CN111690712A - Sample for verifying qualitative detection capability of staphylococcus aureus in mask and preparation method thereof - Google Patents
Sample for verifying qualitative detection capability of staphylococcus aureus in mask and preparation method thereof Download PDFInfo
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- 241000191967 Staphylococcus aureus Species 0.000 title claims abstract description 47
- 238000001514 detection method Methods 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title abstract description 15
- 238000012795 verification Methods 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 21
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- 239000000725 suspension Substances 0.000 claims description 42
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- 230000002452 interceptive effect Effects 0.000 claims description 21
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- 238000007789 sealing Methods 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 11
- 230000001954 sterilising effect Effects 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 8
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- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 7
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 7
- 238000005520 cutting process Methods 0.000 claims description 7
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 7
- 229920000159 gelatin Polymers 0.000 claims description 7
- 239000008273 gelatin Substances 0.000 claims description 7
- 235000019322 gelatine Nutrition 0.000 claims description 7
- 235000011852 gelatine desserts Nutrition 0.000 claims description 7
- 238000002844 melting Methods 0.000 claims description 7
- 229910052751 metal Inorganic materials 0.000 claims description 7
- 239000002184 metal Substances 0.000 claims description 7
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- 239000007788 liquid Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 241000194106 Bacillus mycoides Species 0.000 claims description 5
- 241000194032 Enterococcus faecalis Species 0.000 claims description 5
- 241000588724 Escherichia coli Species 0.000 claims description 5
- 241000186781 Listeria Species 0.000 claims description 5
- 241001147736 Staphylococcus capitis Species 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 5
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- 229940032049 enterococcus faecalis Drugs 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 238000011084 recovery Methods 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 5
- 241000193755 Bacillus cereus Species 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 241000123247 Inonotus Species 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 3
- 230000008018 melting Effects 0.000 claims description 2
- 230000008569 process Effects 0.000 abstract description 9
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- 238000012360 testing method Methods 0.000 abstract description 5
- 244000052616 bacterial pathogen Species 0.000 abstract description 2
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- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
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- 229940049954 penicillin Drugs 0.000 description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
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- 229910052782 aluminium Inorganic materials 0.000 description 2
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- 241001465754 Metazoa Species 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
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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
- C12Q1/02—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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- 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
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/14—Streptococcus; Staphylococcus
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Abstract
The invention discloses a qualitative detection capability verification sample for staphylococcus aureus in a mask and a preparation method thereof. The sample can be used for evaluating the laboratory detection capability, controlling the quality, checking personnel, verifying the method and the like in the detection process, and fills the blank at home and abroad. Meanwhile, the uniformity and the stability of the sample meet the capability verification requirements, and the problem that the viable pathogenic bacteria in the staphylococcus aureus detection sample in the mask can change in the processes of transportation, storage, testing and the like is solved. In addition, the preparation method provided by the invention has simple process and high success rate of preparing samples meeting the requirements.
Description
Technical Field
The invention relates to the technical field of microbiological inspection quality control, in particular to a qualitative detection capability verification sample of staphylococcus aureus in a mask and a preparation method thereof.
Background
The mask belongs to a disposable sanitary product, which is various daily living products that are discarded after being used once, are in direct or indirect contact with a human body and are used for achieving the purposes of physiological sanitation or health care (antibiosis or bacteriostasis) of the human body, is easy to carry and simple to use, is popular with consumers and is an indispensable part of daily life of common people. The disposable sanitary product directly contacts skin or mucous membrane, and the quality of the disposable sanitary product directly influences the body health of a user.
The capability of qualitatively detecting the microorganisms in the disposable sanitary product not only can directly reflect the degree of the microorganism pollution, but also is a necessary means for evaluating raw materials, tool equipment, process flows and the sanitary condition of operators used in the production process, and is a comprehensive basis for sanitary evaluation of the disposable sanitary product.
Due to the particularity of the field of microbiological examination of disposable sanitary products, no organization/organization for preparing a sample for qualitative detection of staphylococcus aureus by taking a mask as a matrix exists at home and abroad, and no related literature report exists.
