CN115747303A - Sample preservation solution, reagent and sample preservation method - Google Patents
Sample preservation solution, reagent and sample preservation method Download PDFInfo
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- CN115747303A CN115747303A CN202211491896.9A CN202211491896A CN115747303A CN 115747303 A CN115747303 A CN 115747303A CN 202211491896 A CN202211491896 A CN 202211491896A CN 115747303 A CN115747303 A CN 115747303A
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- 238000004321 preservation Methods 0.000 title claims abstract description 80
- 239000003761 preservation solution Substances 0.000 title claims abstract description 78
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 10
- 238000000034 method Methods 0.000 title claims abstract description 9
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims abstract description 40
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical group C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims abstract description 40
- 229960003237 betaine Drugs 0.000 claims abstract description 40
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000000243 solution Substances 0.000 claims abstract description 29
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 23
- 230000000415 inactivating effect Effects 0.000 claims abstract description 21
- GHXZTYHSJHQHIJ-UHFFFAOYSA-N Chlorhexidine Chemical compound C=1C=C(Cl)C=CC=1NC(N)=NC(N)=NCCCCCCN=C(N)N=C(N)NC1=CC=C(Cl)C=C1 GHXZTYHSJHQHIJ-UHFFFAOYSA-N 0.000 claims abstract description 19
- 229960003260 chlorhexidine Drugs 0.000 claims abstract description 19
- 239000003093 cationic surfactant Substances 0.000 claims abstract description 8
- 239000003381 stabilizer Substances 0.000 claims abstract description 6
- 239000006172 buffering agent Substances 0.000 claims abstract description 3
- 239000000203 mixture Substances 0.000 claims abstract description 3
- 239000008227 sterile water for injection Substances 0.000 claims description 26
- 239000000523 sample Substances 0.000 claims description 25
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 12
- 239000000872 buffer Substances 0.000 claims description 9
- 239000012530 fluid Substances 0.000 claims description 8
- 150000003856 quaternary ammonium compounds Chemical group 0.000 claims description 8
- 239000008363 phosphate buffer Substances 0.000 claims description 5
- OJIYIVCMRYCWSE-UHFFFAOYSA-M Domiphen bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CCOC1=CC=CC=C1 OJIYIVCMRYCWSE-UHFFFAOYSA-M 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 claims description 4
- 239000012488 sample solution Substances 0.000 claims description 4
- 229960002233 benzalkonium bromide Drugs 0.000 claims description 3
- 229960000686 benzalkonium chloride Drugs 0.000 claims description 3
- KHSLHYAUZSPBIU-UHFFFAOYSA-M benzododecinium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 KHSLHYAUZSPBIU-UHFFFAOYSA-M 0.000 claims description 3
- CADWTSSKOVRVJC-UHFFFAOYSA-N benzyl(dimethyl)azanium;chloride Chemical compound [Cl-].C[NH+](C)CC1=CC=CC=C1 CADWTSSKOVRVJC-UHFFFAOYSA-N 0.000 claims description 3
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 2
- 229960001950 benzethonium chloride Drugs 0.000 claims description 2
- UREZNYTWGJKWBI-UHFFFAOYSA-M benzethonium chloride Chemical compound [Cl-].C1=CC(C(C)(C)CC(C)(C)C)=CC=C1OCCOCC[N+](C)(C)CC1=CC=CC=C1 UREZNYTWGJKWBI-UHFFFAOYSA-M 0.000 claims description 2
- 229960001927 cetylpyridinium chloride Drugs 0.000 claims description 2
- YMKDRGPMQRFJGP-UHFFFAOYSA-M cetylpyridinium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 YMKDRGPMQRFJGP-UHFFFAOYSA-M 0.000 claims description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 2
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- 210000002381 plasma Anatomy 0.000 claims description 2
- 210000002966 serum Anatomy 0.000 claims description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 2
- 210000002700 urine Anatomy 0.000 claims description 2
- 239000003599 detergent Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 27
- 230000001954 sterilising effect Effects 0.000 abstract description 11
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 10
- 244000000010 microbial pathogen Species 0.000 abstract description 8
- 239000003755 preservative agent Substances 0.000 abstract description 5
- 230000002335 preservative effect Effects 0.000 abstract description 5
- 239000002280 amphoteric surfactant Substances 0.000 abstract description 4
- 230000015556 catabolic process Effects 0.000 abstract description 4
- 238000006731 degradation reaction Methods 0.000 abstract description 4
- 208000015181 infectious disease Diseases 0.000 abstract description 4
- 239000000839 emulsion Substances 0.000 abstract description 2
- 230000002779 inactivation Effects 0.000 description 25
- 230000000052 comparative effect Effects 0.000 description 18
- 238000002360 preparation method Methods 0.000 description 13
- 230000000813 microbial effect Effects 0.000 description 10
- 238000001514 detection method Methods 0.000 description 9
- 238000003860 storage Methods 0.000 description 6
- 230000000937 inactivator Effects 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 244000052769 pathogen Species 0.000 description 4
