CN107142304A - Pseudomonas aeruginosa proficiency testing sample and preparation method thereof in medicine - Google Patents
Pseudomonas aeruginosa proficiency testing sample and preparation method thereof in medicine Download PDFInfo
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- CN107142304A CN107142304A CN201710372963.8A CN201710372963A CN107142304A CN 107142304 A CN107142304 A CN 107142304A CN 201710372963 A CN201710372963 A CN 201710372963A CN 107142304 A CN107142304 A CN 107142304A
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Abstract
Pseudomonas aeruginosa proficiency testing sample and preparation method thereof in field of quality control in terms of the invention belongs to microbiologic inhibition tests, more particularly to a kind of medicine.Pseudomonas aeruginosa proficiency testing sample includes object bacteria and background flora in medicine, and the object bacteria is pseudomonas aeruginosa, and the background flora is made up of EHEC, Klebsiella Pneumoniae, staphylococcus aureus, Bacillus cereus.Uniformity of the present invention, stability meet proficiency testing requirement, can at utmost ensure the stability of test sample；The preparation method of sample, technique is simple, and success rate is high.
Description
Technical field
P. aeruginosa in field of quality control in terms of the invention belongs to microbiologic inhibition tests, more particularly to a kind of medicine
Bacterium proficiency testing sample and preparation method thereof.
Background technology
Medicine microbiological Test field is very special.At present, although have accreditation body both at home and abroad and organize the micro- life of medicine
Thing proficiency testing and the large-scale precedent for preparing microorganism proficiency testing sample, but sample is only to contain pseudomonas aeruginosa
Pure bacterial strain, without background bacterium exist, differed greatly with actual sample, i.e., pseudomonas aeruginosa standard sample still belong to blank neck
Domain.
Pseudomonas aeruginosa, also known as Pseudomonas aeruginosa, after intake to human body, can cause the symptoms such as people's diarrhoea, fever.If system
In standby pseudomonas aeruginosa sample, target flora easily changes (breeding and death etc.), may result in the storage life of sample
Short, the uniformity and stability of sample are poor.Therefore, consider the biochemical characteristic of bacterium, choose the metastable flora of property
It is particularly significant to the uniformity and stability for ensureing sample as target flora.The technology of pseudomonas aeruginosa sample preparation
It is required that higher, and the uniformity and stability of sample and lyophilized strain, lyophilized process conditions, freeze drying protectant make
There is close relationship with, medium of rehydration etc..
Pseudomonas aeruginosa index in medicine, directly reflects the hygienic quality and potential pathogenic risk of medicine.If medicine
Pseudomonas aeruginosa is polluted in product, patient direct infection pseudomonas aeruginosa can be caused, or even have fatal risk.Therefore,
Uniformity and all good pseudomonas aeruginosa sample of stability are prepared, randomness that pseudomonas aeruginosa is examined and not can be avoided
Certainty, truly reflects the ability of testing laboratory.This ensures drug safety, very to improving Good Laboratory controlled level
All there is special important meaning as breaking International trade practices, improving China's medicine international competitiveness.
The content of the invention
The purpose of the present invention be overcome the number of bacteria of work total in pseudomonas aeruginosa determination sample transport, storage and
During test etc. clump count the problem of can all change there is provided pseudomonas aeruginosa proficiency testing sample in a kind of medicine,
Uniformity, stability meet proficiency testing requirement, and it is a further object to provide the preparation method of the sample, technique letter
Single, success rate is high.
The technical scheme that is used to achieve the above object of the present invention is：Pseudomonas aeruginosa proficiency testing sample in medicine
Product, it is characterized in that：Including object bacteria and background flora, the object bacteria is pseudomonas aeruginosa (Pseudomonas
Aeruginosa), the background flora is by EHEC (E.coil), Klebsiella Pneumoniae (Klebsiella
Pnenmoniae), staphylococcus aureus (Staphylococcus aureus), Bacillus cereus (Bacillus
Cereus) constitute.
