After sampling, the pierceable membrane 210 is pierced by the piercing part 300 to communicate the first reservoir 101 with the second reservoir 201, for example, the liquid in the second reservoir 201 can flow into the first reservoir 101, thereby collecting and fixing the sample.
Further, by providing the lid body 200 with the liquid inlet 202 and the liquid outlet 203 communicating with the second liquid storage chamber 201, the liquid outlet 203 is covered with the pierceable film 210. So not only can add liquid to second stock solution chamber 201 through annotating liquid mouth 202, make the liquid in the second stock solution chamber 201 flow in first stock solution chamber 101 through liquid outlet 203, and before adding liquid to second stock solution chamber 201, can carry out the leakproofness test to second stock solution chamber 201 earlier, compare collection container 1 among the correlation technique, can improve the sealing reliability of second stock solution chamber 201, the weeping phenomenon of reagent both has been avoided appearing, can avoid external impurity to get into in the second stock solution chamber 201 again and cause the pollution of reagent, be convenient for improve collection container 1's result of use, be convenient for carry out good protection to the sample.
Meanwhile, because the reagent added into the second liquid storage cavity 201 generally has certain toxicity, compared with the collection container 1 in the related art, when the reagent is added into the second liquid storage cavity 201, the operation process of the operator is simple and convenient, and the operator can not directly contact the fixed liquid reagent harmful to the human body, so that the operation safety of the operator can be improved.
In addition, compared with the collection container 1 in the related art, the collection container 1 provided by the invention can save operators for performing large-area sealing operation on the second liquid storage cavity 201 by using a sealing film in the sampling process, so that the sampling operation process is simplified, the sampling convenience is improved, and the working efficiency of the operators is improved.
For example, human body fluids, cells and tissue samples are important for monitoring health conditions and disease diagnosis, and because DNA, RNA and proteins in clinical samples are easily degraded and contaminated by microorganisms when exposed to air, they need to be fixed in time. In some areas that are far from the hospital or have inconvenient transportation, it would be very convenient if the patient could collect the sample by himself and mail it to the hospital. Since reagents used for the fixation and preservation of clinical specimens are toxic to the human body, it is generally desirable that the reagents are individually sealed before use, and that the reagents are prevented from contacting the human body, and can be conveniently opened when used to mix the reagents with the clinical specimens. After the sample is fixed in time, the sample can be kept stable in the transportation process, and the final detection result is not influenced. Specifically, during the collection of the sample, an operator can add a reagent for fixing the sample into the second reservoir 201 through the liquid injection port 202, and then close the liquid injection port 202 to form a sealed cavity. After adding the sample of gathering in first stock solution chamber 101, utilize puncture portion 300 to puncture pierceable membrane 210, make the reagent of second play liquid intracavity flow into first stock solution chamber 101 through liquid outlet 203 in, reagent can mix with the sample from this, is convenient for fix the composition of sample, improves the chemical stability of sample.
Therefore, the collecting container 1 provided by the embodiment of the invention has the advantages of good sealing effect, reliable work and the like.
A collecting container 1 according to an embodiment of the invention is described below with reference to the drawings.
In some embodiments of the present invention, as shown in fig. 1-11, a collection container 1 according to embodiments of the present invention includes a container body 100, a cap body 200, and a puncturing part 300.
Optionally, the penetrable membrane 210 is integrally formed with at least a portion of the cap body 200. This facilitates the production and processing of the cap body 200, simplifies the assembly process of the cap body 200, and improves the production efficiency of the collection container 1.
In some embodiments, as shown in fig. 2, the collecting container 1 further comprises a blocking member 400, and the blocking member 400 is used for blocking the liquid pouring port 202. After reagent is added into the second liquid storage cavity 201, the liquid injection port 202 can be sealed by the plugging piece 400, so that the second liquid storage cavity 201 forms a sealed space, the reagent in the second liquid storage cavity 201 is conveniently stored in a sealed mode, and the storage effect of the reagent is improved.
Specifically, after the liquid is completely added to the second reservoir 201, the liquid inlet 202 and the block piece 400 may be sealed by heat sealing. Therefore, other materials such as glue can be prevented from being mixed into the second reservoir 201.
