CN117969228B - Bacterial liquid diluent, preparation method, reference, kit and application thereof - Google Patents
Bacterial liquid diluent, preparation method, reference, kit and application thereof Download PDFInfo
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 30
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000001768 carboxy methyl cellulose Substances 0.000 claims abstract description 28
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- 238000012360 testing method Methods 0.000 claims description 19
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- 238000004321 preservation Methods 0.000 claims description 11
- 102000004407 Lactalbumin Human genes 0.000 claims description 10
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- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 8
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical group [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 claims description 7
- 239000003945 anionic surfactant Substances 0.000 claims description 6
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- 238000003908 quality control method Methods 0.000 claims description 6
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- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 4
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- NTASFODDPBHBAM-UHFFFAOYSA-N 1-hydroxyethylazanium;chloride Chemical compound [Cl-].CC([NH3+])O NTASFODDPBHBAM-UHFFFAOYSA-N 0.000 description 2
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 2
- 229920000663 Hydroxyethyl cellulose Polymers 0.000 description 2
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- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
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- 239000011780 sodium chloride Substances 0.000 description 2
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- 238000003860 storage Methods 0.000 description 2
- 108020000946 Bacterial DNA Proteins 0.000 description 1
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- CVGSWVDBRMDWPX-UHFFFAOYSA-N methanetriamine;hydrochloride Chemical compound Cl.NC(N)N CVGSWVDBRMDWPX-UHFFFAOYSA-N 0.000 description 1
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- 210000003437 trachea Anatomy 0.000 description 1
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Classifications
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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
- G01N1/38—Diluting, dispersing or mixing samples
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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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 bacterial liquid diluent, a preparation method, a reference substance, a kit and application thereof, and belongs to the technical field of biological detection; adding sodium carboxymethylcellulose into the prepared protein buffer solution, and fully stirring to prepare a component A; placing polysorbate 80 and tris hydrochloride into an ultrasonic stirrer, and stirring to obtain a component B; and fully stirring and mixing the component A and the component B at the temperature of-1-4 ℃. The bacterial liquid diluent provided by the invention can keep the bacterial liquid dispersed and does not affect the culture of the bacterial liquid, and meanwhile, the bacterial liquid diluent provided by the invention has no inhibitor introduction to the extraction and amplification of the rear nucleic acid, and can directly extract or detect the nucleic acid.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a bacterial liquid diluent, a preparation method, a reference, a kit and application thereof.
Background
Fluorescent quantitative PCR is a method in which the amount of a specific product is measured immediately by continuously detecting the change in the intensity of a fluorescent signal during the PCR exponential amplification, and the amount of a target gene is deduced therefrom. Fluorescent quantitative PCR (polymerase chain reaction) is used as a high-efficiency detection method which is rapidly developed in the pathogen detection field, can rapidly and sensitively detect viral DNA, RNA and bacterial DNA, and is increasingly required for detecting whether a body fluid sample is infected with a corresponding pathogen species or not through fluorescent quantitative PCR, however, when a reference product is prepared, the dispersion effect of the bacterial liquid is poor, the bacterial liquid is easy to aggregate after being stored at the temperature of 2-8 ℃, the bacterial liquid is difficult to uniformly disperse after being aggregated, and the storage and the use and the production of the reference product are inconvenient. The hydrophobic nature of the surface of the cell means that the bacteria exhibit an unstable state in polar water, thereby causing a series of changes in the redistribution and arrangement of the cells, resulting in aggregation. Meanwhile, the bacterial liquid is complex and tedious to acquire, and is difficult to disperse due to the fact that the bacterial liquid is aggregated, is easy to aggregate when stored at the temperature of 2-8 ℃, is difficult to uniformly disperse after aggregation, so that the detection efficiency is affected, and the storage and the use and the reference production are inconvenient. The difference between batches is large, which is not beneficial to mass production of reference products.