The sample is using trehalose, skimmed milk power and sterilized water as matrix, and wherein the volume fraction of trehalose is 12%, taken off
The volume fraction of fat milk powder is 0.5%.
The aimed concn of object bacteria is 10 in the sample2CFU/mL, the aimed concn of background flora is 103CFU/mL。
The preparation method of pseudomonas aeruginosa proficiency testing sample in a kind of medicine, it is characterized in that：Bacterium is added including sample
The selection of strain, the preparation of freeze drying protectant, sample are lyophilized, four steps of the uniformity of sample and stability test, wherein sample
Freeze-drying process is：Bacterial strain recovery passage-increasing bacterium-bacteria suspension prepares-lyophilized and packaging-storage, and detailed process is：
(1) bacterial strain recovery passage
Reference culture is inoculated into nutrient agar slant medium, it is recovered and is grown at 36 ± 1 DEG C, and to recovery
Bacterial strain is identified；
(2) bacterium is increased
Object bacteria culture to the logarithmic growth end of term, background bacteria group culture to stationary phase scrapes bacterium colony from inclined-plane, is added to 10mL jelly
The bacterium solution of corresponding single culture is made in dry protective agent；
(3) bacteria suspension is prepared
The bacterium solution that a upper process is obtained is mixed with freeze drying protectant, according to target the aimed concn 10 of bacterium2CFU/mL and background bacterium
The aimed concn 10 of group3CFU/mL prepares bacteria suspension, and the bacteria suspension of object bacteria and the bacteria suspension of 4 background bacterium, the back of the body are prepared respectively
Scape bacterium presses 1:1 volume ratio is mixed to form background flora suspension, then target bacteria suspension and background flora suspension are pressed into 1:1 volume ratio is mixed
Close, be placed in be stirred continuously down on magnetic stirring apparatus and be dispensed into sample bottle, add bottle stopper, but to leave space, reality should not be covered；
(4) freeze and pack
The sample bottle that will be equipped with plastc ring is uncapped and is put into freeze dryer, is freeze-dried 40~50h, is tied when sample is freeze-dried
Shu Hou, directly carries out jumping a queue in case, closing machine, and sample is vacuum state in sample bottle；
(5) store
It is kept in dark place under the conditions of sample is placed on into -18 DEG C, random selected sample carries out uniformity and stably from whole samples
Property experiment, satisfaction require after discharge to participate in the experiment laboratory carry out contrasting between laboralories.
The freeze drying protectant is the sterilized water containing trehalose and skimmed milk power, and wherein volume fraction is respectively：Marine alga
Sugar 12%, skimmed milk power 0.5%.
The uniformity of the sample and stability test are according to CNAS-GL03《Proficiency testing sample homogeneity and stability
Evaluation guide》Carry out.
The selection of strain of the present invention is using target flora of the pseudomonas aeruginosa as pseudomonas aeruginosa sample, with large intestine
Angstrom uncommon bacterium, Klebsiella Pneumoniae, staphylococcus aureus, Bacillus cereus are used as background flora.It is well known that verdigris is false single
Born of the same parents Pseudomonas is in non-fermentative gram-negative bacilli, using pseudomonas aeruginosa as target flora, can at utmost ensure test
The stability of sample, the content of EHEC changes in transport and storage, also will not on statistical significance
Influence the total amount of pseudomonas aeruginosa.
The pseudomonas aeruginosa proficiency testing sample of the present invention has the advantage that characteristic：Microorganism living, quantity are not sent out
Changing, biochemical character do not morph, and set about from the condition for meeting special transport, pass through the research of special process, stability
Verdigris in the sample preparation technology of a set of perfect proficiency testing, entirely appropriate development medicine is formed with the research of uniformity etc. false
The examination of monad (qualitative) project, correct evaluating ability the result.
Brief description of the drawings
Fig. 1 is present invention process flow chart.