Staphylococcus aureus is a common gram-positive bacterium, widely distributed in nature, and can cause infections of humans and animals. After the human body is infected with staphylococcus aureus, local suppurative infection of the skin can be caused, and serious internal organ infection, even septicemia, toxic shock and the like can be caused. Therefore, there is an urgent need for a sample for qualitative detection capability verification of staphylococcus aureus in a mask.
Disclosure of Invention
Aiming at the technical problems, the invention provides a qualitative detection capability verification sample of staphylococcus aureus in a mask and a preparation method thereof, and provides important guarantee for microbiological capability verification of disposable sanitary products.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention firstly provides a qualitative detection capability verification sample of staphylococcus aureus in a mask, wherein the sample comprises a negative sample and a positive sample, the positive sample contains a substrate, target bacteria and interference bacteria, and the negative sample contains the substrate and the interference bacteria; the substrate is a mask, the target bacteria are Staphylococcus aureus (Staphylococcus aureus), the interfering bacteria are one or more of enterococcus faecalis (enterococcus faecalis), Escherichia coli (Escherichia coli), Bacillus mycoides (Bacillus mycoides), Staphylococcus capitis (Staphylococcus capitis), Bacillus cereus (Bacillus cereus) and Listeria inonotus (Listeria innocula).
In the sample verified by the qualitative detection capability of staphylococcus aureus in the mask, the concentration of bacteria in the sample is 103-106cfu/mL。
In the qualitative detection capability verification sample of staphylococcus aureus in the mask, target bacteria and interference bacteria of the positive sample are mixed into mixed bacteria according to the volume ratio of 1:2-3, and the mixed bacteria are added into a matrix.
In the qualitative detection capability verification sample for staphylococcus aureus in the mask, the sample is a freeze-dried sample, and the freeze-dried protective agent contains 5-12% of trehalose, 2-8% of sodium glutamate and 0.5-3% of gelatin.
In the qualitative detection capability verification sample for staphylococcus aureus in the mask, the volume ratio of each strain to the freeze-drying protective agent is 1: 9-3: 7.
In the sample for verifying the qualitative detection capability of staphylococcus aureus in the mask, the matrix treatment method comprises the following steps: taking the disposable mask, removing the ear hanging rubber band and the metal nose support part, cutting into small pieces, resealing with a hot melting sealing machine to obtain the water-absorbing remade mask, and sterilizing the remade mask for later use.
The invention also provides a preparation method of the sample for verifying the qualitative detection capability of staphylococcus aureus in the mask, which comprises the following steps:
s1 strain recovery passage
Recovering each strain, and identifying the recovered strain;
s2, Strain amplification
Culturing the recovered single viable strain at 36 + -1 deg.C for 36h, eluting with sterile normal saline, and gradually diluting to 10% concentration3-106cfu/mL, and mixing with a freeze-drying protective agent according to the proportion of 1: 9-3: 7 to prepare freeze-dried bacterial suspension of corresponding single strain;
s3, preparing bacterial suspension
Positive sample according to target concentration of target bacteria 102-105cfu/mL and target concentration of each of the interfering bacteria 102-105Preparing bacterial suspension by CFU/mL, mixing according to the volume ratio of 1:2-3 of the target bacteria and the interfering bacteria to prepare mixed bacterial suspension, and continuously stirring on a magnetic stirrer;
negative sample interfering bacteria at a target concentration of 10 per bacteria3-106Mixing CFU/mL with equal volume to prepare mixed bacterial suspension, and continuously stirring on a magnetic stirrer;
s4 matrix treatment
Taking a disposable mask, removing a hanging ear rubber band and a metal nose support part, cutting into small pieces, re-sealing by a hot-melting sealing machine to prepare a water-absorbable reproduced mask, and sterilizing the reproduced mask for later use;
s5, freeze-drying
And (3) dropwise adding the mixed bacterial suspension into each mask matrix, and filling the mask matrix into a sample bottle for freeze-drying after the mask completely absorbs the liquid to obtain a freeze-dried sample.
In the preparation method of the sample for verifying the qualitative detection capability of staphylococcus aureus in the mask, in step S1, each strain is respectively inoculated to a TSA culture medium and aerobically cultured for 36 hours at the temperature of 36 +/-1 ℃.