Specifically, as shown in fig. 2, the pouring outlet 202 is located on the side surface of the lid body 200 that faces away from the opening 102. This facilitates the operator to smoothly add reagents into the second reservoir 201.
Specifically, the lower side of the lid body 200 is connected to the container main body 100, and the pouring port 202 is located on the upper surface of the lid body 200.
Alternatively, as shown in fig. 2, the liquid outlet 203 is located on a side surface of the cover body 200 facing the opening 102, and a projection of the liquid outlet 203 in a plane of the opening 102 is located on an inner side of the opening 102. This facilitates the reagent in the second reservoir chamber 201 to flow into the first reservoir chamber 101 through the outlet 203.
Specifically, the lower side of the lid body 200 is connected to the container main body 100, and the liquid outlet 203 is located on the lower side of the lid body 200, and the projection in the plane of the opening 102 is located inside the opening 102.
In some embodiments, as shown in fig. 2, the cover body 200 comprises a cover body 220 and an extension part 230, and the second reservoir 201 is formed in the cover body 220. The extension part 230 is disposed on the cover body 220, a liquid passing channel 231 is disposed in the extension part 230, one end of the liquid passing channel 231 is communicated with the second liquid storage cavity 201, a liquid outlet 203 is formed at the other end, and the pierceable membrane 210 is disposed in the liquid passing channel 231. Therefore, the reagent in the second liquid storage cavity 201 is guided conveniently, the setting area of the pierceable film 210 can be reduced, the pierceable film 210 can be processed conveniently, and the structural strength and the sealing reliability of the second liquid storage cavity 201 are further improved conveniently.
Specifically, as shown in fig. 2, the wall surface of the second reservoir chamber 201 has a guide surface 240 extending circumferentially around the liquid passage 231, and the guide surface 240 extends gradually obliquely downward in a direction approaching the liquid passage 231. Therefore, the reagent can be guided by the guide surface 240, the reagent can flow into the first liquid storage cavity 101 quickly, and the efficiency of the whole sampling operation is improved.
Alternatively, as shown in fig. 2, the puncturing part 300 is provided in the cap body 200, and at least a part of the puncturing part 300 is fitted in the liquid passing channel 231. This facilitates the processing and installation of the puncturing part 300, facilitates the positioning and guiding of the movement of the puncturing part 300, improves the accuracy of the movement of the puncturing part 300, and allows the puncturing part 300 to puncture the pierceable film 210 accurately and smoothly.
Further, as shown in fig. 10, at least one guide groove 310 is formed in the piercing part 300 near the extension part 230, and the guide groove 310 extends along the length direction of the piercing part 300. Therefore, the reagent can be guided by the flow guide groove 310, the flow area of the reagent can be increased, and the flow rate of the reagent can be improved.
Specifically, the guide grooves 310 are provided in plural, and the guide grooves 310 are provided at equal intervals in the circumferential direction of the piercing portion 300.
In some embodiments of the present invention, as shown in fig. 2, an extension 230 is disposed on a side of the cover body 220 facing the opening 102, and the extension 230 is adapted to extend from the opening 102 into the first reservoir 101. Therefore, the reagent in the second liquid storage cavity 201 can be accurately drained into the first liquid storage cavity 101, and the reagent and the sample can be better mixed conveniently.
Specifically, the extension part 230 is provided at the lower side of the cover body 220, and the lower end of the extension part 230 extends into the first reservoir chamber 101 from the opening 102.
In other embodiments of the present invention, as shown in fig. 6, the cap body 220 includes a base 221 and a mounting shell 222, the base 221 is disposed at the opening 102 and connected with the container body 100, and the base 221 has a sampling port 2211. The mounting shell 222 is reversibly disposed on the base 221 to open and close the sampling port 2211, the mounting shell 222 defines the second reservoir 201, and the extending portion 230 is disposed on the mounting shell 222 and extends into the base 221. Can carry out the sample collection again and carry out the operation of fixing to the sample after container main body 100 links to each other with lid 200 like this, not only can further simplify the operation process, improve the efficiency of sampling process, can realize the mixture of sample and second stock solution 201 in the first stock solution chamber 101 fast moreover, be convenient for shorten the sample and store the time in first stock solution chamber 101 alone, prevent that the sample from taking place to pollute or the chemical property of sample changes.
