Sample for verifying qualitative detection capability of pseudomonas aeruginosa in cotton swab and preparation method thereof
阅读说明：本技术 棉签中铜绿假单胞菌定性检测能力验证样品及其制备方法 (Sample for verifying qualitative detection capability of pseudomonas aeruginosa in cotton swab and preparation method thereof ) 是由 张惠媛 刘鑫 李秋阳 石嵩 高欣 于 2020-07-13 设计创作，主要内容包括：本发明公开了一种棉签中铜绿假单胞菌定性检测能力验证样品及其制备方法,首次采用棉签作为基质,添加目标菌和/或干扰菌制备阳性样品和阴性样品,与通常为冻干粉性质的微生物领域能力验证模拟样品不同的是,本发明可以提供一种可以复现检测流程的实物样品,为在一次性使用卫生用品微生物学能力验证的先例。该样品可用于实验室检测能力的评价,检测过程的质量控制、人员考核、方法验证等,填补了国内外空白。同时本发明的样品,均匀性、稳定性符合能力验证要求,避免棉签中铜绿假单胞菌检测样品中活的致病菌在运输、储存和测试等过程中都可发生变化的问题。另外本发明提供的制备方法,工艺简单,制备符合要求的样品成功率高。(The invention discloses a qualitative detection capability verification sample of pseudomonas aeruginosa in a cotton swab 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 sample of the invention has uniformity and stability meeting the capability verification requirements, and the problem that the viable pathogenic bacteria in the pseudomonas aeruginosa detection sample in the cotton swab can change in the processes of transportation, storage, test 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.)
1. The sample for verifying the qualitative detection capability of the pseudomonas aeruginosa in the cotton swab is characterized by comprising a negative sample and a positive sample, wherein the positive sample contains a substrate, target bacteria and interfering bacteria, and the negative sample contains the substrate and the interfering bacteria; the substrate is cotton swabs, the target bacteria are pseudomonas aeruginosa, and the interfering bacteria are one or more of pseudomonas putida, escherichia coli, bacillus proteus vulgaris and bacillus mycoides.
2. The cotton swab of claim 1, wherein the concentration of bacteria in the sample is 103-106cfu/mL。
3. The cotton swab for verifying the qualitative detection capability of the pseudomonas aeruginosa according to claim 1, wherein the target bacteria and the interfering bacteria in the positive sample are mixed into a mixed bacteria according to the volume ratio of 1:2-3, and the mixed bacteria is added into the matrix.
4. The qualitative test capability verification sample of pseudomonas aeruginosa in a cotton swab as claimed in claim 1, wherein the sample is a freeze-dried sample, and the freeze-drying protective agent comprises trehalose with a volume fraction of 8-10%, sodium glutamate with a volume fraction of 3-5%, gelatin with a volume fraction of 0.5-3% and skim milk powder with a volume fraction of 2-5%.
5. The cotton swab for verifying the qualitative detection capability of the pseudomonas aeruginosa according to claim 4, wherein the volume ratio of each strain to the lyoprotectant is 1: 9-3: 7.
6. The qualitative test capability verification sample of pseudomonas aeruginosa in a cotton swab as claimed in claim 1, wherein the cotton swab is made of absorbent cotton and is ready for use after sterilization.
7. The method of preparing a sample for verifying the qualitative detection capability of pseudomonas aeruginosa in a cotton swab as claimed in claim 1, comprising the steps of:
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 cotton swab, wherein the cotton swab is made of absorbent cotton and is used for standby after sterilization;
s5, freeze-drying
And (3) dropwise adding the mixed bacterial suspension into each part of cotton swab matrix, filling the cotton swab matrix into a sample bottle after the cotton swab completely absorbs the liquid, and freeze-drying to obtain a freeze-dried sample.
8. The method for preparing a sample for verifying the qualitative detection capability of pseudomonas aeruginosa in a cotton swab as claimed in claim 1, wherein in step S1, each strain is inoculated into a TSA culture medium and aerobically cultured at 36 ℃ ± 1 ℃ for 36 h.
9. The method for preparing a qualitative detection capability verification sample of pseudomonas aeruginosa in a cotton swab as claimed in claim 1, wherein the lyoprotectant of step S2 comprises trehalose 8-10% by volume, sodium glutamate 3-5% by volume, gelatin 0.5-3% by volume, and skim milk powder 2-5% by volume.
10. The method for preparing a sample for verifying the qualitative detection capability of pseudomonas aeruginosa in a cotton swab as claimed in claim 1, wherein the lyophilization time in the step S5 is 40-50 h.
Technical Field
The invention relates to the technical field of microbiological inspection quality control, in particular to a sample for verifying the qualitative detection capability of pseudomonas aeruginosa in a cotton swab and a preparation method thereof.
Background
The cotton swab belongs to a disposable sanitary product, which is various daily living goods which 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. 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, there is no organization/organization to prepare a sample for qualitative detection of pseudomonas aeruginosa by taking a cotton swab as a matrix at home and abroad, and no related literature report exists.