At present, few researches are performed on bacterial liquid diluents, so that how to keep bacterial liquid dispersed, the detection efficiency is improved, the batch difference is reduced, and theoretical researches are lacking. For example, a quadruple fluorescent quantitative PCR kit for detecting three pathogens of infantile pneumonia disclosed in 23/03/2016 is described in patent document with publication number CN104195266A, and comprises a DNA extract, a quadruple fluorescent PCR reaction solution, an internal reference solution, a negative quality control, a strong positive quality control, a weak positive quality control and five positive quantitative standards. The invention adopts an A11G 10 probe technology, designs four pairs of specific primers and corresponding probes, applies a quadruple fluorescent quantitative PCR technology to simultaneously detect three pathogens and internal references in sputum, optimizes reaction conditions, and establishes a single-tube quadruple PCR method based on the A11G 10 probe technology to simultaneously detect the three pathogens and the internal references in the sputum. The application uses mixed plasmid solution for quality control, and the detection condition of clinical isolates cannot be monitored.
From the above, it can be seen that the difference between the reference lot is reduced in order to effectively control the influence of the detection result. There is a need in the art to develop a diluent for dispersing bacterial fluids that can effectively disperse bacterial fluids, reduce batch-to-batch variation, and be used for bacterial fluid dispersion.
Disclosure of Invention
The invention aims to provide a bacterial liquid diluent, which solves the defects that in the prior art, when reference products are prepared and quality is controlled, the bacterial liquid has poor dispersion effect, is difficult to uniformly disperse after aggregation, is inconvenient to store and use and produce the reference products, easily causes large difference among batches and is unfavorable for mass production of the reference products.
The invention is realized by the following technical scheme that the preparation method of the bacterial liquid diluent comprises the steps of firstly preparing a component A and a component B; the component A comprises sodium carboxymethyl cellulose and a protein buffer solution; placing sodium dodecyl sulfate and ethylene glycol into an ultrasonic stirrer for stirring, wherein the ultrasonic stirring frequency is 95-120 KHz, the stirring time is 45-65 min, then adding lysozyme and/or alpha-lactalbumin, adjusting the ultrasonic stirring frequency to 120-150 KHz, and the stirring time is 1-1.5 h, so as to prepare a protein buffer solution; adding sodium carboxymethylcellulose into the prepared protein buffer solution, fully stirring at 850-900 rpm for 55-75 min to obtain a component A, and then placing the component A in an ice bath for preservation, wherein the temperature of the ice bath is-1-4 ℃; the component B comprises polysorbate 80 and tris hydrochloride; placing polysorbate 80 and tris hydrochloride into an ultrasonic stirrer for stirring, wherein the ultrasonic stirring frequency is 80-100 KHz, and the stirring time is 55-75 min, so as to obtain a component B; and fully stirring and mixing the component A and the component B at the temperature of-1-4 ℃ at the stirring speed of 550-700 rpm for 30-55 min.
In order to achieve the technical purpose of the invention, the second aspect of the invention provides a bacterial liquid diluent, which is prepared according to the preparation method of the bacterial liquid diluent, and comprises sodium carboxymethyl cellulose, polysorbate 80, tris hydrochloride, protein buffer and water; wherein, the mass volume percentage of sodium carboxymethyl cellulose is 0.1-5%, the mass volume percentage of polysorbate 80 is 0.1-5%, the mass volume percentage of tris (hydroxymethyl) aminomethane hydrochloride is 0.06-12.1%, and the tris (hydroxymethyl) aminomethane hydrochloride concentration is 5-1000 mmol/L; the protein buffer includes an anionic surfactant, a polyol, and an amyloid protein; wherein the amyloid is at least one of lysozyme and alpha-lactalbumin, and the concentration of the amyloid is 100 mmol/L; the polyalcohol is glycol, and the concentration of the glycol is 200 mmol/L; the anionic surfactant is sodium dodecyl sulfonate, and the concentration of the sodium dodecyl sulfonate is 10 mmol/L.
The mass volume percentage in the present application refers to the ratio of the mass of the substance to the total volume of the solution in which the substance is located, i.e., the ratio of solute to solution.
Further, the water is sterile purified water.
To achieve the technical purpose of the invention, the third aspect of the invention provides a bacterial solution dilution reference, which comprises the bacterial solution dilution as described above, and an inactivated bacterial solution and/or a simulated sputum matrix diluted by the bacterial solution dilution.
In order to achieve the technical purpose of the invention, the fourth aspect of the invention provides a bacterial liquid diluent kit, which comprises a reaction liquid, a precipitator, a stop solution, a linear standard substance and the bacterial liquid diluent.
Further, the kit may further comprise a bacterial liquid diluent reference as described above.