In the preparation method of the sample for verifying the qualitative detection capability of staphylococcus aureus in the mask, the freeze-drying protective agent in the step S2 contains 5-12% of trehalose, 2-8% of sodium glutamate and 0.5-3% of gelatin by volume.
According to the preparation method of the sample for verifying the qualitative detection capability of staphylococcus aureus in the mask, the freeze-drying time in the step S5 is 40-50 h.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses a qualitative detection capability verification sample for staphylococcus aureus in a mask, which is characterized in that the mask is used as a matrix for the first time, and a positive sample and a negative sample are prepared by adding target bacteria and/or interference bacteria. The sample can be used for evaluating the laboratory detection capability, controlling the quality, checking personnel, verifying the method and the like in the detection process, and fills the blank at home and abroad. Meanwhile, the uniformity and the stability of the sample meet the capability verification requirements, and the problem that the viable pathogenic bacteria in the staphylococcus aureus detection sample in the mask can change in the processes of transportation, storage, testing and the like is solved. In addition, the preparation method provided by the invention has simple process, and the success rate of preparing the sample which meets the requirements of the CNAS-GL 03 'guideline for evaluating uniformity and stability of capability verification sample' is high.
Detailed Description
The invention provides a qualitative detection capability verification sample for staphylococcus aureus in a mask, which comprises a negative sample and a positive sample, wherein the positive sample contains a matrix, target bacteria and interference bacteria, and the negative sample contains a matrix and interference bacteria; the substrate is a mask, the target bacteria are staphylococcus aureus, and the interference bacteria are one or more of enterococcus faecalis, escherichia coli, bacillus mycoides, staphylococcus capitis and listeria inonotus. Staphylococcus aureus (ATCC6538), enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Bacillus mycoides (ATCC 10206), Bacillus cereus (ATCC 11778), Staphylococcus capitis (ATCC35661) and Listeria inokosa (ATCC 33090) adopted by the present invention are all purchased from ATCC, so that the traceability of the strains is ensured, the above strains are only taken as exemplary examples of the embodiment of the present invention, and all the strains can be changed into equivalent strains.
In order to make those skilled in the art better understand the technical solution of the present invention, the present invention will be further described in detail with reference to the following embodiments.
Example 1 preparation of qualitative detection capability verification sample for Staphylococcus aureus in mask
The method comprises the following steps:
s1 strain recovery passage
Respectively inoculating each strain to a TSA culture medium, carrying out aerobic culture at the temperature of 36 +/-1 ℃ for 36h, and identifying the recovered strains;
s2, Strain amplification
Culturing the recovered single viable strain at 36 + -1 deg.C for 36h, culturing target bacteria Staphylococcus aureus to stationary phase, culturing other interference bacteria to end of logarithmic growth phase, culturing bacteria in stationary phase and logarithmic growth end phase with biochemical characteristics more typical than those in logarithmic growth phase, resisting mechanical injury in freeze drying process, eluting with sterile physiological saline, and gradually diluting to bacteria content of 103cfu/mL, mixing with a freeze-drying protective agent according to the proportion of 1:9 to prepare freeze-dried bacterial suspension of corresponding single strain; the freeze-drying protective agent contains 5% of trehalose, 2% of sodium glutamate and 0.5% of gelatin by volume fraction, and is prepared by sterile water for sterilization;
s3, preparing a positive sample by the bacterial suspension according to the target concentration 10 of the target bacteria2cfu/mL and target concentration of each of the interfering bacteria 102Preparing bacterial suspension by cfu/mL, mixing the bacterial suspension and the interfering bacteria according to the volume ratio of 1:2 of the target bacteria to the interfering bacteria to prepare mixed bacterial suspension, and continuously stirring the mixed bacterial suspension on a magnetic stirrer;
negative sample interfering bacteria at a target concentration of 10 per bacteria3cfu/mL are mixed in equal volume to prepare a mixed bacterial suspension, and the mixed bacterial suspension is placed on a magnetic stirrer to be continuously stirred;
s4 matrix treatment
Taking a disposable mask, removing a hanging ear rubber band and a metal nose support part, cutting into small pieces, re-sealing by a hot-melting sealing machine to prepare a water-absorbable reproduced mask, and sterilizing the reproduced mask for later use;
s5, freeze-drying
The mixed bacteria suspension is dripped into each mask matrix, the mask is filled into a sample bottle (penicillin bottle) after liquid is completely absorbed by the mask, the sample bottle is covered and put into a freeze dryer, freeze drying is carried out for 40 hours, after sample freeze drying is finished, in-box plugging is directly carried out, the machine is closed, the sample in the sample bottle is in a vacuum state, and an aluminum cover which can be torn once is pressed outside a rubber plug after shutdown, so that a freeze-dried sample is obtained. The freeze dryer parameters were set as follows: freezing speed is 1 ℃/min; early freezing point-1 deg.C for 15 min; the freezing temperature is-45 ℃; eutectic point-31 ℃; the heating temperature is 15 ℃ for 180 min. Starting the freeze dryer, and directly performing programmed freeze drying process by the machine for 40 h.