Specifically, as shown in fig. 6, the collecting container 1 further includes a filter member 500, and the filter member 500 is disposed in the base 221 and below the liquid outlet 203 (the up-down direction is shown by an arrow a in fig. 1). This allows the filter 500 to be used to filter samples and reagents to remove large particles of contaminants from the samples and reagents.
Specifically, the filter 500 may be a filter screen.
Optionally, the cap 200 is threadably connected to the container body 100. This can improve not only the coupling strength between the cap body 200 and the container body 100 but also the sealing effect at the junction between the cap body 200 and the container body 100.
In some embodiments, as shown in fig. 1, the collection container 1 further comprises a pressing portion 600, the pressing portion 600 is disposed on the cover 200, the pressing portion 600 is connected to the puncturing portion 300, and the pressing portion 600 drives the puncturing portion 300 to puncture the pierceable membrane 210 after being pressed. This not only facilitates the operator to conveniently puncture the pierceable membrane 210 by using the pressing portion 600, but also prevents the pierceable membrane 210 from being pierced by the erroneous operation of the piercing portion 300, thereby further facilitating the improvement of the reliability of the use of the collection container 1.
Specifically, as shown in fig. 2, the pressing portion 600 includes a pressing body 610 and a deformable portion 620, the pressing body 610 is disposed on a side surface of the cover body 200 facing away from the opening 102, and the pressing body 610 is connected to the puncturing portion 300. The deformable portion 620 is located outside the pressing body 610 and surrounds the pressing body 610, and the deformable portion 620 is connected between the cover body 200 and the pressing body. Thus, the operator can conveniently press the membrane, and the pressing part 600 can conveniently drive the puncturing part 300 to move, so that the puncturing part 300 can be conveniently driven to puncture the puncturing membrane 210.
Specifically, the pressing body 610 is provided on the upper surface of the lid body 200.
More specifically, the deformable portion 620 forms a corrugated structure. This facilitates the deformation of the deformable portion 620 by an external force, thereby moving the puncturing part 300.
Specifically, the deformable portion 620 has an initial position and a deformed position, the deformable portion 620 moves between the initial position and the deformed position to move the puncturing part 300, and a movable distance of the deformable portion 620 between the initial position and the deformed position in a moving direction of the puncturing part 300 is greater than a distance between the puncturing part 300 and the pierceable membrane 210 and is smaller than a distance between the puncturing part 300 and the filter 500.
Optionally, the pressing portion 600 further includes a limiting portion, which can limit the moving distance of the deformable portion 620, so as to prevent the piercing portion 300 from piercing the filter 500.
Alternatively, the pressing portion 600 and the puncturing portion 300 may be integrally formed with at least a part of the cap body 200. Therefore, the production and processing of the pressing part 600, the puncturing part 300 and the cover body 200 are facilitated, the assembly process of the collecting container 1 is simplified, and the production efficiency of the collecting container 1 is improved.
Specifically, the container body 100 may be a 50ml or 15ml centrifuge tube.
Optionally, the collection container 1 further includes a sealing upper cover, and after the sample in the first reservoir 101 is mixed with the reagent in the second reservoir 201, the cover 200 may be removed to connect the sealing upper cover to the container body 100 in a sealing manner.
The use of the collecting container 1 according to an embodiment of the invention is described below with reference to the drawings.
According to some embodiments of the present invention, when a sample needs to be collected, a sample is first added into the container body 100, then the container body 100 and the cover 200 are mounted together, and the pressing portion 600 is pressed to drive the puncturing portion 300 to puncture the pierceable membrane 210, so that the reagent in the second reservoir 201 flows into the first reservoir 101, and the mixing of the sample and the reagent is completed, thereby fixing the sample.
According to other embodiments of the present invention, when a sample needs to be collected, the container body 100 and the cover body 200 are mounted together, then the mounting shell 222 is turned over and opened, the sample is added into the container body 100 through the sampling port 2211, the mounting shell 222 is fastened, the pressing portion 600 is pressed to drive the puncturing portion 300 to puncture the pierceable membrane 210, so that the reagent in the second liquid storage cavity 201 flows into the first liquid storage cavity 101, and the mixing of the sample and the reagent is completed, thereby fixing the sample.
