CN105624263A - Standard sample for aminoglycocide resistant staphylococcus aureus detection and preparation method thereof - Google Patents
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Abstract
The invention belongs to the field of microbiological examination and quarantine, in particular to a standard sample for aminoglycocide resistant staphylococcus aureus detection. The standard sample contains a freeze-dried bacterial mixture and a freeze-drying protective additive and is vacuum-packed with penicillin bottles, wherein the bacterial mixture comprises aminoglycocide resistant staphylococcus aureus. The successful preparation of the standard sample provided by the invention has a very important practical significance in comprehensive and thorough development of solutions to the preparation technology and the stability guarantee technology of food-borne pathogenic bacteria resistant standard samples and active development of food-borne pathogenic bacteria resistance detection standard samples in China to fill a blank in the detection field; besides, as food safety has been paid more and more attention, carrying out food-borne pathogenic bacteria resistance detection becomes increasingly significant, and standard samples are important elements for quality control of laboratories, so that food-borne pathogenic bacteria resistant standard samples have a broad market, as well as greater economic benefits and social benefits.
Description
Technical field
The invention belongs to Micro biological Tests quarantine field, particularly streptococcus aureus detection standard model.
Background technology
Food safety is great public health problem, is the important factor of restriction China Economic development. And the food origin disease that microbial contamination causes, it is problem the most outstanding in China's food safety. In recent years, along with developing rapidly of the adjustment of food industries structure, economic globalization and international trade, the prevention and control of China's food origin disease face a series of new problem and challenge.
Sensitivity testing to antibacterials (antimicrobialsusceptibilitytest, AST) it is bacterial drug resistance monitoring and the important means of epidemiology survey and resistance mechanism research, comprises disk diffusion method, meat soup/agar dilution, self-reacting device detection method, E-test method etc. The widely used one method of disk diffusion method teacher clinical labororatory, the disk diffusion method that WHO recommends is K-B method (improvement Kirby-Bauer method), the U.S. clinical Laboratory Standard council (CLSI) is annual all to carry out supplementing correction to its standard according to new result of study, and current NCCLS/CLSI standard affects the widest in the world.
At present, streptococcus aureus and intestinal bacteria resistance are detected by domestic main employing disk diffusion method, but it is more special for the E. coli detection producing ESBLs, mainly contain for detecting the method for ESBLs: two scraps of paper work in coordination with diffusion process (double-disksynergytest, DDS), the disk diffusion method (inhibitor-potentiateddiscdiffusiontest) that enzyme inhibitors strengthens, broth dilution method (the inhibitorpotentiatedbrothdilutedtest that enzyme inhibitors strengthens, IPBDT), three-dimensional test method (three-dimensionaltests, TD), head spore nitre saliva Fen competition analysis test (nitrocefincompetitionassayNCA), Etest method and Vitek test method etc. these cardinal principles are that ESBLs is hydrolyzed third generation head and embraces the detection method that the activity of bacterium element can suppress by enzyme inhibitors, are applicable to the detection of ESBLs in Klebsiella Pneumoniae, Klebsiella oxytoca and escherichia coli, are the standard methods detecting ESBLs at present. currently used SN/T1944-2007 " in animal and goods thereof the mensuration disk diffusion method of bacterial drug resistance " examination criteria, bacterium is carried out interpretation whether resistance by the method mainly recommended based on CLSI and resistance break.
Summary of the invention
It is an object of the invention to overcome the situation that in current food, pathogenic bacterium resistance standard model is in short supply, needs according to real work and present status in China have carried out the development of pathogenic bacterium resistance standard model in food, homogeneity and having good stability, meet inspection and quarantine requirement, it is to increase inspection and quarantine level and efficiency.
The technical scheme that the present invention adopts for achieving the above object is: aminoglycoside resistant streptococcus aureus detection standard model; sample contains bacterial mixture and the lyophilized vaccine of freeze-drying; adopting cillin bottle vacuum packaging, wherein bacterial mixture comprises aminoglycoside resistant streptococcus aureus.
Described standard model every bottle sample-loading amount is 1 gram, on average containing 2.0-4.0 �� 10 in every part of sample6Cfu object bacteria.