S6, packaging and storing
The lyophilized samples were stored at a temperature of 4 ℃.
Example 2 preparation of qualitative detection capability verification sample for Staphylococcus aureus in mask
The method comprises the following steps:
s1 strain recovery passage
Respectively inoculating each strain to a TSA culture medium, carrying out aerobic culture at the temperature of 36 +/-1 ℃ for 36h for resuscitation, and identifying the resuscitated strains;
s2, Strain amplification
Culturing the recovered single viable strain at 36 + -1 deg.C for 36h, eluting with sterile normal saline, and gradually diluting to 10% concentration5cfu/mL, mixing with a freeze-drying protective agent according to the proportion of 2:8 to prepare freeze-dried bacterial suspension of corresponding single strain; the freeze-drying protective agent contains trehalose with the volume fraction of 8%, sodium glutamate with the volume fraction of 5% and gelatin with the volume fraction of 1%, and is prepared by sterile water for sterilization;
s3, preparing bacterial suspension
Positive sample according to target concentration of target bacteria 104cfu/mL and target concentration of each of the interfering bacteria 104Preparing bacterial suspension by cfu/mL, mixing the bacterial suspension and the interfering bacteria according to the volume ratio of 1:2 of the target bacteria to the interfering bacteria to prepare mixed bacterial suspension, and continuously stirring the mixed bacterial suspension on a magnetic stirrer;
negative sample interfering bacteria at a target concentration of 10 per bacteria4cfu/mL are mixed in equal volume to prepare a mixed bacterial suspension, and the mixed bacterial suspension is placed on a magnetic stirrer to be continuously stirred;
s4 matrix treatment
Taking a disposable mask, removing a hanging ear rubber band and a metal nose support part, cutting into small pieces, re-sealing by a hot-melting sealing machine to prepare a water-absorbable reproduced mask, and sterilizing the reproduced mask for later use;
s5, freeze-drying
The mixed bacteria suspension is dripped into each mask matrix, after the mask completely absorbs liquid, the liquid is filled into a sample bottle (penicillin bottle) and then is covered and put into a freeze dryer, the freeze dryer parameters are as in embodiment 1, freeze drying is carried out for 45 hours, after the sample freeze drying is finished, in-box plugging is directly carried out, the machine is closed, the sample in the sample bottle is in a vacuum state, and an aluminum cover which can be torn off at one time is pressed outside a rubber plug after shutdown, so that a freeze-dried sample is obtained.
S6, packaging and storing
The lyophilized samples were stored at a temperature of 4 ℃.