In order to achieve the technical object of the present invention, a fifth aspect of the present invention provides an application of a bacterial liquid diluent kit, and the kit as described above may have any of the following applications: (1) use in vaccine development; (2) use in microbial enumeration; (3) use in a fluorescent PCR assay; (4) use in detecting pathogenic bacteria in a sample; (5) use in an antibiotic susceptibility test; (6) use in quality control of pharmaceutical products.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. The bacterial liquid diluent is creatively prepared, so that stable diffusion and aggregation of bacterial liquid can be well realized, the bacterial liquid diluent can be stored for 1 year at the temperature of 2-8 ℃ and still has good bacterial liquid aggregation preventing effect and good stability;
2. The bacterial liquid diluent provided by the invention can keep the bacterial liquid dispersed and does not affect the culture of the bacterial liquid, and meanwhile, the bacterial liquid diluent provided by the invention has no inhibitor introduction to the extraction and amplification of the rear nucleic acid, and can directly extract or detect the nucleic acid;
3. After the bacteria liquid is subjected to gradient dilution by adopting the bacteria liquid diluent provided by the invention, the bacteria liquid is better subjected to linear regression analysis after detection, which shows that the bacteria liquid diluent can effectively avoid aggregation of bacteria.
Drawings
The accompanying drawings, which are included to provide a further understanding of embodiments of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the principles of the application. In the drawings:
FIG. 1 is a test chart of culture versus PCR provided in example 4 of the present invention;
FIG. 2 is a linear graph of the bacterial liquid dilution gradient provided in example 6 of the present invention;
FIG. 3 is a schematic diagram of a bacterial suspension dilution gradient dilution PCR provided in example 6 of the present invention;
FIG. 4 is a linear plot of the gradient dilution of physiological saline provided in example 6 of the present invention;
FIG. 5 is a gradient dilution PCR chart of physiological saline provided in example 6 of the present invention;
FIG. 6 is a linear plot of the gradient dilution of purified water provided in example 6 of the present invention;
FIG. 7 is a purified water gradient dilution PCR map provided in example 6 of the present invention.
Detailed Description
For the purpose of making 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 with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to. The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth. The term "about" as used herein means a range of + -20% of the numerical values thereafter. In some embodiments, the term "about" means a range of ±10% of the numerical value following that. In some embodiments, the term "about" means a range of ±5% of the numerical value following that. The term "matrix" as used herein refers to secretions of the respiratory tract (bronchi, trachea, larynx, nose) or exudates within the alveoli, or mimics thereof. The term "reference" as used herein refers to a substance of known content in clinical medical testing that is of defined character on the same substrate as the actual specimen.
Example embodiment 1,
The embodiment provides a preparation method of a bacterial liquid diluent, which comprises the steps of preparing a component A and a component B.
The A component can comprise sodium carboxymethyl cellulose and protein buffer solution.
Firstly, sodium dodecyl sulfate and ethylene glycol are placed into an ultrasonic stirrer to be stirred, wherein the ultrasonic stirring frequency is 95-120 KHz, and the stirring time is 45-65 min.
And then adding lysozyme and alpha-lactalbumin, adjusting the ultrasonic stirring frequency to be 120-150 KHz, and stirring for 1-1.5 h to prepare the protein buffer solution.
Adding sodium carboxymethylcellulose into the prepared protein buffer solution, fully stirring at 850-900 rpm for 55-75 min to obtain a component A, and then placing the component A in an ice bath for preservation, wherein the temperature of the ice bath is-1-4 ℃.
The B component may include polysorbate 80 and tris hydrochloride.
And (3) placing polysorbate 80 and tris hydrochloride into an ultrasonic stirrer for stirring, wherein the ultrasonic stirring frequency is 80-100 KHz, and the stirring time is 55-75 min, so as to obtain the component B.
Fully stirring and mixing the component A and the component B at the temperature of-1-4 ℃ at the stirring speed of 550-700 rpm for 30-55 min; preparing bacterial liquid diluent.
Example embodiment 2,
The present exemplary embodiment provides a bacterial liquid diluent prepared according to the preparation method of the above exemplary embodiment 1, including sodium carboxymethyl cellulose, polysorbate 80, tris hydrochloride, a protein buffer, and water.