- 230000001717 pathogenic effect Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 239000008055 phosphate buffer solution Substances 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 229940027983 antiseptic and disinfectant quaternary ammonium compound Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000002563 ionic surfactant Substances 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- 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
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a sample preservation solution, a reagent and a method for preserving a sample, and relates to the technical field of biological engineering. It comprises the following steps: the water-soluble emulsion comprises a stabilizing agent, an inactivating agent, a buffering agent and water, wherein the stabilizing agent is selected from betaine, and the inactivating agent is selected from one or a mixture of at least two of chlorhexidine and cationic surfactants. The sample preservative solution provided by the invention can reduce the infection and degradation risks of a sample to be detected and can effectively inactivate pathogenic microorganisms. The betaine is an amphoteric surfactant, has a good sterilization effect, and can be matched with an inactivating agent to synergistically improve the sterilization effect.
Description
Technical Field
The invention relates to the technical field of biological engineering, in particular to a sample preservation solution, a reagent and a sample preservation method.
Background
The traditional detection methods of pathogenic microorganisms mainly comprise morphological detection, microorganism culture, smear microscopy, antigen-antibody detection and nucleic acid detection, which are still widely applied clinically, however, the detection methods in clinical laboratories have different limitations due to own methodologies. With the development of molecular biology technology, pathogenic molecular diagnosis is gradually applied to clinical application.
Pathogenic metagenome sequencing (mNGS) is a high-throughput sequencing technology which directly extracts nucleic acid from a specimen of an infected focus part of a patient to realize pathogen species identification without depending on culture or presetting pathogens. Compared with the traditional clinical laboratory detection method, the pathogenic mNGS detects the nucleic acid sequence of pathogenic microorganisms based on the nucleic acid level, can break through the limitations of different pathogen types, comprehensively covers thousands or even tens of thousands of pathogens without bias, and simultaneously identifies various pathogenic microorganisms such as bacteria, fungi, viruses, tuberculosis, parasites and the like.
Because the sample to be detected contains certain pathogenic microorganisms, and certain infection and degradation risks exist due to the fact that the sample needs to be stored and transported under objective conditions after being collected, the development of a storage solution which is beneficial to storage and can effectively inactivate the pathogenic microorganisms is imperative.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a sample preservation solution, a reagent and a sample preservation method so as to be convenient for preservation and transportation of a sample to be detected.
The invention is realized by the following steps:
in a first aspect, the present invention provides a sample preservation solution comprising: the water-soluble emulsion comprises a stabilizing agent, an inactivating agent, a buffering agent and water, wherein the stabilizing agent is selected from betaine, and the inactivating agent is selected from one or a mixture of at least two of chlorhexidine and cationic surfactants.
The betaine is an amphoteric surfactant, has a good sterilization effect, and can be matched with an inactivating agent to synergistically improve the sterilization effect. Moreover, betaines and inactivators are preferred because they are effective at killing microbial cells at lower concentrations. Further, for the inactivation treatment of microbial cells, chlorhexidine and a cationic surfactant may be used alone or, if necessary, two or more thereof may be combined and mixed together.
The buffer is used to adjust the pH of the sample preservation solution.
In a preferred embodiment of the present invention, the concentration of betaine in the sample preservation solution is 1-10% (w/v); at too low a concentration, the desired effective effect of inactivating the microbial cells is not achieved. On the other hand, if the concentration is too high, problems such as waste liquid treatment after treatment, inactivation of industrial enzymes, etc. are caused, so in view of the stability of the microbial cells used, the inventors have screened the optimum concentration of betaine, and found that the microbial cells can be effectively inactivated at the above concentration range, and the stability of the sample at-20 ℃ to 4 ℃ can be maintained, particularly the stability of the sample at-20 ℃ and 4 ℃.
For example, the concentration of betaine in the sample preservation solution is 1%,2%,3%,4%,5%,6%,7%,8%,9% or 10%.