A sample collection kit according to an embodiment of the present invention is described below. A sample collection kit according to an embodiment of the invention comprises a collection container 1 according to the above-described embodiment of the invention, a pipette, a collection cup, a glove, facial tissue, and wet tissue.
Other constructions and operations of the collecting container 1 according to embodiments of the present invention are known to a person skilled in the art and will not be described in detail here.
According to some embodiments of the invention, a fixed reagent is disposed in the second reservoir chamber, the fixed reagent comprising a reagent carrying an aldehyde group. Therefore, in the using process, the fixing reagent can be used for fixing the collected biological sample, and when the reagent carrying aldehyde groups is adopted, the sample can be effectively fixed, so that pathogens such as viruses or bacteria in the sample can not have infectivity or toxicity any more, and the operation is safe. In addition, through adopting this collection container, can avoid human contact fixed reagent effectively to avoid fixed reagent to the injury or the negative effects of human body. According to an embodiment of the invention, the pathogens include bacteria and viruses. According to an embodiment of the invention, the virus comprises an RNA virus. According to an embodiment of the invention, the RNA virus comprises at least one of a hiv virus, a hepatitis c virus, a encephalitis b virus, an influenza virus, a rhinovirus, a poliovirus, a coxsackie virus, a dengue virus, a rotavirus, a tobacco mosaic virus, a coronavirus, a marburg virus, an ebola virus. According to an embodiment of the invention, the coronavirus comprises at least one of a SARS virus, a MERS virus and 2019-nCoV.
Generally, nucleic acids, particularly RNA, contained in viruses are easily degraded, and in many cases, there are no conditions under which RNA detection is not performed immediately in many cases, and viral nucleic acids can be protected from degradation to a certain extent by using the collection container according to an embodiment of the present invention. In general, RNA is preserved at a low temperature such as liquid nitrogen, or by adding a protective solution. The existing RNA protective solution can be stored for only 3-7 days at normal temperature, so that the RNA protective solution is not suitable for long-distance transportation and can not be used for long-term storage of a sample bank. In addition, many viruses are extremely transmissible or infectious, and require very strict precautions to avoid infection, thus the virus sampling, nucleic acid extraction and analysis processes are complicated to operate. The collection container of the present invention can provide a simple, safe and effective method for inactivating and preserving pathogens, such as viruses, which can provide great help for medical staff and researchers. According to the embodiment of the present invention, the fixing agent provided in the collection container can effectively and rapidly inactivate viruses, and can preserve viral nucleic acids at normal temperature for a long period of time, for example, several months or even years.
According to some embodiments of the invention, the reagent bearing aldehyde groups comprises at least one selected from the group consisting of formaldehyde, glutaraldehyde and paraformaldehyde. Thus, the nucleic acid or protein can be protected by effectively causing the nucleic acid or protein to undergo a cross-linking reaction with the fixing agent using the fixing agent, and can be stored for a long period of time, for example, at room temperature for at least one month. These cross-linked products can subsequently be subjected to a de-cross-linking treatment by a de-cross-linking reagent in order to detect the corresponding nucleic acids or proteins.
It should be noted that, according to the embodiments of the present invention, the biological sample that can be used is preferably a liquid biological sample, including but not limited to blood, body fluid, tissue fluid, tears, stool, sputum, urine, saliva, semen, nasal discharge, pleural fluid, ascites, prostatic fluid, hair, skin, epithelial tissue, etc., lavage fluid for alveoli, bladder, stomach, intestine, uterus or nasal cavity, a suspension containing cells, bacteria, fungi or viruses obtained by collecting a sample by puncturing, brushing or scraping in a liquid, and a cell, bacteria, fungi or virus sample cultured in a liquid.