Wherein, the mass volume percentage of sodium carboxymethyl cellulose is 0.1-5%, the mass volume percentage of polysorbate 80 is 0.1-5%, the mass volume percentage of tris (hydroxymethyl) aminomethane hydrochloride is 0.06-12.1%, the tris (hydroxymethyl) aminomethane hydrochloride concentration is 5-1000mmol/L, and the water is sterile purified water.
For example, the mass volume percent of sodium carboxymethyl cellulose may be 0.2, 0.5, 2.2, 3.6, 4.7%; the mass volume percentage of polysorbate 80 can be 0.3, 0.8, 2.4, 3.8, 4.2%; the concentration of the hydroxymethyl aminomethane hydrochloride can be 6, 100, 230, 380, 790 and 930 mmol/L.
The protein buffer may include an anionic surfactant, a polyol, and an amyloid protein.
Wherein, the amyloid can be at least one of lysozyme and alpha-lactalbumin, and the concentration of the amyloid can be 100 mmol/L;
The polyol can be ethylene glycol, and the concentration of the ethylene glycol can be 200 mmol/L;
the anionic surfactant may be sodium dodecyl sulfonate, which may have a concentration of 10 mmol/L.
Example embodiment 3,
The present exemplary embodiment provides a bacterial fluid dilution reference, comprising,
The bacterial fluid dilutions described in example 2 above, as well as inactivated bacterial fluid and/or simulated sputum matrix diluted by the bacterial fluid dilutions.
Example embodiment 4,
The present exemplary embodiment provides a bacterial liquid diluent kit, which includes a reaction liquid, a precipitant, a stop solution, a linear standard, and the bacterial liquid diluent as described in exemplary embodiment 2. The kit may also include a bacterial fluid dilution reference as described in exemplary embodiment 3.
In order to better understand one bacterial fluid dilution provided in this embodiment, the following further clarifies the process with reference to the drawings and examples.
EXAMPLE 1,
And the dispersion preservation effect of bacterial liquid dilutions with different concentrations is achieved.
Experimental group set up in this example:
Experiment group 1: sodium carboxymethylcellulose was 1% by mass and volume, polysorbate 80 was 2% by mass and volume, and tris hydrochloride was 5mmol/L in concentration, and 100mL was prepared using sterile purified water.
Experiment group 2: sodium carboxymethylcellulose was 1% by mass and volume, polysorbate 80 was 1% by mass and volume, and tris hydrochloride was 1000mmol/L in concentration, and 100mL was prepared using sterile purified water.
Experiment group 3: sodium carboxymethylcellulose was 1% by mass and volume, polysorbate 80 was 1% by mass and volume, and tris hydrochloride was 100mmol/L in concentration, and 100mL was prepared using sterile purified water.
Experiment group 4: sodium carboxymethylcellulose 1% by mass/volume, polysorbate 80 1% by mass/volume, a concentration of tris hydrochloride 100mmol/L, and a protein buffer comprising alpha-lactalbumin 100mmol/L, ethylene glycol 200 mmol/L, and sodium dodecyl sulfonate 10 mmol/L were prepared using sterile purified water for 100mL.
Through the experiment, when the concentration of the tris (hydroxymethyl) aminomethane hydrochloride is 100mmol/L, the dispersion preservation effect is good.
Through the experiment, when the mass volume percentage of the sodium carboxymethyl cellulose is 1%, the dispersion preservation effect is better.
Through the experiment, when the mass volume percentage of polysorbate 80 is 1%, the dispersion preservation effect is good.
Through the experiment, the dispersion preservation effect of the bacterial liquid diluent added with the protein buffer solution is best.
EXAMPLE 2,
In the embodiment, the bacterial liquid diluent prepared by the preparation method in the exemplary embodiment 1 is used for carrying out PCR detection after the bacterial liquid is subjected to gradient dilution and placed for 6 months under the environmental condition of 8-15 ℃.
The preparation method is characterized in that the preparation method is placed for 2 months under the environmental condition of 8-15 ℃, the dispersion preservation effect is good, after extraction, fluorescent quantitative PCR detection is carried out, the cycle number is equivalent, and the difference is not more than 1. The final bacterial liquid stability is shown in Table 1.
TABLE 1 bacterial liquid stability
The bacterial liquid diluent added with the protein buffer solution has better temperature tolerance, can stably maintain the stability of the protein, prevent denaturation or inactivation, and can help reduce experimental variation and improve the accuracy and repeatability of the result by maintaining a more stable experimental environment.