In an alternative embodiment, the concentration of betaine in the sample preservation solution is 5-10% (w/v). Under the concentration, the sample preservation solution has good preservation effect at the temperature of minus 20 ℃ and 4 ℃, and the sample has good stability and inactivation effect.
In a preferred embodiment of the present invention, the concentration of the inactivating agent in the sample preservation solution is 0.01-0.1% (w/v).
When an ionic surfactant is used, the addition of this solution at high concentrations during the inactivation treatment may cause bacterial lysis, rupture and aggregation of the microbial cells. It is effective to add the surfactant at a low concentration.
In an alternative embodiment, the concentration of the inactivating agent in the sample preservation solution is 0.05-0.1% (w/v). Slightly increasing the concentration of the inactivating agent in the sample preservation solution helps to improve the inactivating effect of the sample preservation solution.
In a preferred embodiment of the present invention, the cationic surfactant is a quaternary ammonium compound.
In an alternative embodiment, the quaternary ammonium compounds include, without limitation: at least one of benzalkonium chloride, benzalkonium bromide, benzethonium chloride, cetylpyridinium chloride, dodecyldimethyl phenoxyethyl ammonium bromide and hexadecyl trimethyl ammonium bromide.
In a preferred embodiment of the present invention, the buffer is selected from phosphate buffer;
in an alternative embodiment, the phosphate buffer is selected from a disodium hydrogen phosphate solution, a sodium dihydrogen phosphate solution, a dipotassium hydrogen phosphate solution, or a potassium dihydrogen phosphate solution.
In a preferred embodiment of the present invention, the pH of the sample storage solution is 6.5 to 8.0. The inventor proves that the pH of the sample preservation solution has little influence on the stability of the sample solution, but has obvious influence on the inactivation effect. Has good inactivation effect under the pH condition.
In an alternative embodiment, the sample preservation solution has a pH of 7.0-8.0, which is more effective in inactivation.
In an alternative embodiment, the sample preservation solution has a pH of 7.5 to 7.8, which is more effective in inactivation.
In an alternative embodiment, the water of the sample preservation solution is sterile water for injection.
In an alternative embodiment, the sample is blood, serum, plasma or urine.
In a preferred embodiment of the present invention, the specimen preservation solution includes: 1 to 10 percent of betaine, 0.01 to 0.05 percent of chlorhexidine and the balance of sterilized water for injection, and the pH value of the sample preservation solution is adjusted to 7.5 by a buffer. Under the condition, the inactivation effect of the sample preservation solution is good, and the stability of the sample solution after the sample preservation solution is added is good.
In an alternative embodiment, the sample preservation solution comprises: 1-10% of betaine, 0.01-0.1% of quaternary ammonium compound and the balance of sterilized water for injection, and the pH value of the sample preservation solution is adjusted to 7.5 by using a buffer. Under the condition, the inactivation effect of the sample preservation solution is good, and the stability of the sample solution after the sample preservation solution is added is good.
In an alternative embodiment, the sample preservation solution comprises: betaine with the concentration of 1-10%, chlorhexidine with the concentration of 0.01-0.05%, quaternary ammonium compound with the concentration of 0.01-0.05% and the balance of water for sterilization and injection, and the pH value of the sample preservation solution is adjusted to 7.5 by a buffer.
In a second aspect, the present invention also provides a reagent or a kit, wherein the reagent or the kit contains the above-mentioned sample preservation solution. The sample preservation solution provided by the invention can be used for a detection reagent or a kit.
In a third aspect, the invention also provides a sample liquid, which comprises a sample and the sample preservation liquid.
In a fourth aspect, the present invention also provides a method for preserving a sample, comprising the steps of: the sample to be preserved is mixed with the above-mentioned sample preservation solution.
In an alternative embodiment, the preservation of the sample is performed at-20 ℃ to 4 ℃. For example, the temperature is between-20 ℃ and-4 ℃.
The invention has the following beneficial effects:
the sample preservative solution provided by the invention can reduce the infection and degradation risks of a sample to be detected and can effectively inactivate pathogenic microorganisms. The betaine is an amphoteric surfactant, has a good sterilization effect, and can be matched with an inactivating agent to synergistically improve the sterilization effect. Moreover, the betaine and the inactivator can effectively kill the microbial cells at lower concentration. Further, for the inactivation treatment of microbial cells, chlorhexidine and a cationic surfactant may be used alone or in combination of two or more thereof.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a sample preservation solution, and the preparation method comprises the following steps: taking a measuring flask with 1g of betaine and 0.01g to 100ml of chlorhexidine, adding a proper amount of water to dissolve, adding water to a scale, uniformly mixing, and adjusting the pH value to 7.5 by using a disodium hydrogen phosphate solution.
