Qualitative detection capability verification sample for coliform in cotton swab and preparation method thereof
阅读说明：本技术 棉签中大肠菌群定性检测能力验证样品及其制备方法 (Qualitative detection capability verification sample for coliform in cotton swab and preparation method thereof ) 是由 张惠媛 石嵩 高璟瑜 张捷 马师良 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大肠菌群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,避免棉签中大肠菌群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 coliform group in 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 uniformity and the stability of the sample meet the capability verification requirements, and the problem that the live pathogenic bacteria in the coliform group detection sample in the cotton swab 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.)
1. The qualitative detection capability verification sample of coliform bacteria in the cotton swab 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 matrix is cotton swabs, the target bacteria is Escherichia coli, and the interfering bacteria is one or two of enterococcus faecalis and Bacillus mycoides.
2. The swab coliform bacteria qualitative test capability verification sample of claim 1, wherein the bacteria concentration in the sample is 103-106cfu/mL。
3. The qualitative detection capability verification sample of coliform bacteria in the cotton swab as claimed in claim 1, wherein the target bacteria and the interfering bacteria in 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.
4. The qualitative detection capability verification sample of coliform bacteria 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 8-10% by volume, sodium glutamate 3-5% by volume, gelatin 0.5-3% by volume and skim milk powder 2-5% by volume.
5. The qualitative detection capability verification sample of coliform bacteria in a cotton swab as claimed in 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 coliform bacteria in a cotton swab as claimed in claim 1, wherein said cotton swab is made of absorbent cotton, and is sterilized for use.
7. The method for preparing a sample for verifying the qualitative detection capability of coliform bacteria in a cotton swab as set forth in claim 1, wherein the method comprises 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 coliform bacteria in a cotton swab as set forth in claim 1, wherein each of the strains 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 coliform bacteria in a swab as set forth in claim 1, wherein the lyoprotectant in step S2 includes trehalose in an amount of 8-10% by volume, sodium glutamate in an amount of 3-5% by volume, gelatin in an amount of 0.5-3% by volume, and skim milk powder in an amount of 2-5% by volume.
10. The method for preparing a sample for verifying the qualitative detection capability of coliform bacteria in a swab of claim 1, wherein the lyophilization time in step S5 is 40-50 h.
Technical Field
The invention relates to the technical field of microbiological inspection quality control, in particular to a qualitative detection capability verification sample of coliform bacteria 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, no organization/organization exists at home and abroad to prepare a sample for qualitative detection of coliform group by taking a cotton swab as a matrix, and no related literature report exists.
The coliform group is a group of gram-negative bacillus-free bacteria which is aerobic and facultative anaerobic, can decompose lactose to produce acid and gas at 37 ℃ for 24h, is mainly derived from human and animal feces, and can be generally used as an index bacteria for water body feces pollution. The bacteria of this bacterial group may include Escherichia coli, Citrobacter, Klebsiella aerogenes, Enterobacter cloacae, and the like. Among them, Escherichia coli is a typical indicator bacterium of the Escherichia coli group as fecal contamination, and the presence or absence of fecal contamination in food is mainly indicated by the detection of the Escherichia coli group. The number of coliform bacteria indicates the degree of fecal pollution and reflects the degree of harm to human health. The feces are human intestinal excretions, wherein the feces of healthy people and the feces of intestinal patients or bacteria carriers exist, so that the feces have the pollution of some intestinal pathogenic bacteria (such as salmonella, shigella and the like) besides normal bacteria, and the possibility of the pollution of the intestinal pathogenic bacteria exists in the sanitary product, so that the threat of poisoning and epidemic diseases of the sanitary product latent to human health can be presumed. Therefore, there is an urgent need for a sample for verifying the qualitative detection capability of coliform bacteria in a mask.
Disclosure of Invention
Aiming at the technical problems, the invention provides a qualitative detection capability verification sample of coliform bacteria in a cotton swab 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 coliform bacteria in a cotton swab, wherein the sample comprises a negative sample and a positive sample, the positive sample contains a matrix, target bacteria and interference bacteria, and the negative sample contains a matrix and interference bacteria; the matrix is cotton swab, the target bacteria is Escherichia coli (Escherichia coli), and the interfering bacteria is one or two of enterococcus faecalis (enterococcus faecalis) and Bacillus mycoides (Bacillus mycoides).
In the qualitative detection capability verification sample of the coliform group in the cotton swab, the concentration of bacteria in the sample is 103-106cfu/mL。
In the qualitative detection capability verification sample of the coliform group 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 coliform in the cotton swab, the sample is a freeze-dried sample, and the freeze-drying protective agent 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.
In the qualitative detection capability verification sample for the coliform bacteria in the cotton swab, the volume ratio of each strain to the freeze-drying protective agent is 1: 9-3: 7.
In the qualitative detection capability verification sample for coliform bacteria in the cotton swab, the cotton swab is made of absorbent cotton and is used for standby after sterilization.
The invention also provides a preparation method of the qualitative detection capability verification sample for coliform bacteria 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 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 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 coliform group in the cotton swab, each strain is respectively inoculated to the TSA culture medium in the step S1, and aerobic culture is carried out for 36 hours at the temperature of 36 +/-1 ℃.
In the preparation method of the qualitative detection capability verification sample for coliform 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.
In the preparation method of the sample for verifying the qualitative detection capability of the coliform group 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 coliform bacteria in a cotton swab, which is characterized in that the cotton swab 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 live pathogenic bacteria in the coliform group detection sample in the cotton swab 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 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 coliform bacteria in a cotton swab, 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 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 matrix is cotton swabs, the target bacteria is Escherichia coli, and the interfering bacteria is one or two of enterococcus faecalis and Bacillus mycoides. Escherichia coli (ATCC 25922), enterococcus faecalis (ATCC 29212) and Bacillus mycoides (ATCC 10206) adopted by the invention are purchased from ATCC, so that the traceability of the strains is ensured, and the strains are only taken as exemplary examples of the embodiment 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.