EXAMPLE 3,
In the bacterial liquid dilution liquid used in this example, no protein buffer was added to the bacterial liquid dilution liquid in this example, as compared with the bacterial liquid dilution liquid in example 2.
In this example, the stability of the bacterial liquid stored in the bacterial liquid dilution of the present invention after 12 months was tested at 2 to 8 ℃.
Experiment group 1: the mass volume percentage of sodium carboxymethyl cellulose is 2.5%, the mass volume percentage of polysorbate 80 is 3.5%, when the mass volume percentage of tris (hydroxymethyl) aminomethane hydrochloride is 0.1% and the concentration of tris (hydroxymethyl) aminomethane hydrochloride is 500 mmol/L, the bacterial liquid is preserved for 12 months at 2-8 ℃, the dispersion preservation effect is good, the fluorescence quantitative PCR detection is carried out after extraction, the cycle number is equivalent, and the difference is not more than 1. The final bacterial liquid stability is shown in Table 2.
TABLE 2 bacterial liquid stability
As can be seen from comparative examples 2 and 3, the bacterial liquid diluted solution added with the protein buffer solution has a better dilution effect and a better dispersion preservation effect than those of the bacterial liquid diluted solution without the protein buffer solution, and can stably maintain the stability of the protein and prevent denaturation or inactivation.
EXAMPLE 4,
And (3) culturing the bacterial liquid diluted bacterial liquid and pure water diluted bacterial liquid, and performing PCR contrast verification.
Preparation of bacterial liquid diluent of the example: sodium carboxymethylcellulose is 1% by mass/volume, polysorbate 80 is 1% by mass/volume, the concentration of hydroxymethyl aminomethane hydrochloride is 0.06% by mass/volume, the concentration of tris aminomethane hydrochloride is 100 mmol/L, and a protein buffer solution comprising alpha-lactalbumin at a concentration of 100 mmol/L, ethylene glycol at a concentration of 200 mmol/L and sodium dodecyl sulfonate at a concentration of 10 mmol/L is added, and 100mL is prepared using sterile purified water.
The bacterial liquid diluent and pure water are used for preparing bacterial liquid with the same concentration respectively. And culturing at the same time, and extracting according to the instruction of the extraction kit after culturing for 3 days. The test was performed using fluorescent PCR, the PCR primers being provided in the kit.
The results show that after the bacterial solutions diluted by pure water and bacterial solution dilution are respectively cultured and extracted, fluorescence quantitative PCR detection is carried out, the cycle number is equivalent, and the difference is not more than 1. Table 3 shows the effect of the bacterial suspension dilution in this example on the culture, FIG. 1 shows a test chart of the comparative PCR in this example, and it can be seen from Table 3 and FIG. 1 that the bacterial suspension dilution of the present invention does not affect the culture.
TABLE 3 influence of bacterial dilution on culture
EXAMPLE 5,
The bacterial liquid dilution preparation of this example was the same as example 4, except that in this example, the mass volume percentage of sodium carboxymethylcellulose was 0.1%, the mass volume percentage of polysorbate 80 was 0.1%, the mass volume percentage of tris hydrochloride was 1%, the concentration of tris hydrochloride was 5 mmol/L, and a protein buffer including alpha-lactalbumin at a concentration of 100 mmol/L, ethylene glycol at a concentration of 200 mmol/L, and sodium dodecyl sulfate at a concentration of 10 mmol/L was added, and 100mL was prepared using sterile purified water.
The bacterial liquid diluent and pure water are used for preparing bacterial liquid with the same concentration respectively. And culturing at the same time, and extracting according to the instruction of the extraction kit after culturing for 3 days. The test was performed using fluorescent PCR, the PCR primers being provided in the kit.
The results show that after the bacterial solutions diluted by pure water and bacterial solution dilution are respectively cultured and extracted, fluorescence quantitative PCR detection is carried out, the cycle number is equivalent, and the difference is not more than 0.9.
EXAMPLE 5,
The bacterial liquid dilution preparation of this example was the same as example 4, except that in this example, the mass volume percentage of sodium carboxymethylcellulose was 3.5%, the mass volume percentage of polysorbate 80 was 5%, the mass volume percentage of tris hydrochloride was 12.1%, the concentration of tris hydrochloride was 1000mmol/L, and a protein buffer including 100 mmol/L of alpha-lactalbumin, 200 mmol/L of ethylene glycol, and 10 mmol/L of sodium dodecyl sulfate was added, and 100mL was prepared using sterile purified water.