Example 2
The embodiment provides a sample preservation solution, and the preparation method comprises the following steps: taking a measuring flask with the volume of 1g of betaine and 0.01g to 100ml of chlorhexidine, adding a proper amount of sterile water for injection to dissolve, adding the sterile water for injection to scale, uniformly mixing, and adjusting the pH value to 7.5 by using a disodium hydrogen phosphate solution.
Example 3
The embodiment provides a sample preservation solution, and the preparation method comprises the following steps: taking 10g of betaine and 0.01g to 100ml of chlorhexidine, adding a proper amount of sterile water for injection to dissolve, adding the sterile water for injection to a scale, uniformly mixing, and adjusting the pH value to 7.5 by using a disodium hydrogen phosphate solution.
Example 4
The embodiment provides a sample preservation solution, and the preparation method comprises the following steps: taking 10g of betaine and 0.05g of chlorhexidine to a 100ml measuring flask, adding a proper amount of sterile water for injection to dissolve, adding the sterile water for injection to scale, uniformly mixing, and adjusting the pH value to 7.5 by using a disodium hydrogen phosphate solution.
Example 5
The embodiment provides a sample preservation solution, and the preparation method comprises the following steps: taking 10g of betaine and 0.05g of chlorhexidine to 100ml measuring flask, adding a proper amount of sterile water for injection for dissolution, adding the sterile water for injection to scale, mixing uniformly, and adjusting the pH value to 7.0 by using a phosphate buffer solution.
Example 6
The embodiment provides a sample preservation solution, and the preparation method comprises the following steps: taking 10g of betaine and 0.05g of chlorhexidine to a 100ml measuring flask, adding a proper amount of sterile water for injection to dissolve, adding the sterile water for injection to scale, uniformly mixing, and adjusting the pH value to 6.5 by using a phosphate buffer solution.
Example 7
The embodiment provides a sample preservation solution, and the preparation method comprises the following steps: taking 10g of betaine and 0.05g of chlorhexidine to a 100ml measuring flask, adding a proper amount of sterile water for injection to dissolve, adding the sterile water for injection to scale, uniformly mixing, and adjusting the pH value to 8.0 by using a phosphate buffer solution.
Example 8
The embodiment provides a sample preservation solution, and the preparation method comprises the following steps: taking 10g of betaine and 0.5g to 100ml of benzalkonium chloride, adding a proper amount of sterile water for injection to dissolve, adding the sterile water for injection to a scale, uniformly mixing, and adjusting the pH value to 7.5 by using a disodium hydrogen phosphate solution.
Example 9
The embodiment provides a sample preservation solution, and the preparation method comprises the following steps: taking 10g of betaine and 0.5g of benzalkonium bromide to 100ml of measuring flask, adding a proper amount of sterile water for injection for dissolution, adding the sterile water for injection to a scale, uniformly mixing, and adjusting the pH value to 7.5 by using a disodium hydrogen phosphate solution.
Example 10
The embodiment provides a sample preservation solution, and the preparation method comprises the following steps: taking 10g of betaine and 0.05g of dodecyl dimethyl phenoxyethyl ammonium bromide to 100ml of measuring flask, adding a proper amount of sterile water for injection to dissolve, adding the sterile water for injection to scale, uniformly mixing, and adjusting the pH value to 7.5 by using a disodium hydrogen phosphate solution.
Example 11
The embodiment provides a sample preservation solution, and the preparation method comprises the following steps: taking 10g of betaine, 0.05g of chlorhexidine and 0.05g of dodecyl dimethyl phenoxyethyl ammonium bromide to 100ml of measuring flask, adding a proper amount of sterilized injection water for dissolving, adding the sterilized injection water to scale, uniformly mixing, and adjusting the pH value to 7.5 by using a disodium hydrogen phosphate solution.
Comparative example 1
Compared with the embodiment 1, the sample preservation solution provided by the comparative embodiment does not contain the inactivating agent, and the preparation method of the sample preservation solution comprises the following steps: taking a measuring flask with the volume of 1g to 100ml of betaine, adding a proper amount of water to dissolve the betaine to a scale, uniformly mixing, and adjusting the pH value to 7.5 by using a disodium hydrogen phosphate solution.
