WO2023116477A1 - 一种用于检测口罩细菌过滤效率的方法 - Google Patents

一种用于检测口罩细菌过滤效率的方法 Download PDF

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WO2023116477A1
WO2023116477A1 PCT/CN2022/138248 CN2022138248W WO2023116477A1 WO 2023116477 A1 WO2023116477 A1 WO 2023116477A1 CN 2022138248 W CN2022138248 W CN 2022138248W WO 2023116477 A1 WO2023116477 A1 WO 2023116477A1
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bacterial
mask
aerosol
filtration efficiency
sample
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付钊
李挥
周玉岩
冀亚坤
李聪
陈明
安丽康
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河北省药品医疗器械检验研究院(河北省化妆品检验研究中心)
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/14—Streptococcus; Staphylococcus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
    • G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)

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  • the invention belongs to the technical field of mask detection, in particular to a method for detecting the bacterial filtration efficiency of a mask.
  • Medical masks and medical surgical masks play an important role in clinical and epidemic prevention. They are used to cover the user's mouth, nose and lower collar, and provide a physical barrier to prevent the direct penetration of pathogenic microorganisms, body fluids, particles, etc. Among them, bacterial filtration efficiency, as an important indicator of medical masks and medical surgical masks, has attracted much attention in its inspection and testing. Bacterial filtration efficiency; BFE refers to the percentage of mask material that filters out bacteria-containing suspended particles at a specified flow rate. With the outbreak of the new crown epidemic in 2020, masks have become an effective weapon against the epidemic in the early stage of epidemic prevention, and the bacterial filtration efficiency, as an important indicator of the protection level of masks, has attracted much attention.
  • the existing biological test method for bacterial filtration efficiency is: the Staphylococcus aureus, which is configured at a concentration of 5 ⁇ 10 5 CFU/mL, is atomized and sprayed to the On the mask fixed on the sampler, the aerosol was collected using a six-stage Anderson sampler placed on a tryptic soy agar plate. Incubate the collected positive quality control, negative quality control and sample agar plates at (37 ⁇ 2)°C for (48 ⁇ 4) h, then count the colony forming units (positive wells) formed by the bacterial particle aerosol, and Convert this to the possible number of impacted EUT particles using the Positive Well Conversion Table. The converted values are used to determine the average level of bacterial particle aerosol delivered to the test sample. Finally, the converted data is used to calculate the bacterial filtration efficiency of the mask through the formula.
  • the inspection takes a long time. It takes at least 2 days from the beginning of the inspection to the result, which greatly affects the inspection speed. Even though the EU standard has changed the incubation time from 48 hours to 24 hours, compared with other inspection items of masks, the time taken is still long.
  • the Anderson sampler is prone to errors. Because of the wall loss of the Anderson sampler, the particles slipping from the collection surface and being broken, the above situations will cause errors in the experimental results and affect the reliability of the experimental results.
  • the calculation is cumbersome.
  • the original data In order to calculate the bacterial filtration efficiency, the original data must be converted using the positive hole conversion table, and each medium plate is counted, and the amount of data is large.
  • the purpose of the present invention is to address the above problems and provide a new method for detecting the bacterial filtration efficiency of a mask.
  • the present invention has the following characteristics: it greatly saves time and cost and increases the reliability of experimental data.
  • the present invention provides the following technical solutions: a method for detecting bacterial filtration efficiency of a mouth mask, comprising the following steps:
  • the bacterial aerosol average particle diameter (MPS) of ejection should be 3.0 ⁇ 0.3 ⁇ m;
  • the geometric standard deviation of bacterial aerosol distribution should not If it exceeds 1.5, with a flow rate of 1mL/min, spray the aerosol through the pipeline vertically to the mask fixed on the sampler, spray for 1min, then stop spraying, spray sterile air for 1min, and the aerosol enters the impact through the mask
  • each collector uses 20mL sterile peptone water to collect the aerosol that penetrates the sample, and the gas flow rate through the sampler is controlled at 28.3L/min, and a total of three parallel tests are carried out;
  • Aseptic operation counts the peptone water in the collector with an electronic bacteria counter, and draws respectively the number of bacteria in the peptone water added with the mask sample, i.e. the bacterial penetration value, and the number of bacteria in the peptone water without the mask sample, That is, the positive control value;
  • This method can reduce the systematic error caused by relying on the Anderson sampler and increase the reliability of the experimental data.
  • This method can reduce the workload of the test personnel, simplify the work flow and operation, improve the reliability of the test results, and enhance the authenticity of the data.
  • TSA tryptone soy agar
  • TB tryptone soy broth
  • B Staphylococcus aureus CMCC
  • Sample pretreatment Before the test, place the sample in an environment with a temperature of (21 ⁇ 5)°C and a relative humidity of (85 ⁇ 5)% for at least 4 hours.
  • Atomize the suspension of Staphylococcus aureus, and the average particle diameter (MPS) of the sprayed bacterial aerosol should be 3.0 ⁇ 0.3 ⁇ m; the geometric standard deviation of the bacterial aerosol distribution should not exceed 1.5, with 1mL/min
  • the flow rate will be vertically sprayed through the pipeline aerosol to the mask fixed on the sampler, sprayed for 1min, then stop spraying, and spray sterile air for 1min.
  • the aerosol enters the impact liquid collector through the mask, and each collector uses 20mL sterile peptone water to collect the aerosol that penetrates the sample, and the gas flow rate through the sampler is controlled at 28.3L/min. A total of three parallel experiments were carried out.
  • Staphylococcus aureus CMCC (B) 26003 in 50 mL of tryptone liquid medium, shake culture at 37°C for 24 hours, then dilute the above culture with 1.5% peptone to a concentration of 5 ⁇ 10 5 CFU/mL bacterial suspension.
  • the air sample in the aerosol chamber is collected for 2 minutes as a negative quality control. During this process, the bacterial suspension cannot be delivered to the nebulizer.
  • the aseptic operation counts the peptone water in the collector with a bacterial counter to obtain the number of bacteria in the peptone water with the mask sample added, that is, the bacterial penetration value, and the number of bacteria in the peptone water without the mask, That is, the positive control value.
  • the method of the present invention detects this batch of masks, and the bacterial filtration efficiency is 99.8%, while the bacterial filtration efficiency of this batch of masks by the original method is 99.9%, and the detection results have no significant difference.
  • Bacteria counters can use cell flow counters or use electronic bacteria counters, or other instruments that can be used to count individual bacteria in liquids.