Pseudomonas aeruginosa (p. aeruginosa), or pseudomonas aeruginosa, is a less pathogenic but highly drug resistant bacterium. Is widely existed in nature, and is a kind of bacteria which is common to wound infection. Can cause suppurative lesion. The disease after infection is named because the pathogens such as pus and exudate are green. Pseudomonas aeruginosa (p. aeruginosa) belongs to pseudomonas, widely distributed in the nature and normal human skin, intestinal tract and respiratory tract, and is one of the more common clinical opportunistic pathogens. Therefore, a sample for verifying the qualitative detection capability of pseudomonas aeruginosa in the mask is urgently needed.
Disclosure of Invention
Aiming at the technical problems, the invention provides a sample for verifying the qualitative detection capability of pseudomonas aeruginosa in a cotton swab and a preparation method thereof, and provides important guarantee for the 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 verification sample of the qualitative detection capability of pseudomonas aeruginosa in a cotton swab, 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 a substrate and interference bacteria; the substrate is cotton swabs, the target bacteria are pseudomonas aeruginosa (P.aeruginosa), and the interfering bacteria are one or more of pseudomonas putida (P.putica), Escherichia coli (Escherichia coli), Bacillus vulgaris (Proteus vulgaris) and Bacillus mycoides (Bacillus mycoides).
In the verification sample of the qualitative detection capability of the pseudomonas aeruginosa in the cotton swab, the concentration of bacteria in the sample is 103-106cfu/mL。
In the verification sample of the qualitative detection capability of the pseudomonas aeruginosa in the cotton swab, the target bacteria and the 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 the matrix.
In the qualitative detection capability verification sample for the pseudomonas aeruginosa in the cotton swab, the sample is a freeze-dried sample, and the freeze-drying protective agent contains 8-10% of trehalose, 3-5% of sodium glutamate, 0.5-3% of gelatin and 2-5% of skim milk powder by volume.
In the verification sample for the qualitative detection capability of the pseudomonas aeruginosa in the cotton swab, the volume ratio of each strain to the freeze-drying protective agent is 1: 9-3: 7.
In the verification sample for the qualitative detection capability of the pseudomonas aeruginosa in the cotton swab, the cotton swab is made of absorbent cotton, and the cotton swab is sterilized for later use.
The invention also provides a preparation method of the sample for verifying the qualitative detection capability of the pseudomonas aeruginosa in the cotton swab, 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 diluting step by stepReleased to a 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 cotton swab, wherein the cotton swab is made of absorbent cotton and is used for standby after sterilization;
s5, freeze-drying
And (3) dropwise adding the mixed bacterial suspension into each part of cotton swab matrix, filling the cotton swab matrix into a sample bottle after the cotton swab completely absorbs the liquid, and freeze-drying to obtain a freeze-dried sample.
In the preparation method of the sample for verifying the qualitative detection capability of the pseudomonas aeruginosa in the cotton swab, in the 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 method for preparing the sample for verifying the qualitative detection capability of the pseudomonas aeruginosa in the cotton swab, the freeze-drying protective agent in the step S2 contains 8-10% of trehalose by volume fraction, 3-5% of sodium glutamate by volume fraction, 0.5-3% of gelatin by volume fraction and 2-5% of skim milk powder by volume fraction.
According to the preparation method of the sample for verifying the qualitative detection capability of the pseudomonas aeruginosa in the cotton swab, 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 pseudomonas aeruginosa in a cotton swab, which is different from a capability verification simulation sample in the field of microorganisms with freeze-dried powder properties in that the cotton swab is used as a substrate, and a target bacterium and/or an interference bacterium are added to prepare a positive sample and a negative sample for the first time. 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 sample of the invention has uniformity and stability meeting the capability verification requirements, and the problem that the viable pathogenic bacteria in the pseudomonas aeruginosa detection sample in the cotton swab can change in the processes of transportation, storage, test and the like is solved. In addition, the preparation method provided by the invention has simple process, and the success rate of obtaining the sample required by CNAS-GL 03 & lt & gt evaluation guidelines for uniformity and stability of capability verification sample & gt is high.
Detailed Description
The invention provides a qualitative detection capability verification sample of pseudomonas aeruginosa in a cotton swab, which comprises a negative sample and a positive sample, wherein the positive sample contains a substrate, target bacteria and interfering bacteria, and the negative sample contains the substrate and the interfering bacteria; the target bacteria are main detection bacteria of the examination sample, and the interference bacteria are bacteria with similar biochemistry or similar shape of the target bacteria. Can accurately separate and distinguish target bacteria and background bacteria as main examination targets.
Wherein the substrate is cotton swabs, the target bacteria is pseudomonas aeruginosa, and the interfering bacteria is one or more of pseudomonas putida, escherichia coli, proteus vulgaris and bacillus mycoides. The pseudomonas aeruginosa (ATCC27853) adopted by the invention, wherein the interference bacteria are pseudomonas putida (CICC 21624), escherichia coli (ATCC 25922), proteus vulgaris (ATCC 49132) and bacillus mycoides (ATCC 10206) which are all purchased from ATCC and CICC (China Industrial microorganism culture Collection center), thereby ensuring the traceability of the strains, and the strains are only taken as exemplary examples of the implementation mode of the invention and can be replaced by 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.