The bacterial liquid diluent and pure water are used for preparing bacterial liquid with the same concentration respectively. And culturing for 4 days, and extracting according to the instruction of the extraction kit. The test was performed using fluorescent PCR, the PCR primers being provided in the kit.
The results show that after the bacterial solutions diluted by pure water and bacterial solution dilution are respectively cultured and extracted, fluorescence quantitative PCR detection is carried out, the cycle number is equivalent, and the difference is not more than 0.95.
EXAMPLE 6,
In this example, the bacterial liquid dilution, physiological saline and purified water provided by the invention are subjected to PCR detection after being placed at 2-8 ℃ for 1 week.
The bacterial liquid diluent preparation of this example is the same as example 4; the simulated sputum matrix used in this example was prepared as follows: the mass volume percentage of sodium alginate is 0.5%, the mass volume percentage of sodium carboxymethyl cellulose is 0.5%, the mass volume percentage of hydroxyethyl cellulose is 0.1%, the mass volume percentage of potato soaked powder is 2%, the mass volume percentage of sodium chloride is 0.9%, the concentration of magnesium chloride is 1 mmol/L, and the mass volume percentage of mucin is 2%. The formulation was performed using sterile purified water for 100mL.
In the experimental group 1, inactivated Aspergillus niger is diluted in a gradient way by using a bacterial liquid diluent, the mixture is placed for 1 week at the temperature of 2-8 ℃, and an Aspergillus niger solution is added into simulated sputum according to the concentration of 1%, so that reference substances with different concentrations are prepared, wherein the final concentrations of the reference substances are as follows: 1000000cfu/mL, 100000cfu/mL, 10000cfu/mL, 1000cfu/mL, each concentration was subjected to PCR detection.
Table 4 shows the bacterial liquid gradient dilution test values of experimental group 1, fig. 2 shows a bacterial liquid dilution gradient dilution linear chart, and fig. 3 shows a bacterial liquid dilution gradient dilution PCR chart.
TABLE 4 bacterial liquid gradient dilution test values
Experiment group 2. Aspergillus niger inactivated by gradient dilution with physiological saline is placed at 2-8deg.C for 1 week, and Aspergillus niger solution is added into simulated sputum according to 1% concentration to prepare reference substances with different concentrations, wherein the final concentrations of the reference substances are as follows: 1000000cfu/mL, 100000cfu/mL, 10000cfu/mL, 1000cfu/mL, each concentration was subjected to PCR detection.
Table 5 shows the gradient dilution test values of aspergillus niger inactivated by gradient dilution with physiological saline in experimental group 2, fig. 4 shows a linear plot of the gradient dilution with physiological saline, and fig. 5 shows a PCR plot of the gradient dilution with physiological saline.
TABLE 5 physiological saline gradient dilution test values
Experiment group 3. Aspergillus niger inactivated by gradient dilution with purified water, standing at 2-8deg.C for 1 week, adding Aspergillus niger solution into simulated sputum according to 1% concentration, and preparing reference substances with different concentrations, wherein the final concentrations of the reference substances are as follows: 1000000cfu/mL, 100000cfu/mL, 10000cfu/mL, 1000cfu/mL, each concentration was subjected to PCR detection.
Table 6 shows the gradient dilution test values of inactivated aspergillus niger diluted with purified water gradient in experimental group 3, fig. 6 shows a linear plot of purified water gradient dilution, and fig. 7 shows a PCR plot of purified water gradient dilution.
TABLE 6 gradient dilution test values for purified water
The experiment shows that the reference product prepared by the bacterial liquid diluted by the bacterial liquid diluent provided by the invention has better linearity and good dispersion effect.
EXAMPLE 7,
The present example provides a method of preparing a bacterial fluid dilution,
Firstly, sodium dodecyl sulfate and ethylene glycol are placed into an ultrasonic stirrer for stirring, wherein the ultrasonic stirring frequency is 96KHz, and the stirring time is 47min.
Then adding lysozyme, adjusting the ultrasonic stirring frequency to be 125 KHz and the stirring time to be 1h, and preparing the protein buffer solution.
Adding sodium carboxymethylcellulose into the prepared protein buffer, stirring thoroughly at 855rpm for 59min, and storing the prepared mixture A in ice bath at-1deg.C.