The method for making of aminoglycoside resistant streptococcus aureus detection standard model of the present invention: detailed process is as follows:
The activation of the first step bacterial strain, Zengjing Granule and purifying:
Reference culture used derives from USS bacterial classification collection institute (AmericanTypeCultureCollection), reference culture ATCC51329, aminoglycoside resistant streptococcus aureus reference culture carries out bacterial strain activation, Zengjing Granule, carries out biochemical identification and drug sensitive test;
2nd step sample preparation:
Adopt vacuum lyophilization mode to prepare, sterile purified water will add protective material, again boil sterilizing; The erythromycin resistance streptococcus aureus of activation is added after cooling, mixed even; Divide and it is filled in aseptic dry cillin bottle, vacuum lyophilization;
3rd step Bacteria Identification and resistance measure:
The standard model getting preparation is identified, through adopting biochemical identification and drug sensitive test analysis, the standard model prepared by confirmation is aminoglycoside resistant streptococcus aureus standard model;
4th step uniformity test:
Adopt and add aminoglycoside resistant streptococcus aureus in the base and form full-page proof, then fully mixed even and the method for packing prepares 100 parts of standard models; According to conventional result of study, in fully mixed even full-page proof, object bacteria obeys Poisson's distribution, and in full-page proof, object bacteria addition is 2.0-4.0 �� 108Cfu, on average containing 2.0-4.0 �� 10 in every part of sample6Cfu object bacteria; Calculate according to Poisson's distribution, containing 2.0-4.0 �� 10 in each sample6The probability of cfu object bacteria is 99.81%; Object bacteria amount in every bottle of sample is controlled in 2.0-4.0 �� 106Between cfu, the homogeneity of sample is ensured;
5th step stability test:
The stability of this standard model through the mensuration of 2 years, aminoglycoside resistant streptococcus aureus standard model in dry conditions, lucifuge, sealing, refrigerated storage, stable in 2 years.
Described standard model carries out the 6th step uncertainty evaluation after stability test:
According to GUM[4], it is determined that the uncertainty of measurement model of aminoglycoside resistant streptococcus aureus content W; The stardard uncertairty degree that the synthetic standards uncertainty of W is far longer than in bottle between ununiformity and bottle that ununiformity is brought, therefore, it is possible to ignore in bottle between ununiformity and bottle ununiformity to the impact of standard model definite value uncertainty; The aminoglycoside resistant streptococcus aureus wherein added is 100% pure substance, and its uncertainty can also be ignored;
Getting Coverage factor k=2, in standard model, aminoglycoside resistant streptococcus aureus content expansion uncertainty is 0.11%.
Concrete technique prepared by described 2nd step sample is as follows:
A., between fixed temperature and humidity, aseptic technique, preparation is containing 2.0-4.0 �� 108The full-page proof of cfu, mixes even above-mentioned sample by specific mode; The mixing of described specific mode sample is that sample is placed in clean sterile bag, and mixed instrument pats mixing, then joins in the overall bean powder aqueous solution by mixed even good above-mentioned solution, repeats mixing step;
B. being positioned in pilot lyophilizer by biased sample cryogenic vacuum lyophilize is prepared into freeze-dried mixed powder;
C. freeze-dried mixed powder sample is sub-packed in the 7mL Brown Glass Brown glass bottles and jars only of the cleaning of 160 DEG C of dry heat treatment 2h and seals lid sealing, every bottle of in-built freeze-dried mixed powder sample 1g. The sample that finally all packing are good is through 60CO4kGy irradiation 1h;
D. 100 bottles of standard models containing aminoglycoside resistant streptococcus aureus are obtained. Unique identification sticked on by sample after bottled, refrigerated storage.
Lyophilize in described b.:
The bacterium liquid packing 0.5mL/ bottle that will prepare in a step, places serum cap, but fortress is not tight, is placed in pilot lyophilizer cryogenic vacuum lyophilize, is prepared into freeze-dried mixed powder;
Refrigerating process is as follows:
1) the precooling stage: baffle temperature 5 DEG C;
2) in the freezing stage: arranging baffle temperature is 10 DEG C, and each stage reduces by 10 DEG C, to-50 DEG C, each temperature stage is respectively 1h working time;
3) primary drying phase:Baffle temperature-50 DEG C, working time 0.5h, vacuum tightness 0.2mbar;Baffle temperature-40 DEG C, working time 7h, vacuum tightness 0.4mbar;Baffle temperature-35 DEG C, working time 7h, vacuum tightness 0.6mbar;Baffle temperature-25 DEG C, working time 5h, vacuum tightness 0.8mbar;Baffle temperature-15 DEG C, working time 4h, vacuum tightness 1.0mbar;Baffle temperature-5 DEG C, working time 2h, vacuum tightness 1.2mbar;
4) the redrying stage:Baffle temperature 0 DEG C, working time 3h, vacuum tightness 0.0010mbar;Baffle temperature 15 DEG C, working time 3h, vacuum tightness 0.0010mbar;Baffle temperature 25 DEG C, working time 3h, vacuum tightness 0.0010mbar.