Fig. 2 is the stability test result figure of sample pseudomonas aeruginosa under the conditions of 4 DEG C.
Fig. 3 is the stability test result figure of sample at different temperatures.
Embodiment
Below in conjunction with the accompanying drawings and specific embodiment is described in further detail to the present invention, but the invention is not limited in tool
Body embodiment.
Embodiment 1
Pseudomonas aeruginosa proficiency testing sample in medicine, including object bacteria and background flora, the object bacteria are that verdigris is false single
Born of the same parents bacterium (Pseudomonas aeruginosa), the background flora is by EHEC (E.coil), Klebsiella Pneumoniae
(Klebsiella pnenmoniae), staphylococcus aureus (Staphylococcus aureus), Bacillus cereus
(Bacillus cereus) is constituted.
The sample is using trehalose, skimmed milk power and sterilized water as matrix, and wherein trehalose volume fraction is 12%, degreasing
Milk powder volume fraction is 0.5%.
The aimed concn of object bacteria is 10 in the sample2CFU/mL, the aimed concn of background flora is 103CFU/mL。
Embodiment 2
As shown in figure 1, in a kind of embodiment 1 in medicine pseudomonas aeruginosa proficiency testing sample preparation method, including sample
Add that the selection of bacterial strain, the preparation of freeze drying protectant, sample be lyophilized, four steps of the uniformity of sample and stability test, tool
Body step is as follows：
1st, sample adds the selection of bacterial strain
According to object bacteria：Pseudomonas aeruginosa (Pseudomonas aeruginosa), background flora：EHEC
(E.coil), Klebsiella Pneumoniae (Klebsiella pnenmoniae), staphylococcus aureus (Staphylococcus
Aureus), Bacillus cereus (Bacillus cereus) selection standard bacterial strain, all reference cultures are purchased from government and specified
Mechanism, and with bacterial strain certificate, it is ensured that the traceability of bacterial strain.
2nd, the preparation of freeze drying protectant
Sample is using trehalose, skimmed milk power and sterilized water as matrix (volume fraction：Trehalose 12%, skimmed milk power 0.5%) match somebody with somebody
Freeze drying protectant processed.
3rd, sample is freezed
(1) bacterial strain recovery passage
Reference culture is inoculated into nutrient agar, it is recovered and is grown at 36 ± 1 DEG C, and to the bacterial strain of recovery
Identified；
(2) bacterium is increased
Object bacteria culture to the logarithmic growth end of term, background bacteria group culture to stationary phase scrapes bacterium colony from inclined-plane, is added to 10mL jelly
The bacterium solution of corresponding single culture is made in dry protective agent；
(3) bacteria suspension is prepared
The bacterium solution that a upper process is obtained is mixed with freeze drying protectant, according to target the aimed concn 10 of bacterium2CFU/mL and background bacterium
The aimed concn 10 of group3CFU/mL prepares bacteria suspension.To ensure the bacterial strain containing above aimed concn in obtained final sample,
According to object bacteria 10 in the present embodiment2CFU/mL prepares the bacteria suspension of object bacteria；Background bacterium presses 103CFU/mL prepares 4 background bacterium
Bacteria suspension, by 1:1 volume ratio is mixed, and forms background flora suspension；Background flora suspension presses 1 with target bacteria suspension:1 volume ratio
Mixing, obtains plastc ring, and bacterial strain content is checked using turbidimetry；It is placed in be stirred continuously down on magnetic stirring apparatus and takes 1.0mL
It is dispensed into sample bottle (cillin bottle), adds bottle stopper, but to leave space, reality should not be covered
(4) freeze and pack
The sample bottle that will be equipped with plastc ring is uncapped and is put into freeze dryer, and freeze drier parameter is by following setting：
Chilling rate (Cooling Rate) 0.5 DEG C/min
- 1 DEG C of 15min of early stage cold point (Incipient Freezing Point)
- 40 DEG C of cryogenic temperature (Cooling temperature)
- 29 DEG C of eutectic point (Melting Point Eutectic temperature)
20 DEG C of 120min of heating-up temperature (Heating temperature)
Start freeze drier, machine is directly entered the freezing dry process of sequencing, whole freeze-drying process 40h.