In a second aspect of the invention, the invention proposes a kit comprising, according to an embodiment of the invention: the collection container as described above, and a fixed reagent disposed in the second reservoir chamber of the collection container. As mentioned above, the kit can be used for biological sample collection and immobilization treatment, so that the immobilized reagent can perform immobilization treatment on the collected biological sample in the using process, and when the reagent carrying aldehyde group is used, the sample can be effectively immobilized, so that pathogens such as viruses or bacteria in the sample can not have infectivity or toxicity any more, and the operation is safe. In addition, through adopting this collection container, can avoid human contact fixed reagent effectively to avoid fixed reagent to the injury or the negative effects of human body. It should be noted that the features and advantages described above in relation to the collection container apply equally to the kit and are not described in further detail here.
According to an embodiment of the present invention, the kit may further include: a de-crosslinking reagent comprising at least one of citric acid, citrate, EDTA, and Tris. Referring to FIG. 12, these cross-linked products may be subjected to a de-cross-linking treatment by a de-cross-linking reagent to detect the corresponding nucleic acids or proteins.
According to an embodiment of the present invention, further comprising: a detection reagent adapted to perform at least one of in situ hybridization, immunohistochemistry, ELISA, western blot, nucleic acid extraction, PCR reaction, nucleic acid sequencing.
According to an embodiment of the present invention, the viral nucleic acid may be preserved by using a formaldehyde solution, such as formalin. After formalin is added to a sample, the formaldehyde molecules form cross-links between nucleic acids and proteins, effectively inactivating viruses, and preventing nucleic acid degradation. Further, the nucleic acid sample may be preserved in formalin for long-distance transportation or long-term preservation. In addition, according to the embodiment of the present invention, when RNA detection is required, the precipitate may be collected by high-speed centrifugation, the supernatant may be discarded, and then the sample may be subjected to high-temperature (70-100 ℃) treatment using the aforementioned decrosslinking reagent, and the crosslinks between nucleic acids and proteins may be opened. In addition, the nucleic acid can be extracted by a conventional Trizol method or other methods, or can be directly subjected to in situ hybridization detection.
When the kit provided by the embodiment of the invention is used for processing the sample, the operation is simple and easy, special training is not required, and the nucleic acid, particularly RNA, can be stored for a long time at normal temperature. Conventional methods can be used for extraction and detection of nucleic acids, particularly RNA, after being subjected to decrosslinking. In addition, formalin can effectively inactivate viruses, and the de-crosslinking operation is performed at high temperature without contacting active viruses, so that the method can be performed in a common biological laboratory without special protection.
The use of the collection container of the present invention is illustrated and explained below by specific examples, which are intended to be illustrative and not limiting as to the scope of the invention.
EXAMPLE 1 Collection, immobilization and in situ hybridization assay of human urine samples
Adding a fixing reagent containing formaldehyde and PBS into a fixing solution storage box (a second liquid storage cavity) of the collection container, collecting urine, pouring the urine into a collection pipe of the fixing device through a funnel structure, covering the fixing solution storage box, and pressing a button to release the fixing solution. And after the stationary liquid is released, unscrewing the stationary liquid storage box, covering the cover tightly, turning upside down and uniformly mixing, and storing at room temperature.
After 24 hours of storage, the urine samples were mixed well and a portion of the samples were removed, and the remaining samples were stored at room temperature. The collection tube containing the urine sample is put into a centrifuge for 10000 Xg centrifugation for 30 minutes, the liquid is discarded, and the cell sediment is collected. PBS is added into the cell sediment for centrifugal washing twice, the cells are resuspended by PBS, the cell suspension is dripped on a glass slide with positive charge, and the glass slide is dried at 40 ℃.
And (3) adopting a probe aiming at the GAPDH gene intron sequence, carrying out pretreatment and in-situ hybridization on the sample according to the operation method of an RNAscope kit, carrying out signal amplification and hematoxylin nucleus staining, and analyzing the expression condition of the gene by using microscope imaging.
After one month of storage, the cells in the remaining samples were collected and subjected to in situ hybridization in the same manner as described above, and the expression of the genes was analyzed by microscopic imaging. The results are shown in FIG. 13, in which FIG. 13(a) is the expression of the gene in the urine sample stored for 24 hours, and FIG. 13(b) is the expression of the gene in the urine sample stored for one month. As shown in fig. 13, the signal of each allele expression appears as a red dot in the nucleus, and the expression of GAPDH gene was detected in most nuclei in the samples stored for 24 hours and one month, indicating that the RNA molecules in the samples were well fixed after 24 hours and not significantly degraded within one month.
