Qualitative detection capability verification sample for hemolytic streptococcus in cotton swab and preparation method thereof
阅读说明：本技术 棉签中溶血性链球菌定性检测能力验证样品及其制备方法 (Qualitative detection capability verification sample for hemolytic streptococcus in cotton swab and preparation method thereof ) 是由 石嵩 高欣 吕雨莎 张惠媛 万晓楠 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溶血性链球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,避免棉签中溶血性链球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 hemolytic streptococcus 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 uniformity and the stability of the sample meet the capability verification requirements, and the problem that the viable pathogenic bacteria in the hemolytic streptococcus 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 the hemolytic streptococcus 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 the matrix and the interference bacteria; the matrix is cotton swabs, the target bacteria is beta hemolytic streptococcus, and the interference bacteria is one or more of Listeria lnokhei, enterococcus faecalis, Escherichia coli, staphylococcus capitis and Bacillus mycoides.
2. The qualitative test for the ability to detect hemolytic streptococci in a cotton swab of claim 1, wherein the concentration of bacteria in the sample is 103-106cfu/mL。
3. The qualitative detection capability verification sample of hemolytic streptococcus in cotton swab of 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 the matrix.
4. The qualitative test capability verification sample of hemolytic streptococcus in cotton swab of 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 bovine serum albumin with a volume fraction of 2-5%.
5. The qualitative detection capability verification sample of hemolytic streptococcus in cotton swab of claim 4, wherein the volume ratio of each strain to the freeze-drying protective agent is 1: 9-3: 7.
6. The qualitative test capability verification sample of hemolytic streptococcus in cotton swab of claim 1, wherein the cotton swab is made of absorbent cotton, and the cotton swab is sterilized for later use.
7. The method for preparing the qualitative detection capability verification sample of hemolytic streptococcus in cotton swab of 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 102-105cfu/mL ofPreparing bacterial suspension, mixing the target bacteria and the interfering bacteria according to the volume ratio of 1:2-3 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 hemolytic streptococcus in a cotton swab of claim 1, wherein in step S1, each strain is inoculated to a Columbia blood agar culture medium, the target bacteria are anaerobically cultured at a temperature of 36 ℃ ± 1 ℃ for 36 hours, and the interfering bacteria are aerobically cultured at a temperature of 36 ℃ ± 1 ℃ for 36 hours.
9. The method for preparing a sample for verifying the qualitative detection capability of hemolytic streptococcus in a cotton swab of claim 1, wherein the lyoprotectant in step S2 comprises 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 bovine serum albumin in an amount of 2-5% by volume.
10. The method for preparing a sample for verifying the qualitative detection capability of Streptococcus hemolyticus in a cotton swab according to claim 1, wherein the lyophilization time in step S5 is 40-50 hours.
Technical Field
The invention relates to the technical field of microbiological inspection quality control, in particular to a qualitative detection capability verification sample of hemolytic streptococcus 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 has yet prepared a sample for qualitative detection of hemolytic streptococcus using a cotton swab as a matrix at home and abroad, and no related literature reports exist.
Hemolytic streptococcus (β -hemolytic streptococcus) is widely distributed in nature, exists in water, air, dust, feces, and oral cavity, nasal cavity, and throat of healthy people and animals, and can be infected by direct contact, air spray transmission, or wound through skin and mucosa, and contaminated food such as milk, meat, egg, and their products can also infect human. Hemolytic streptococcus often causes pyogenic inflammation of skin, subcutaneous tissue, respiratory tract infections, fulminant epidemic of epidemic pharyngitis and allergic reactions such as neonatal sepsis, bacterial endocarditis, scarlet fever and rheumatic fever, glomerulonephritis. Therefore, a sample for verifying the qualitative detection capability of hemolytic streptococcus in the mask is urgently needed.
Disclosure of Invention
Aiming at the technical problems, the invention provides a qualitative detection capability verification sample of hemolytic streptococcus in a cotton swab and a preparation method thereof, and provides important guarantee for microbiological capability verification of disposable hygienic 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 hemolytic streptococcus 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 swabs, the target bacteria are beta-hemolytic streptococcus (beta-hemolytic streptococcus), and the interfering bacteria are one or more of Listeria lnonensis (Listeria monocytogenes), Enterococcus faecalis (Enterococcus faecalis), Escherichia coli (Escherichia coli), Staphylococcus capitis (Staphylococcus capitis), and Bacillus mycoides (Bacillus mycoides).
In the sample verified by the qualitative detection capability of the hemolytic streptococcus in the cotton swab, the concentration of bacteria in the sample is 103-106cfu/mL。
In the qualitative detection capability verification sample of the hemolytic streptococcus 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 hemolytic streptococcus 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 bovine serum albumin.
In the qualitative detection capability verification sample for the hemolytic streptococcus 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 the hemolytic streptococcus 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 qualitative detection capability verification sample of the hemolytic streptococcus 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 qualitative detection capability verification sample of the hemolytic streptococcus in the cotton swab, in the step S1, each strain is respectively inoculated to a Columbia blood agar culture medium, the target bacteria are subjected to anaerobic culture for 36 hours at the temperature of 36 +/-1 ℃, and the interfering bacteria are subjected to aerobic culture for 36 hours at the temperature of 36 +/-1 ℃.
In the preparation method of the qualitative detection capability verification sample of the hemolytic streptococcus 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 bovine serum albumin by volume fraction.
According to the preparation method of the qualitative detection capability verification sample of the hemolytic streptococcus 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 qualitative detection capability verification sample for the hemolytic streptococcus in the cotton swab is prepared by taking the cotton swab as a matrix and adding target bacteria and/or interference bacteria to prepare a positive sample and a negative sample for the first time, and is different from a capability verification simulation sample in the field of microorganisms with freeze-dried powder properties. 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 hemolytic streptococcus 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 hemolytic streptococcus 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 the matrix and the 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 substrate is cotton swabs, the target bacteria is beta hemolytic streptococcus, and the interfering bacteria is one or more of Listeria lnokhei, enterococcus faecalis, Escherichia coli, Staphylococcus capitis and Bacillus mycoides. The beta hemolytic streptococcus (CMCC 32210) adopted by the invention is purchased from CMCC (China medical bacteria collection management center), and Listeria lnonix (ATCC 33090), enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), staphylococcus capitis (ATCC35661) and bacillus mycoides (ATCC 10206) are purchased from ATCC (American type culture Collection) and CMCC (China medical bacteria collection management center), so that the traceability of the strain is ensured, and the strain is only taken as an exemplary example of the implementation mode of the invention and can be replaced by an equivalent strain.
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.