Comparative example 2
Compared with example 1, the sample preservation solution provided by the comparative example does not contain betaine, and the preparation method of the sample preservation solution comprises the following steps: taking a measuring flask of 0.01g to 100ml of chlorhexidine, adding a proper amount of water to dissolve, adding water to scale, mixing uniformly, and adjusting the pH value to 7.5 by using a disodium hydrogen phosphate solution.
Comparative example 3
Compared with example 1, the sample storage solution provided in this comparative example was sterilized water for injection containing phosphate buffer at pH 7.5.
Experimental example 1
Stability survey
The same blood was selected, divided into 16 portions on average, each 5ml portion, and decomposed and added with 1ml of the preservation solution of examples 1-11 and comparative examples 1-3, 2 portions without any preservation solution, wherein 1 portion is blank control and is sterile water, the other portion is initial control, and the initial control is OD value detection of the blood sample before preservation to obtain the value of OD260/OD280. All samples were stored at-20 ℃ and 4 ℃ and sampled for OD detection for 5 days, 10 days and 20 days, respectively. Detecting OD260/OD280.
The statistical results of the mean values are shown in tables 1 and 2, and the results show that: comparing examples 1 and 2, it can be seen that changing the type of water in the preservation solution has little effect on the stability of the sample at-20 ℃ and 4 ℃; comparing examples 1 and 3, it is clear that increasing the betaine concentration helps to improve the stability of the samples at 4 ℃, especially after 20 days of storage, while the stability of the samples at-20 ℃ is not affected much.
It can be seen from comparison of examples 3 and 4 that increasing the concentration of the same type of inactivator had little effect on the storage stability of the samples at-20 ℃ and 4 ℃. It can be seen from comparative examples 4,5,6 and 7 that the pH of the specimen preservation solution has little influence on the stability of the specimen at-20 ℃ and 4 ℃.
It can be seen from comparative examples 4,8,9,10 and 11 that the substitution of the inactivating agent of the specimen preservation solution with other inactivating agents of the present invention has little effect on the stability of the specimen at-20 ℃ and 4 ℃.
Furthermore, the absence of betaine alone and the absence of inactivator both resulted in samples with poor stability at-20 ℃ and 4 ℃, especially in comparative example 2, which did not contain betaine, which was very poor.
The UV results are as follows
TABLE 1 preservation solution OD260/OD280 at-20 deg.C
TABLE 2 OD260/OD280 preservation solution at 4 ℃
Experimental example 2
The experimental example was used for examining the inactivation effect.
Cutting filter paper into round pieces with the diameter of 1cm by adopting a paper sheet method, sterilizing at 120 ℃, respectively soaking the round pieces into the preserving solutions of the embodiments 1-11 and the comparative example 3 after sterilization, simultaneously setting sterile water as a blank control group for standby, respectively eluting experimental bacteria (staphylococcus aureus and escherichia coli) by using 5ml of nutrient broth agar to prepare a bacteria mixed solution, averagely smearing the bacteria solution on a nutrient agar plate, inverting the incubator at 37 ℃ for 10min, using sterile tweezers to paste the soaked round pieces in the middle of the plate, culturing for 24h in the incubator at 37 ℃, taking out the round pieces and observing the diameter of an inhibition zone.
The zone of inhibition results are shown in table 3.
TABLE 3 results of zone of inhibition
| Preserving fluid for use | Staphylococcus aureus inhibiting circle diameter (mm) | Diameter of Escherichia coli bacteria ring (mm) |
| Comparative example 1 | 6.0 | 6.1 |
| Example 1 | 10.7 | 10.0 |
| Comparative example 2 | 8.6 | 9.2 |
| Example 2 | 8.4 | 8.9 |
| Example 3 | 8.1 | 8.8 |
| Example 4 | 11.2 | 12.1 |
| Example 5 | 7.8 | 7.5 |
| Example 6 | 7.2 | 6.9 |
| Example 7 | 13.4 | 14.1 |
| Example 8 | 13.1 | 13.7 |
| Example 9 | 14.3 | 13.9 |
| Example 10 | 14.8 | 15.1 |
| Example 11 | 18.5 | 19.7 |
| Comparative example 3 | 5.9 | 6.2 |
| Blank control | 5.6 | 5.8 |
The results in Table 3 show that the inactivation effect of the specimen preservative fluid is not significantly affected by changing the type of water in the preservative fluid as seen in comparative examples 1 and 2. It can be seen from comparison of examples 1 and 3 that increasing the betaine concentration has little effect on the inactivation of the sample preservative fluid.