Example 2 fixation and immunohistochemical detection of human mesenchymal Stem cell culture samples
Culturing human mesenchymal stem cells in MEM medium, removing the medium, suspending the cells with trypsin, terminating the trypsin reaction with serum-containing medium, and centrifuging to collect cell precipitate. PBS was added to the cell pellet and washed twice by centrifugation, and the cells were resuspended in PBS. Using a 15ml sample collection and fixation device with the same fixation reagents as in example 1 in the fixative reservoir, the cell suspension was added to the collection tube, the fixative reservoir was closed, and the fixative was released by pressing the button. And after the stationary liquid is released, unscrewing the stationary liquid storage box, covering the cover tightly, turning upside down and uniformly mixing, and storing at room temperature.
After 24 hours of storage, the cell samples were mixed and a portion of the sample was removed and the remaining samples were stored at room temperature. The collection tube containing the cell sample is placed in a centrifuge for centrifugation to collect the cell pellet. PBS is added into the cell sediment for centrifugal washing twice, the cells are resuspended by PBS, the cell suspension is dripped on a glass slide with positive charge, and the glass slide is dried at 40 ℃.
Treating a sample slide in 10mM sodium citrate solution at 95 ℃ for 10 minutes to untie cross-linking between proteins, incubating the sample slide for 1 hour at room temperature by using 5% BSA to block a cell sample, then adding an alpha-SMA antibody, incubating the sample slide for 16 hours at 4 ℃, washing the antibody by using PBS, adding a secondary antibody with HRP, incubating the sample slide for 1 hour at room temperature, washing the antibody by using PBS, adding DAB color developing solution, staining cell nuclei by using hematoxylin after color development is finished, and analyzing the expression condition of the alpha-SMA protein by using microscope imaging.
After one month of storage, cells from the remaining samples were collected and tested for immunohistochemistry in the same manner as described above, and the expression of α -SMA protein was analyzed by microscopic imaging. The results are shown in FIG. 14, in which FIG. 14(a) shows the expression of the gene in the cell sample stored for 24 hours, and FIG. 14(b) shows the expression of the gene in the cell sample stored for one month. As shown in FIG. 14, the α -SMA protein in the cytoplasm was stained brown, and in most cells, the α -SMA protein was detected in the samples stored for 24 hours and one month, indicating that the protein molecules in the samples could be well immobilized after 24 hours and no significant degradation occurred within one month.
Example 3 fixation and PCR detection of human bronchial Brush cell samples
Lung tumor cells are brushed from the trachea of a lung cancer patient by using a bronchus brush, 5ml of erythrocyte lysate is added into a collection pipe of a 15ml sample collection and fixing device in advance, and the brush head is rinsed in the liquid for several times and then taken out. The fixative solution storage box was covered and the same fixative solution as in example 1 was released by pressing the button. And after the stationary liquid is released, unscrewing the stationary liquid storage box, covering the cover tightly, turning upside down and uniformly mixing, and storing at room temperature.
After 24 hours of storage, the samples were mixed and a portion of the samples were removed and the remaining samples were stored at room temperature. The collection tube containing the cell sample is placed in a centrifuge for centrifugation to collect the cell pellet. PBS was added to the cell pellet and washed twice by centrifugation. The cross-linking between DNA and protein was broken by treatment in 10mM sodium citrate solution at 95 ℃ for 10 minutes. Use of
Genomic DNA was extracted from the QIAamp DNA FFPE Tissue DNA extraction kit, and then PCR was performed on different fragments of the EGFR gene using primers for the EGFR gene. The PCR product was subjected to agarose gel electrophoresis.
After one month of storage, the cells in the remaining sample were collected and genomic DNA was extracted for PCR in the same manner as described above. The results are shown in FIG. 15, in which FIG. 15(a) shows the expression of the gene in the sample stored for 24 hours, and FIG. 15(b) shows the expression of the gene in the sample stored for one month. As shown in FIG. 15, the amplification product of EGFR gene was detected in most of the samples stored for 24 hours and one month, indicating that the DNA molecules in the samples were well immobilized after 24 hours and were not significantly degraded within one month.