Then placing polysorbate 80 and tris hydrochloride into an ultrasonic stirrer for stirring, wherein the ultrasonic stirring frequency is 85 KHz, and the stirring time is 57min, so as to prepare a mixture B.
Fully stirring and mixing the two mixtures at 0 ℃ at 560rpm for 35min; preparing bacterial liquid diluent.
EXAMPLE 8,
The present example also provides a method of preparing a bacterial fluid dilution,
Firstly, sodium dodecyl sulfate and ethylene glycol are placed into an ultrasonic stirrer for stirring, wherein the ultrasonic stirring frequency is 110KHz, and the stirring time is 58min.
Then adding lysozyme, adjusting the ultrasonic stirring frequency to be 135 KHz and the stirring time to be 1.2 hours, and preparing the protein buffer solution.
Adding sodium carboxymethylcellulose into the prepared protein buffer, stirring thoroughly at 880rpm for 65min, and storing the prepared mixture A in ice bath at 2deg.C.
Then placing polysorbate 80 and tris hydrochloride into an ultrasonic stirrer for stirring, wherein the ultrasonic stirring frequency is 90 KHz, and the stirring time is 66min, so as to prepare a mixture B.
Fully stirring and mixing the two mixtures at 1 ℃ at 680rpm for 45min; preparing bacterial liquid diluent.
EXAMPLE 9,
The present example also provides a method of preparing a bacterial fluid dilution,
Firstly, sodium dodecyl sulfate and ethylene glycol are placed into an ultrasonic stirrer for stirring, wherein the ultrasonic stirring frequency is 118KHz, and the stirring time is 64min.
Then adding lysozyme, adjusting the ultrasonic stirring frequency to be 150 KHz and the stirring time to be 1.5 hours, and preparing the protein buffer solution.
Adding sodium carboxymethylcellulose into the prepared protein buffer, stirring thoroughly at 900rpm for 75min, and storing the prepared mixture A in ice bath at 4deg.C.
Then placing polysorbate 80 and tris hydrochloride into an ultrasonic stirrer for stirring, wherein the ultrasonic stirring frequency is 100 KHz, and the stirring time is 75min, so as to prepare a mixture B.
Fully stirring and mixing the two mixtures at 4 ℃ at the stirring speed of 700rpm for 55min; preparing bacterial liquid diluent.
EXAMPLE 10,
In the example, the two bacterial liquid dilutions provided by the invention are respectively subjected to gradient bacterial liquid dilution, and after being placed for 2 weeks at 8-15 ℃, PCR detection is carried out. Wherein, the preparation of the A bacterial liquid diluent is the same as in example 3, and the preparation of the B bacterial liquid diluent is the same as in example 8.
The preparation of the a bacterial liquid diluent of this example is the same as that of example 3, and the preparation of the B bacterial liquid diluent is the same as that of example 8; the simulated sputum matrix used in this example was prepared as follows: the mass volume percentage of sodium alginate is 0.5%, the mass volume percentage of sodium carboxymethyl cellulose is 0.5%, the mass volume percentage of hydroxyethyl cellulose is 0.1%, the mass volume percentage of potato soaked powder is 2%, the mass volume percentage of sodium chloride is 0.9%, the concentration of magnesium chloride is 1 mmol/L, and the mass volume percentage of mucin is 2%. The formulation was performed using sterile purified water for 100mL.
In the experimental group 1, inactivated Aspergillus niger is diluted by a bacterial liquid diluent A in a gradient way, the mixture is placed for 2 weeks at the temperature of 8-15 ℃, and an Aspergillus niger solution is added into simulated sputum according to the concentration of 1%, so that reference products with different concentrations are prepared, wherein the final concentrations of the reference products are respectively as follows: 1000000cfu/mL, 100000cfu/mL, 10000cfu/mL, 1000cfu/mL, PCR detection was performed for each concentration. Table 7 shows the bacterial liquid gradient dilution test values of experimental group 1.