Example 3 preparation of qualitative detection capability verification sample for Staphylococcus aureus in mask
The method comprises the following steps:
s1 strain recovery passage
Respectively inoculating each strain to a TSA culture medium, carrying out aerobic culture at the temperature of 36 +/-1 ℃ for 36h, and identifying the recovered strains;
s2, Strain amplification
Culturing the recovered single viable strain at 36 + -1 deg.C for 36h, eluting with sterile normal saline, and gradually diluting to 10% concentration6cfu/mL, mixing with a freeze-drying protective agent according to the proportion of 3:7 to prepare freeze-dried bacterial suspension of corresponding single strain; the freeze-drying protective agent contains 12% of trehalose, 8% of sodium glutamate and 3% of gelatin by volume fraction, and is prepared by sterile water for sterilization;
s3, preparing bacterial suspension
Positive sample according to target concentration of target bacteria 105cfu/mL and target concentration of each of the interfering bacteria 105Preparing bacterial suspension by cfu/mL, mixing the bacterial suspension and the interfering bacteria according to the volume ratio of 1:3 of the target bacteria to the interfering bacteria to prepare mixed bacterial suspension, and continuously stirring the mixed bacterial suspension on a magnetic stirrer;
negative sample interfering bacteria at a target concentration of 10 per bacteria6cfu/mL are mixed in equal volume to prepare a mixed bacterial suspension, and the mixed bacterial suspension is placed on a magnetic stirrer to be continuously stirred;
s4 matrix treatment
Taking a disposable mask, removing a hanging ear rubber band and a metal nose support part, cutting into small pieces, re-sealing by a hot-melting sealing machine to prepare a water-absorbable reproduced mask, and sterilizing the reproduced mask for later use;
s5, freeze-drying
And (3) dropwise adding the mixed bacterial suspension into each mask matrix, filling the mask matrix into a sample bottle (penicillin bottle) after the mask completely absorbs liquid, covering the sample bottle with a cover, and putting the sample bottle into a freeze dryer, wherein the freeze dryer has the parameters shown in the embodiment 1, freeze-drying for 50 hours, directly plugging the sample bottle after the sample freeze-drying is finished, closing the freeze dryer, and keeping the sample in the sample bottle in a vacuum state to obtain a freeze-dried sample.
S6, packaging and storing
The lyophilized samples were stored at a temperature of 4 ℃.
Example 4 Staphylococcus aureus uniformity and stability verification in a mask
Uniformity and stability checks of target bacteria in a sample are the main methods to verify the effectiveness of the sample preparation process. Examination of sample uniformity and stability was performed according to CNAS-GL 03 "guidelines for evaluation of uniformity and stability of samples for capability verification".
The uniformity and stability detection method of the obtained sample and the result are as follows:
10 samples of example 1 were randomly selected, respectively, and the target bacteria were detected by the test method of the second method of GB 4789.10-2016 (national food safety Standard for food microbiology test, Staphylococcus aureus test), and Staphylococcus aureus was tested in 2 × 10 samples under repeated conditions. The data of the results were statistically processed by ANOVA, and the statistical procedure and results are shown in Table 1.
TABLE 1 detection results of Staphylococcus aureus in samples
TABLE 2 statistical results of the analysis of variance of Staphylococcus aureus in the samples
And (4) conclusion: critical value of F0.05(9,10)3.02. The calculated F value was 1.14 and the sample F value < the critical value, indicating that at a significant level of 0.05, the distribution of the target bacteria in the sample was uniform. The homogeneity results of the resulting samples of examples 2 and 3 are similar to example 1.
Stability two types of tests were used: one is the stability test at storage temperature (4 ℃) and the other is the stability test at elevated temperature (simulating the transport conditions of the sample), three temperature points were chosen, 25 ℃, 36 ℃ and 42 ℃. The samples in the embodiment 1 are randomly taken, the samples are detected periodically, 3 samples are tested according to different storage time at different temperature points, and when the detection value of the sample in the stability test is consistent with the specified value, the stability of the capacity verification sample is in accordance with the requirement, and meanwhile, the longest storage time in accordance with the requirement of the capacity verification sample under different temperature conditions is determined. The stability test results are shown in table 3 below.
TABLE 3 stability test results
And (4) conclusion: the sample is stored at the low temperature of 4 ℃ for 120 days and at the high temperature of 42 ℃ for 20 days, the detection results of the sample all meet the requirements, and the stability is good.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: it is to be understood that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof, but such modifications or substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A staphylococcus aureus qualitative detection capability verification sample in a mask is characterized by comprising a negative sample and a positive sample, wherein the positive sample contains a matrix, target bacteria and interference bacteria, and the negative sample contains a matrix and interference bacteria; the substrate is a mask, the target bacteria are staphylococcus aureus, and the interference bacteria are one or more of enterococcus faecalis, escherichia coli, bacillus mycoides, bacillus cereus, staphylococcus capitis and listeria inonotus.