The preparation of standard model of the present invention completes, to the comprehensively deep technology of preparing researching and solving pathogenic bacterium resistance standard model in food and stability guarantee technology, actively develop the development of pathogenic bacterium Resistance detection standard model in China's food, replenish the blank of this fields of measurement, there is very important realistic meaning. In addition people are to the concern of food safety aspect and attention, and in conducting food pathogenic bacterium Resistance detection work expansion day by day and importance, standard model is again the important step of Good Laboratory control work, food pathogenic resistance standard model has wide market, has bigger economic benefit and social benefit.
Accompanying drawing explanation
Fig. 1 is aminoglycoside resistant streptococcus aureus preparation of standard sample process flow sheet of the present invention.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail, but the present invention is not limited to specific embodiment.
Embodiment 1 prepares erythromycin aminoglycoside resistant streptococcus aureus standard model
As shown in Figure 1, specific embodiment is as follows:
The activation of the first step bacterial strain, Zengjing Granule and purifying:
Reference culture used derives from USS bacterial classification collection institute (AmericanTypeCultureCollection), and reference culture ATCC51329, aminoglycoside resistant streptococcus aureus reference culture is according to GB/T4789.40-2008[1]Carry out bacterial strain activation, Zengjing Granule, carry out biochemical identification and drug sensitive test;
2nd step sample preparation:
Adopt vacuum lyophilization mode to prepare, sterile purified water will add protective material, again boil sterilizing; The erythromycin resistance streptococcus aureus of activation is added after cooling, mixed even; Divide and it is filled in aseptic dry cillin bottle, vacuum lyophilization;
Instrument and reagent
Freeze drier, sterilizing milk powder, trehalose, Brown Glass Brown glass bottles and jars only, biochemical susceptibility assessing instrument
Operation steps
A., between fixed temperature and humidity, aseptic technique, preparation is containing 2.0 �� 108The full-page proof of cfu, mixes even above-mentioned sample by specific mode;
B. being positioned in pilot lyophilizer by biased sample cryogenic vacuum lyophilize (carrying out according to specific freeze-drying program) is prepared into freeze-dried mixed powder;
C. freeze-dried mixed powder sample is sub-packed in the 7mL Brown Glass Brown glass bottles and jars only of the cleaning of 160 DEG C of dry heat treatment 2h and seals lid sealing, every bottle of in-built freeze-dried mixed powder sample 1g. The sample that finally all packing are good is through 60CO4kGy irradiation 1h;
D. 100 bottles of standard models containing aminoglycoside resistant streptococcus aureus are obtained. Unique identification sticked on by sample after bottled, refrigerated storage.
3rd step Bacteria Identification and resistance measure:
Get the standard model of preparation, key lab is detected with transgenic plant product and microorganism detection laboratory, Heilungkiang Entry-Exit Inspection and Quarantine Bureau technique center is identified by the national microorganism in Liaoning Entry-Exit Inspection and Quarantine Bureau technique center, through adopting GB4789.10-2010 " national food safety standard food microbiological analysis streptococcus aureus ", SN/T1944-2007 " in animal and goods thereof the mensuration disk diffusion method of bacterial drug resistance " industry standard, carry out Bacteria Identification and drug sensitive test analysis, standard model prepared by confirmation is aminoglycoside resistant streptococcus aureus standard model,
4th step uniformity test:
Adopt and add aminoglycoside resistant streptococcus aureus in the base and form full-page proof, then fully mixed even and the method for packing prepares 100 parts of standard models; According to conventional result of study, in fully mixed even full-page proof, object bacteria obeys Poisson's distribution, and in full-page proof, object bacteria addition is 2.0 �� 108Cfu, on average containing 2.0 �� 10 in every part of sample6Cfu object bacteria. Calculate according to Poisson's distribution, containing 2.0 �� 10 in each sample6The probability of cfu object bacteria is 99.81%. Namely only there will be containing 2.0 �� 10 less than 1 bottle of sample in 100 bottles of samples6Below cfu or 2.0 �� 106The situation of more than cfu object bacteria. Object bacteria amount in every bottle of sample is controlled in 2.0 �� 106Between cfu, the homogeneity of sample is ensured.
