阅读说明：本技术 棉签中金黄色葡萄球菌定性检测能力验证样品及其制备方法 (Sample for verifying qualitative detection capability of staphylococcus aureus in cotton swab and preparation method thereof ) 是由 石嵩 马师良 张惠媛 张文玲 刘鑫 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金黄色葡萄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,避免棉签中金黄色葡萄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 staphylococcus aureus 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 living pathogenic bacteria in the staphylococcus aureus 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 for staphylococcus aureus 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 interfering bacteria, and the negative sample contains a matrix and interfering bacteria; the matrix is cotton swabs, the target bacteria is staphylococcus aureus, and the interfering bacteria are one or more of enterococcus faecalis, escherichia coli, bacillus mycoides, staphylococcus capitis and listeria inonotus.

2. The qualitative detection capability verification sample of staphylococcus aureus in a cotton swab as claimed in claim 1, wherein the concentration of bacteria in the sample is 10³-10⁶cfu/mL。

3. The qualitative detection capability verification sample for staphylococcus aureus in a 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 a volume ratio of 1:2-3, and the mixed bacteria are added into a matrix.

4. The qualitative detection capability verification sample of staphylococcus aureus 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 5-12%, sodium glutamate with a volume fraction of 2-8% and gelatin with a volume fraction of 0.5-3%.

5. The qualitative detection capability verification sample for staphylococcus aureus in a cotton swab as claimed in claim 4, wherein the volume ratio of each strain to the freeze-drying protective agent is 1: 9-3: 7.

6. The qualitative detection capability verification sample for staphylococcus aureus 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 for preparing the qualitative detection capability verification sample of staphylococcus aureus in the cotton swab as claimed in claim 1, wherein the method 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 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 staphylococcus aureus in a cotton swab as claimed in claim 1, wherein each strain is inoculated into a TSA culture medium in step S1, and aerobic culture is carried out at 36 ℃ ± 1 ℃ for 36 h.

9. The method for preparing a qualitative detection capability verification sample of staphylococcus aureus in a cotton swab as claimed in claim 1, wherein the lyoprotectant of step S2 comprises trehalose at 5-12% by volume, sodium glutamate at 2-8% by volume, and gelatin at 0.5-3% by volume.

10. The method for preparing a sample for verifying the qualitative detection capability of staphylococcus aureus in a cotton swab as claimed in claim 1, wherein the freeze-drying 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 qualitative detection capability verification sample of staphylococcus aureus 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 staphylococcus aureus by taking a cotton swab as a matrix at home and abroad, and no related literature reports exist.

Staphylococcus aureus is a common gram-positive bacterium, widely distributed in nature, and can cause infections of humans and animals. After the human body is infected with staphylococcus aureus, local suppurative infection of the skin can be caused, and serious internal organ infection, even septicemia, toxic shock and the like can be caused. Therefore, a qualitative detection capability verification sample for staphylococcus aureus in a cotton swab is urgently needed.

Disclosure of Invention

Aiming at the technical problems, the invention provides the qualitative detection capability verification sample of the staphylococcus aureus in the cotton swab and the preparation method thereof, and provides important guarantee for the microbiological capability verification of the disposable sanitary product.

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 staphylococcus aureus 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 Staphylococcus aureus (Staphylococcus aureus), the interference bacteria are one or more of Enterococcus faecalis (Enterococcus faecalis), Escherichia coli (Escherichia coli), Bacillus mycoides (Bacillus mycoides), Bacillus cereus (Bacillus cereus), Staphylococcus capitis (Staphylococcus capitis) and Listeria lnnococcus (Listeria innocula).

In the sample verified by the qualitative detection capability of staphylococcus aureus in the cotton swab, the concentration of bacteria in the sample is 10³-10⁶cfu/mL。

In the qualitative detection capability verification sample of staphylococcus aureus 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 staphylococcus aureus in the cotton swab, the sample is a freeze-dried sample, and the freeze-drying protective agent contains 5-12% of trehalose, 2-8% of sodium glutamate and 0.5-3% of gelatin.

In the qualitative detection capability verification sample for staphylococcus aureus 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 staphylococcus aureus 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 staphylococcus aureus 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 staphylococcus aureus in the cotton swab, in 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 preparation method of the sample for verifying the qualitative detection capability of staphylococcus aureus in the cotton swab, the freeze-drying protective agent in the step S2 contains 5-12% of trehalose, 2-8% of sodium glutamate and 0.5-3% of gelatin.

The preparation method of the sample for verifying the qualitative detection capability of staphylococcus aureus in the cotton swab has the step S5 that the freeze-drying time is 40-50 hours.

Compared with the prior art, the invention has the beneficial effects that:

the invention discloses a qualitative detection capability verification sample for staphylococcus aureus in a cotton swab, which is characterized in that the cotton swab is used as a matrix for the first time, a positive sample and a negative sample are prepared by adding a target bacterium and/or an interference bacterium, and the qualitative detection capability verification sample 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 living pathogenic bacteria in the staphylococcus aureus 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 staphylococcus aureus 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 matrix is cotton swabs, the target bacteria is staphylococcus aureus, and the interfering bacteria are one or more of enterococcus faecalis, escherichia coli, bacillus mycoides, staphylococcus capitis and listeria inonotus. Staphylococcus aureus (ATCC6538), enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Bacillus mycoides (ATCC 10206), Bacillus cereus (ATCC 11778), Staphylococcus capitis (ATCC 35661) and Listeria lnokhei (ATCC33090) adopted by the invention are all purchased from ATCC, so that the traceability of the strains is ensured, the strains are only taken as exemplary examples of the embodiment of the invention, and all the strains 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.