TABLE 7 bacterial liquid gradient dilution test values
Experiment group 2: the inactivated Aspergillus niger is diluted in a gradient way by using a B bacterial liquid diluent, the mixture is placed for 2 weeks at the temperature of 8-15 ℃, and the Aspergillus niger solution is added into simulated sputum according to the concentration of 1%, so that reference substances with different concentrations are prepared, wherein the final concentrations of the reference substances are as follows: 1000000cfu/mL, 100000cfu/mL, 10000cfu/mL, 1000cfu/mL, PCR detection was performed for each concentration. Table 8 shows the bacterial fluid gradient dilution test values of experimental group 2. The dilution of the B-cell solution used in the test group was the same as that used in example 8.
TABLE 8 bacterial liquid gradient dilution test values
As can be seen from comparison of the gradient dilution test values of the experimental groups 1 and 2, the bacterial liquid diluent added with the protein buffer solution has better temperature tolerance, and meanwhile, the experimental result can also indicate that the bacterial liquid diluent prepared after the protein buffer solution component of the invention is added can effectively protect the protein produced by microorganisms from being degraded, thereby maintaining the bioactivity of the protein. Meanwhile, the bacterial liquid diluent added with the protein buffer solution can help simulate the ionic strength and the pH value of the natural environment or in-vivo environment of microorganisms while ensuring the dilution effect, thereby providing an environment closer to the physiological condition in the experiment, effectively controlling the influence of the detection result and reducing the difference between reference batches.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (6)
1. The preparation method of the bacterial liquid diluent is characterized by comprising the steps of firstly preparing a component A and a component B;
the component A comprises sodium carboxymethyl cellulose and a protein buffer solution;
placing sodium dodecyl sulfate and ethylene glycol into an ultrasonic stirrer for stirring, wherein the ultrasonic stirring frequency is 95-120 KHz, the stirring time is 45-65 min, then adding lysozyme and/or alpha-lactalbumin, adjusting the ultrasonic stirring frequency to 120-150 KHz, and the stirring time is 1-1.5 h, so as to prepare a protein buffer solution;
Adding sodium carboxymethylcellulose into the prepared protein buffer solution, fully stirring at 850-900 rpm for 55-75 min to obtain a component A, and then placing the component A in an ice bath for preservation, wherein the temperature of the ice bath is-1-4 ℃; the component B comprises polysorbate 80 and tris hydrochloride;
Placing polysorbate 80 and tris hydrochloride into an ultrasonic stirrer for stirring, wherein the ultrasonic stirring frequency is 80-100 KHz, and the stirring time is 55-75 min, so as to obtain a component B;
And fully stirring and mixing the component A and the component B at the temperature of-1-4 ℃ at the stirring speed of 550-700 rpm for 30-55 min.
2. A bacterial liquid diluent, which is characterized by being prepared by the preparation method of claim 1 and comprising sodium carboxymethyl cellulose, polysorbate 80, tris hydrochloride, protein buffer and water;
Wherein the mass volume percentage of the sodium carboxymethyl cellulose is 0.1-5%,
The mass volume percentage of polysorbate 80 is 0.1-5%,
The weight volume percentage of the tris (hydroxymethyl) aminomethane hydrochloride is 0.06-12.1%, and the concentration of the tris (hydroxymethyl) aminomethane hydrochloride is 5-1000 mmol/L;
The protein buffer includes an anionic surfactant, a polyol, and an amyloid protein;
Wherein the amyloid is at least one of lysozyme and alpha-lactalbumin, and the concentration of the amyloid is 100 mmol/L; the polyalcohol is glycol, and the concentration of the glycol is 200 mmol/L; the anionic surfactant is sodium dodecyl sulfonate, and the concentration of the sodium dodecyl sulfonate is 10 mmol/L.
3. A bacterial fluid dilution reference comprising the bacterial fluid dilution of claim 2, and an inactivated bacterial fluid and/or simulated sputum matrix diluted by the bacterial fluid dilution.
4. A bacterial fluid dilution kit comprising a reaction fluid, a precipitant, a stop fluid, a linear standard, and the bacterial fluid dilution of claim 2.
5. The bacterial suspension dilution kit according to claim 4, further comprising a bacterial suspension dilution reference as defined in claim 3.
6. The use of the bacterial liquid diluent kit according to any one of claims 4 to 5, wherein the bacterial liquid diluent kit has the following uses:
(1) Application in vaccine development;
(2) Use in microbial enumeration;
(3) Application in fluorescent PCR test;
(4) Use in detecting pathogenic bacteria in a sample;
(5) Use in antibiotic susceptibility testing;
(6) The application in quality control of medical products.
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