Comparing examples 3 and 4, it can be seen that increasing the concentration of the same type of inactivating agent has a greater effect on the inactivation of the sample preservation solution, and the higher the concentration, the better the inactivation. As can be seen from comparative examples 4,5,6 and 7, the inactivation effect was more excellent at pH 7.5 and 8.0 in the specimen-preservation solution.
Comparative examples 4,8,9,10 and 11 show that the inactivation of the sample preservation solution is replaced by other inactivation agents, and the inactivation effect of the sample preservation solution has a certain influence, especially the addition of two inactivation agents can significantly improve the inactivation effect.
In addition, the inactivation effect of the sample preservation solution is obviously reduced by adding no inactivating agent, especially the inactivation effect of the comparative example 3 without adding betaine and the inactivating agent is extremely poor.
In conclusion, the sample preservation solution provided by the invention can reduce the infection and degradation risks of the sample to be detected and can effectively inactivate pathogenic microorganisms. Betaine is an amphoteric surfactant, has a good sterilization effect, and can synergistically improve the sterilization effect by being matched with an inactivating agent. Moreover, the betaine and the inactivator can effectively kill the microbial cells at lower concentration. Further, for the inactivation treatment of microbial cells, chlorhexidine and a cationic surfactant may be used alone or in combination of two or more thereof.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A specimen preservation fluid, comprising: the detergent comprises a stabilizing agent, a deactivating agent, a buffering agent and water, wherein the stabilizing agent is selected from betaine, and the deactivating agent is selected from one or a mixture of at least two of chlorhexidine and a cationic surfactant.
2. The sample preservation solution according to claim 1, wherein the concentration of betaine in the sample preservation solution is 1-10% (w/v);
preferably, the concentration of betaine in the sample preservation solution is 50% (w/v).
3. The sample preservation solution according to claim 1 or 2, wherein the concentration of the inactivating agent in the sample preservation solution is 0.01-0.1% (w/v);
preferably, the concentration of the inactivating agent in the sample preservation solution is 0.05-0.1% (w/v).
4. The specimen preservation solution according to claim 3, wherein the cationic surfactant is a quaternary ammonium compound;
preferably, the quaternary ammonium compound is selected from: at least one of benzalkonium chloride, benzalkonium bromide, benzethonium chloride, cetylpyridinium chloride, dodecyldimethyl phenoxyethyl ammonium bromide and hexadecyl trimethyl ammonium bromide.
5. The specimen preservation solution according to claim 1, wherein the buffer is selected from a phosphate buffer;
preferably, the phosphate buffer is selected from a disodium hydrogen phosphate solution, a sodium dihydrogen phosphate solution, a dipotassium hydrogen phosphate solution, or a potassium dihydrogen phosphate solution.
6. The sample preservation fluid according to claim 1, wherein the pH of the sample preservation fluid is 6.5 to 8.0;
preferably, the pH of the sample preservation solution is 7.0-8.0;
preferably, the pH of the sample preservation solution is 7.5-7.8;
preferably, the water of the sample preservation solution is sterilized water for injection;
preferably, the sample is blood, serum, plasma or urine.
7. The sample preservation fluid according to claim 1, characterized in that it comprises: 1-10% of betaine, 0.01-0.05% of chlorhexidine and the balance of sterilized water for injection, and the pH value of the sample preservation solution is adjusted to 7.5 by a buffer;
preferably, the sample preservation solution comprises: 1-10% of betaine, 0.01-0.1% of quaternary ammonium compound and the balance of sterilized water for injection, and the pH value of the sample preservation solution is adjusted to 7.5 by a buffer;
preferably, the sample preservation solution comprises: 1 to 10 percent of betaine, 0.01 to 0.05 percent of chlorhexidine, 0.01 to 0.05 percent of quaternary ammonium compound and the balance of sterilized water for injection, and the pH value of the sample preservation solution is adjusted to 7.5 by a buffer.
8. A reagent or a kit comprising the specimen preservation solution according to any one of claims 1 to 7.
9. A sample solution comprising a sample and the sample-retaining solution according to any one of claims 1 to 7.
10. A method of preserving a sample, comprising the steps of: mixing a sample to be preserved with a sample preservation solution according to any one of claims 1 to 7;
preferably, the preservation of the sample is performed at-20 ℃ to 4 ℃.
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