After sample freeze-drying terminates, directly carry out jumping a queue in case；Closing machine.The not tight cillin bottle of plug is rejected,
Sample is vacuum state in cillin bottle；
(5) store
It is kept in dark place under the conditions of sample is placed on into -18 DEG C, and the sample of preservation suitably manage and detect, from whole sample
Random selected sample carries out uniformity and stability test in product, and satisfaction requires that after discharge carries out laboratory monitoring to laboratory of participating in the experiment
Compare.
4th, the uniformity of sample and stability test
Uniformity and stability inspection to object bacteria in sample are the main methods of verification sample preparation process validity.Check
Sample homogeneity and stability are according to CNAS-GL03《Proficiency testing sample homogeneity and estimation of stability guide》Carry out.
Gained sample homogeneity and Detection of Stability method and result are as follows：
12 samples are randomly selected respectively, and using SN/T 1897-2007 methods, the verdigris of 2 × 12 parts of samples is tested in repeat condition
Pseudomonad.Result data carries out statistical disposition with one-way analysis of variance (ANOV), and statistic procedure and result are as follows：
The variance analysis formula of table 1
In upper table,
The sample homogeneity testing result of table 2
The sample homogeneity of table 3 tests the results of analysis of variance
Conclusion：Under 95% fiducial probability, compared with influence of the other factors to test result, the inhomogeneities of sample is to connect
Receive.
Using two kinds of stability test：One kind is the stability test at short temperature (4 DEG C), another
Kind it is the stability test at high temperature (traffic condition of analog sample), from three temperature spots, respectively 20 DEG C,
36 DEG C and 45 DEG C.Periodic detection sample, tests 3 samples, by 2 × 3 parts of samples for the different temperature points different holding time
As a result average (logarithmic transformed after) and uniformity test results contrast, between absolute difference divided by test plan in ability comment
Valency is with sane standard deviation S*, it is that principle determines to meet proficiency testing sample requirement under condition of different temperatures that ratio, which is less than 0.3,
The most long holding time.Stability test result is shown in Fig. 2 and Fig. 3.
Embodiment 3
In medicine described in the present embodiment each step of the preparation method of pseudomonas aeruginosa proficiency testing sample with implementation
Identical in example 2, different technical parameters are：Strain cultures select nutrient agar slant medium；During bacteria suspension is prepared, mesh
The concentration of object bacteria in bacteria suspension is marked according to 5 × 102CFU/mL is prepared, and the concentration of each background bacterium is according to 10 in background flora suspension3
CFU/mL is prepared；Freeze-drying process 45h.
Embodiment 4
In medicine described in the present embodiment each step of the preparation method of pseudomonas aeruginosa proficiency testing sample with implementation
Identical in example 3, different technical parameters are：During bacteria suspension is prepared, in target bacteria suspension the concentration of object bacteria according to 4.5 ×
102CFU/mL is prepared；Freeze-drying process 50h.
Claims (6)
1. pseudomonas aeruginosa proficiency testing sample in medicine, it is characterized in that：Including object bacteria and background flora, the object bacteria
For pseudomonas aeruginosa, the background flora is by EHEC, Klebsiella Pneumoniae, staphylococcus aureus, waxy brood cell
Bacillus constitutes.