5th step stability test
The stability of this standard model through the mensuration of 2 years, aminoglycoside resistant streptococcus aureus standard model in dry conditions, lucifuge, sealing, refrigerated storage, stable in 2 years.
6th step uncertainty evaluation:
Average
This standard sample adopts and adds aminoglycoside resistant streptococcus aureus formation full-page proof in the base, then fully mixed even and the method for packing prepares 100 parts of standard models, according to conventional result of study, in fully mixed even full-page proof, object bacteria obeys Poisson's distribution, and in full-page proof, object bacteria addition is 2.0 �� 108Cfu, on average containing 2.0 �� 10 in every part of sample6Cfu object bacteria.
Uncertainty evaluation
According to GUM, it is determined that the uncertainty of measurement model of aminoglycoside resistant streptococcus aureus content W. The stardard uncertairty degree that the synthetic standards uncertainty of W is far longer than in bottle between ununiformity and bottle that ununiformity is brought, therefore, ununiformity can be ignored in bottle between ununiformity and bottle on the impact of standard model definite value uncertainty, the aminoglycoside resistant streptococcus aureus added is 100% pure substance, and its uncertainty can also be ignored;
Getting Coverage factor k=2, in standard model, aminoglycoside resistant streptococcus aureus content expansion uncertainty is 0.11%.
The expression of standard model definite value
In this standard sample, aminoglycoside resistant streptococcus aureus content (/g) is: 2.0 �� 106cfu��0.11%��
Finally carry out packing and store
Label substance: title, lot number, content, weight, validity period and preparation unit.
Packaging: 1g fills, and internal packing is Brown Glass Brown glass bottles and jars only, the hard box of outer packaging plastic cement.
Storage: under drying conditions, lucifuge, sealing, storage at normal temperature, stable in 2 years.
Embodiment 2
Preparing erythromycin aminoglycoside resistant streptococcus aureus standard model, specific embodiment is identical with embodiment 1, the difference is that object bacteria addition 4.0 �� 10 in full-page proof8cfu��
Claims (6)
1. aminoglycoside resistant streptococcus aureus detection standard model; it is characterized in that; standard model contains bacterial mixture and the lyophilized vaccine of freeze-drying, adopts cillin bottle vacuum packaging, and wherein bacterial mixture comprises aminoglycoside resistant streptococcus aureus.
2. aminoglycoside resistant streptococcus aureus detection standard model according to claim 1, is characterized in that, described standard model every bottle sample-loading amount is 1 gram, on average containing 2.0-4.0 �� 10 in every part of sample6Cfu object bacteria.
3. the method for making of aminoglycoside resistant streptococcus aureus detection standard model, is characterized in that, detailed process is as follows:
The activation of the first step bacterial strain, Zengjing Granule and purifying:
Reference culture used derives from USS bacterial classification and collects institute, reference culture ATCC51329, and aminoglycoside resistant streptococcus aureus reference culture carries out bacterial strain activation, Zengjing Granule, carries out biochemical identification and drug sensitive test;
2nd step sample preparation:
Adopt vacuum lyophilization mode to prepare, sterile purified water will add protective material, again boil sterilizing; The erythromycin resistance streptococcus aureus of activation is added after cooling, mixed even; Divide and it is filled in aseptic dry cillin bottle, vacuum lyophilization;
3rd step Bacteria Identification and resistance measure:
The standard model getting preparation is identified, through adopting biochemical identification and drug sensitive test analysis, the standard model prepared by confirmation is aminoglycoside resistant streptococcus aureus standard model;
4th step uniformity test:
Adopt and add aminoglycoside resistant streptococcus aureus in the base and form full-page proof, then fully mixed even and the method for packing prepares 100 parts of standard models; According to conventional result of study, in fully mixed even full-page proof, object bacteria obeys Poisson's distribution, and in full-page proof, object bacteria addition is 2.0-4.0 �� 108Cfu, on average containing 2.0-4.0 �� 10 in every part of sample6Cfu object bacteria; Calculate according to Poisson's distribution, containing 2.0-4.0 �� 10 in each sample6The probability of cfu object bacteria is 99.81%; Object bacteria amount in every bottle of sample is controlled in 2.0-4.0 �� 106Between cfu, the homogeneity of sample is ensured;
5th step stability test
The stability of this standard model through the mensuration of 2 years, aminoglycoside resistant streptococcus aureus standard model in dry conditions, lucifuge, sealing, refrigerated storage, stable in 2 years.