In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the invention and to simplify the description, and are not intended to indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and are therefore not to be considered limiting of the invention. Furthermore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless otherwise specified. In the description of the present invention, the first feature being "on" or "under" the second feature may include the first and second features being in direct contact, and may also include the first and second features being in contact with each other not directly but through another feature therebetween.
In the description of the invention, "above", "over" and "above" a first feature in a second feature includes the first feature being directly above and obliquely above the second feature, or simply means that the first feature is higher in level than the second feature.
In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples" or the like mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (39)
1. A method of processing a biological sample, comprising:
(1) immobilizing a biological sample with an immobilization reagent, the immobilization reagent being adapted to inactivate pathogens in the biological sample and the immobilization reagent being adapted to form cross-linked products with proteins or nucleic acid molecules in the biological sample; and
(2) subjecting the immobilized product obtained in step (1) to a decrosslinking treatment using a decrosslinking reagent to decrosslink the crosslinked product to obtain the nucleic acid molecule or protein suitable for the detection.
2. The method of claim 1, wherein the immobilized reagent comprises a reagent carrying an aldehyde group.
3. The method of claim 2, wherein the aldehyde group-bearing reagent comprises at least one of formaldehyde, glutaraldehyde, and paraformaldehyde.
4. The method of claim 2, wherein the immobilizing reagent comprises at least one selected from the group consisting of:
(a) a solution containing calcium carbonate and formalin;
(b) formalin solution with pH value of 7.0-7.5;
(c) a paraformaldehyde solution with a pH of 7.0-7.5;
(d) a solution containing glutaraldehyde and formaldehyde;
(e) a solution containing formaldehyde, mercuric chloride and sodium acetate;
(f) a solution containing acetic acid and formaldehyde;
(g) solution containing picric acid, acetic acid and formaldehyde
(h) A solution containing picric acid and formaldehyde; and
(i) a solution comprising periodate, lysine and paraformaldehyde.
5. The method of claim 1, wherein the de-crosslinking reagent comprises at least one of citric acid, citrate, EDTA, and Tris.
6. The method according to claim 1, wherein the decrosslinking treatment is performed at a temperature higher than room temperature.
7. The method according to claim 1, wherein the decrosslinking treatment is performed at a temperature of 70 to 100 ℃.
8. The method of claim 1, wherein the nucleic acid molecule comprises at least one of DNA and RNA.
9. The method of claim 8, wherein said nucleic acid sample suitable for said detecting contains RNA molecules no more than 300nt in length.
10. The method of claim 8, wherein the pathogens comprise bacteria and viruses.
11. The method of claim 10, wherein the virus comprises an RNA virus.
12. The method of claim 11, wherein the RNA virus comprises at least one of a hiv virus, a hepatitis c virus, a encephalitis b virus, an influenza virus, a rhinovirus, a poliovirus, a coxsackievirus, a dengue virus, a rotavirus, a tobacco mosaic virus, a coronavirus, a marburg virus, an ebola virus.
13. The method of claim 12, wherein the coronavirus comprises at least one of SARS virus, MERS virus, and 2019-nCoV.
14. The method of claim 1, wherein the biological sample comprises at least one of lavage fluid of alveoli or bladder or stomach or intestine or uterus or nasal cavity, suspension containing cells or bacteria or fungi or viruses obtained by rinsing in liquid after collection of the sample by puncturing or brushing or scraping, cells or bacteria or fungi or viruses cultured in liquid, blood, body fluid, interstitial fluid, tears, stool, sputum, urine, saliva, semen, nasal discharge, pleural fluid, ascites, prostatic fluid, hair, skin, epithelial tissue.
15. The method according to claim 1, wherein after step (1) and before step (2), the immobilized product is stored at room temperature.
16. The method of claim 15, further comprising, after step (2):
isolating the nucleic acid molecule from the nucleic acid sample.
17. The method of claim 1, further comprising, after step (2):
the nucleic acid molecules are detected by in situ hybridization using a probe.