2. The qualitative detection capability verification sample of staphylococcus aureus in mask according to claim 1, wherein the concentration of bacteria in the sample is 103-106cfu/mL。
3. The mask staphylococcus aureus qualitative detection capability verification sample as claimed in claim 1, wherein the target bacteria and the interfering bacteria of the positive sample are mixed into mixed bacteria according to a volume ratio of 1:2-3, and the mixed bacteria are added into a matrix.
4. The mask staphylococcus aureus qualitative detection capability verification sample according to claim 1, wherein the sample is a freeze-dried sample, and the freeze-drying protective agent comprises trehalose at a volume fraction of 5-12%, sodium glutamate at a volume fraction of 2-8%, and gelatin at a volume fraction of 0.5-3%.
5. The mask Staphylococcus aureus qualitative detection capability verification sample according to claim 4, wherein the volume ratio of each strain to the freeze-drying protective agent is 1: 9-3: 7.
6. The qualitative detection capability verification sample of staphylococcus aureus in a mask according to claim 1, wherein the matrix is processed by the following steps: taking the disposable mask, removing the ear hanging rubber band and the metal nose support part, cutting into small pieces, resealing with a hot melting sealing machine to obtain the water-absorbing remade mask, and sterilizing the remade mask for later use.
7. The method for preparing a sample for verifying the qualitative detection capability of staphylococcus aureus in a mask according to claim 1, comprising the following steps:
s1 strain recovery passage
Recovering each strain, and identifying the recovered strain;
s2, Strain amplification
Culturing the recovered single viable strain at 36 + -1 deg.C for 36h, eluting with sterile normal saline, and gradually diluting to bacteria concentration of 103-106cfu/mL, and mixing with a freeze-drying protective agent according to the proportion of 1: 9-3: 7 to prepare freeze-dried bacterial suspension of corresponding single strain;
s3, preparing bacterial suspension
Positive sample according to target concentration of target bacteria 102-105cfu/mL and target concentration of each of the interfering bacteria 102-105Preparing bacterial suspension by cfu/mL, mixing the bacterial suspension and the interfering bacteria according to the volume ratio of 1:2-3 of the target bacteria to the interfering bacteria to prepare mixed bacterial suspension, and continuously stirring the mixed bacterial suspension on a magnetic stirrer;
negative sample interfering bacteria at a target concentration of 10 per bacteria3-106cfu/mL are mixed in equal volume to prepare a mixed bacterial suspension, and the mixed bacterial suspension is placed on a magnetic stirrer to be continuously stirred;
s4 matrix treatment
Taking a disposable mask, removing a hanging ear rubber band and a metal nose support part, cutting into small pieces, re-sealing by a hot-melting sealing machine to prepare a water-absorbable reproduced mask, and sterilizing the reproduced mask for later use;
s5, freeze-drying
And (3) dropwise adding the mixed bacterial suspension into each mask matrix, and filling the mask matrix into a sample bottle for freeze-drying after the mask completely absorbs the liquid to obtain a freeze-dried sample.
8. The method for preparing a sample for verifying the qualitative detection capability of staphylococcus aureus in a mask according to claim 1, wherein each strain is inoculated to a TSA culture medium in step S1 and aerobically cultured at 36 ℃ ± 1 ℃ for 36 hours.
9. The method for preparing a sample for verifying the qualitative detection capability of staphylococcus aureus in a mask according to claim 1, wherein the lyoprotectant of step S2 comprises trehalose at a volume fraction of 5-12%, sodium glutamate at a volume fraction of 2-8%, and gelatin at a volume fraction of 0.5-3%.
10. The method for preparing a sample for verifying the qualitative detection capability of staphylococcus aureus in a mask according to claim 1, wherein the lyophilization time in step S5 is 40-50 h.
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| WO2023116477A1 (en) * | 2021-12-22 | 2023-06-29 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Method for detecting bacterial filtration efficiency of mask |
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| WO2023116477A1 (en) * | 2021-12-22 | 2023-06-29 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Method for detecting bacterial filtration efficiency of mask |
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