4. the method for making of aminoglycoside resistant streptococcus aureus detection standard model according to claim 3, is characterized in that, described standard model carries out the 6th step uncertainty evaluation after stability test:
According to GUM, it is determined that the uncertainty of measurement model of aminoglycoside resistant streptococcus aureus content W; The stardard uncertairty degree that the synthetic standards uncertainty of W is far longer than in bottle between ununiformity and bottle that ununiformity is brought, therefore, it is possible to ignore in bottle between ununiformity and bottle ununiformity to the impact of standard model definite value uncertainty; The aminoglycoside resistant streptococcus aureus wherein added is 100% pure substance, and its uncertainty can also be ignored;
Getting Coverage factor k=2, in standard model, aminoglycoside resistant streptococcus aureus content expansion uncertainty is 0.11%.
5. the method for making of aminoglycoside resistant streptococcus aureus detection standard model according to claim 3 or 4, is characterized in that, concrete technique prepared by described 2nd step sample is as follows:
A., between fixed temperature and humidity, aseptic technique, preparation is containing 2.0-4.0 �� 108The full-page proof of cfu, mixed even above-mentioned sample;
B. being positioned in pilot lyophilizer by biased sample cryogenic vacuum lyophilize is prepared into freeze-dried mixed powder;
C. being sub-packed in the 7mL Brown Glass Brown glass bottles and jars only of the cleaning of 160 DEG C of dry heat treatment 2h by freeze-dried mixed powder sample and seal lid sealing, every bottle of in-built freeze-dried mixed powder sample 1g, the sample that finally all packing are good is through 60CO4kGy irradiation 1h;
D. obtaining 100 bottles of standard models containing aminoglycoside resistant streptococcus aureus, unique identification sticked on by the sample after bottled, refrigerated storage.
6. the method for making of aminoglycoside resistant streptococcus aureus detection standard model according to claim 5, is characterized in that, lyophilize in described b.:
The bacterium liquid packing 0.5mL/ bottle that will prepare in a step, places serum cap, but fortress is not tight, is placed in pilot lyophilizer cryogenic vacuum lyophilize, is prepared into freeze-dried mixed powder;
Refrigerating process is as follows:
1) the precooling stage: baffle temperature 5 DEG C;
2) in the freezing stage: arranging baffle temperature is 10 DEG C, and each stage reduces by 10 DEG C, to-50 DEG C, each temperature stage is respectively 1h working time;
3) primary drying phase:Baffle temperature-50 DEG C, working time 0.5h, vacuum tightness 0.2mbar;Baffle temperature-40 DEG C, working time 7h, vacuum tightness 0.4mbar;Baffle temperature-35 DEG C, working time 7h, vacuum tightness 0.6mbar;Baffle temperature-25 DEG C, working time 5h, vacuum tightness 0.8mbar;Baffle temperature-15 DEG C, working time 4h, vacuum tightness 1.0mbar;Baffle temperature-5 DEG C, working time 2h, vacuum tightness 1.2mbar;
4) the redrying stage:Baffle temperature 0 DEG C, working time 3h, vacuum tightness 0.0010mbar;Baffle temperature 15 DEG C, working time 3h, vacuum tightness 0.0010mbar;Baffle temperature 25 DEG C, working time 3h, vacuum tightness 0.0010mbar.
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| CN107190048A (en) * | 2017-05-24 | 2017-09-22 | 中检科（北京）实验室能力评价有限公司 | Staphylococcus aureus proficiency testing sample and preparation method thereof in medicine |
| CN107236784A (en) * | 2017-06-28 | 2017-10-10 | 中国检验检疫科学研究院 | Staphylococcus aureus standard sample and preparation method thereof in milk powder |
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| CN115410653A (en) * | 2022-08-31 | 2022-11-29 | 吉林金域医学检验所有限公司 | Bacterial drug resistance monitoring method and device, computer equipment and storage medium |
| CN115410653B (en) * | 2022-08-31 | 2023-10-17 | 吉林金域医学检验所有限公司 | Bacterial drug resistance monitoring method, device, computer equipment and storage medium |
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