18. A collection container, comprising:
a container body defining a first reservoir chamber therein having an opening;
the cover body is movably arranged at the opening, a second liquid storage cavity is defined in the cover body, the cover body is provided with a liquid injection port and a liquid outlet which are communicated with the second liquid storage cavity, and a pierceable film covers the liquid outlet;
the puncture part is arranged on the container body or the cover body and used for puncturing the pierceable film so as to allow the first liquid storage cavity to be communicated with the second liquid storage cavity.
19. The collection container of claim 18, wherein the pierceable membrane is integrally formed with at least a portion of the cap.
20. A collection container according to claim 18, further comprising a closure for closing the pour opening.
21. The collection container according to claim 18, wherein the pouring outlet is located on a side surface of the lid body facing away from the opening.
22. The collection container according to claim 18, wherein the liquid outlet is located on a side surface of the lid body facing the opening, and a projection of the liquid outlet in a plane of the opening is located inside the opening.
23. The collection container of claim 18, wherein the cap comprises:
the second liquid storage cavity is formed in the cover body;
the extension portion is arranged on the cover body, a liquid passing channel is arranged in the extension portion, one end of the liquid passing channel is communicated with the second liquid storage cavity, the other end of the liquid passing channel forms the liquid outlet, and the pierceable membrane is arranged in the liquid passing channel.
24. The collection container of claim 23, wherein the wall of the second reservoir has a guide surface extending circumferentially around the flow channel, the guide surface extending gradually downwardly in a direction approaching the flow channel.
25. The collection container of claim 23, wherein the piercing portion is disposed on the cap, and at least a portion of the piercing portion fits within the liquid-passing channel.
26. The collection container of claim 23, wherein the piercing portion is provided with at least one channel adjacent to the extension portion, the channel extending along the length of the piercing portion.
27. The collection container of any one of claims 23-26, wherein the extension is disposed on a side of the cap body facing the opening, the extension adapted to extend from the opening into the first reservoir.
28. The collection container of any one of claims 23-26, wherein the cap body comprises:
the base is arranged at the opening and is connected with the container main body, and the base is provided with a sampling port;
the installation shell is arranged on the base in a turnover mode so as to open and close the sampling port, the installation shell limits the second liquid storage cavity, and the extending portion is arranged on the installation shell and extends into the base.
29. The collection container of claim 28, further comprising a filter element disposed within the base and below the liquid outlet.
30. The collection container of any one of claims 18-27, wherein the cap is threadably connected to the container body.
31. The collection container of claim 18, further comprising:
and the pressing part is arranged on the cover body and is connected with the puncture part, and the pressing part drives the puncture part to puncture the puncture membrane after being pressed down.
32. The collection container of claim 31, wherein the press portion comprises:
the pressing body is arranged on the surface of one side of the cover body, which is far away from the opening, and the pressing body is connected with the puncture part;
a deformable portion located outside the pressing body and surrounding the pressing body, the deformable portion being connected between the cover and the pressing body.
33. The collection container of claim 32, wherein the deformable portion forms a corrugated structure.
34. The collection container of claim 31, wherein the pressing portion, the puncturing portion, and at least a portion of the cap are integrally formed.
35. The collection container of claim 18, wherein a fixed reagent is disposed in the second reservoir, the fixed reagent comprising a reagent carrying an aldehyde group.
36. The collection container of claim 35, wherein the aldehyde group-bearing reagent comprises at least one selected from the group consisting of formaldehyde, glutaraldehyde and paraformaldehyde.
37. A kit, comprising:
the collection container of any one of claims 18-36, and
a fixed reagent disposed in the second reservoir chamber of the collection container.
38. The kit of claim 37, further comprising:
a de-crosslinking reagent comprising at least one of citric acid, citrate, EDTA, and Tris.
39. The kit of claim 37, further comprising:
a detection reagent adapted to perform at least one of in situ hybridization, immunohistochemistry, ELISA, western blot, nucleic acid extraction, PCR reaction, nucleic acid sequencing.
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| CN202010377054.5A CN113621682A (en) | 2020-05-07 | 2020-05-07 | Biological sample processing method, collection container and kit |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010377054.5A CN113621682A (en) | 2020-05-07 | 2020-05-07 | Biological sample processing method, collection container and kit |
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