2. pseudomonas aeruginosa proficiency testing sample in medicine according to claim 1, it is characterized in that：The sample is with sea
Algae sugar, skimmed milk power and sterilized water are matrix, and the volume fraction of wherein trehalose is that the volume fraction of 12%, skimmed milk power is
0.5%。
3. pseudomonas aeruginosa proficiency testing sample in medicine according to claim 1 or 2, it is characterized in that：The sample
The aimed concn of middle object bacteria is 102 CFU/mL, the aimed concn of background flora is 103 CFU/mL。
4. the preparation method of pseudomonas aeruginosa proficiency testing sample in medicine according to claim 1, it is characterized in that：Bag
Include the sample addition selection of bacterial strain, the preparation of freeze drying protectant, lyophilized sample, four steps of the uniformity of sample and stability test
Suddenly, wherein sample freeze-drying process is：Bacterial strain recovery passage-increasing bacterium-bacteria suspension prepares-lyophilized and packaging-storage, specific mistake
Cheng Wei：
（1）Bacterial strain recovery passage
Reference culture is inoculated into nutrient agar slant medium, it is recovered and is grown at 36 ± 1 DEG C, and to recovery
Bacterial strain is identified；
（2）Increase bacterium
Object bacteria culture to the logarithmic growth end of term, background bacteria group culture to stationary phase scrapes bacterium colony from inclined-plane, is added to 10mL jelly
The bacterium solution of corresponding single culture is made in dry protective agent；
（3）Bacteria suspension is prepared
The bacterium solution that a upper process is obtained is mixed with freeze drying protectant, according to target the aimed concn 10 of bacterium2CFU/mL and background bacterium
The aimed concn 10 of group3 CFU/mL prepares bacteria suspension, and the bacteria suspension of object bacteria and the bacteria suspension of 4 background bacterium, the back of the body are prepared respectively
Scape bacterium presses 1:1 volume ratio is mixed to form background flora suspension, then target bacteria suspension and background flora suspension are pressed into 1:1 volume ratio is mixed
Close, be placed in be stirred continuously down on magnetic stirring apparatus and be dispensed into sample bottle, add bottle stopper, but to leave space, reality should not be covered；
（4）Lyophilized and packaging
The sample bottle that will be equipped with plastc ring is uncapped and is put into freeze dryer, is freeze-dried 40~50h, is tied when sample is freeze-dried
Shu Hou, directly carries out jumping a queue in case, closing machine, and sample is vacuum state in sample bottle；
（5）Storage
It is kept in dark place under the conditions of sample is placed on into -18 DEG C, random selected sample carries out uniformity and stably from whole samples
Property experiment, satisfaction require after discharge to participate in the experiment laboratory carry out contrasting between laboralories.
5. the preparation method of pseudomonas aeruginosa proficiency testing sample in medicine according to claim 4, it is characterized in that：Institute
Freeze drying protectant is stated for the sterilized water containing trehalose and skimmed milk power, wherein volume fraction is respectively：Trehalose 12%, degreasing
Milk powder 0.5%.
6. the preparation method of pseudomonas aeruginosa proficiency testing sample in medicine according to claim 4, it is characterized in that：Institute
State sample uniformity and stability test according to CNAS-GL03《Proficiency testing sample homogeneity and estimation of stability guide》
Carry out.
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Cited By (6)
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| CN109868238A (en) * | 2019-02-28 | 2019-06-11 | 浙江宝录检测技术有限公司 | Bacillus subtilis employment and suitability test (E & ST) bacterial strain and preparation method thereof |
| CN109971673A (en) * | 2019-02-28 | 2019-07-05 | 浙江宝录检测技术有限公司 | Clostridium sporogenes employment and suitability test (E & ST) bacterial strain and preparation method thereof |
| CN109971672A (en) * | 2019-02-28 | 2019-07-05 | 浙江宝录检测技术有限公司 | Pseudomonas aeruginosa employment and suitability test (E & ST) bacterial strain and preparation method thereof |
| CN111662949A (en) * | 2020-07-13 | 2020-09-15 | 北京海关技术中心 | Sample for verifying qualitative detection capability of pseudomonas aeruginosa in mask and preparation method thereof |
| CN111690711A (en) * | 2020-07-13 | 2020-09-22 | 北京海关技术中心 | Sample for verifying qualitative detection capability of pseudomonas aeruginosa in cotton swab and preparation method thereof |
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