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Abstract

一种用于检测口罩细菌过滤效率的方法，属于口罩检测技术领域。该方法包括以下步骤：(1)样品预处理；(2)试验用细菌悬液制备：(3)金黄色葡萄球菌菌悬液雾化成气溶胶喷出，以1mL/min的流量，将通过管路的气溶胶垂直喷送到固定在采样器上的口罩上，喷送1min，再停止喷雾，喷送无菌空气1min，气溶胶经过口罩进入冲击式液体采集器中，每个采集器用20mL无菌蛋白胨水收集穿透过样品的气溶胶，将通过采样器的气体流速控制在28.3L/min的条件下；(4)无菌操作将采集器中的蛋白胨水用电子细菌计数器计数，分别得出加有口罩样品的蛋白胨水中的细菌数量，即细菌穿透值，以及不加口罩的蛋白胨水中的细菌数量，即阳性对照值；(5)按公式计算结果。

Description

一种用于检测口罩细菌过滤效率的方法 技术领域
本发明属于口罩检测技术领域，具体是一种用于检测口罩细菌过滤效率的方法。
背景技术
医用口罩和医用外科口罩在临床和防疫中具有重要作用，用于覆盖住使用者的口、鼻及下领，为防止病原体微生物、体液、颗粒物等的直接透过提供物理屏障。其中细菌过滤效率作为医用口罩和医用外科口罩的重要指标，在其检验检测中备受关注。细菌过滤效率bacterial filtration efficiency；BFE是指在规定流量下，口罩材料对含菌悬浮粒子滤除的百分数。2020年新冠疫情爆发，口罩在防疫初期成为了抗击疫情的有效利器，而细菌过滤效率作为口罩防护水平的重要指标，备受关注。现有的细菌过滤效率生物检验方法是：将配置成5×10 5CFU/mL浓度的金黄色葡萄球菌，通过试验系统，在气体流速为28.3L/min的条件下，雾化并喷送到固定在采样器上的口罩上，利用放置有胰蛋白酶大豆琼脂平板的六级安德森采样器进行气溶胶的采集。将采集到的阳性质控、阴性质控和试样琼脂平板在(37±2)℃培养(48±4)h，然后对细菌颗粒气溶胶形成的菌落形成单位(阳性孔)进行计数，并使用阳性孔转换表将其转换为可能的撞击EUT粒数。转换后的数值用于确定输送到试验样品上的细菌颗粒气溶胶的平均水平。最后利用转换后的数据经公式计算得出口罩的细菌过滤效率。
现有的口罩细菌过滤效率检验方法存在以下不足：
1、检验所需时间长。从检验开始到结果出来至少需要2天的时间，这极大的影响了检验速度。即使欧盟标准已经把培养时间由48小时修改为24小时，但相较于口罩的其他检项，所用时间依然偏长。
2、安德森采样器容易引起误差。因为安德森采样器存在壁损失，颗粒从 采集面滑脱和被打碎等情况，以上情况都会对实验结果产生误差，影响实验结果的可靠性。
3、采样步骤复杂，所需琼脂平板也较多。采集器的安装和拆卸都需要多次进行，增加了操作人员的劳动强度和试验成本。
计算繁琐。为了计算出细菌过滤效率，必须将原始数据利用阳性孔转化表进行数据转换，并对每一块培养基平板进行计数，数据量较大。
发明内容
本发明的目的是针对以上问题，提供了一种用于检测口罩细菌过滤效率的新方法，本发明具有以下特点：极大的节省了时间成本、增加实验数据的可靠性。
为实现上述目的，本发明提供如下技术方案：一种用于检测口罩细菌过滤效率的方法，包括以下步骤：
(1)将样品放置在温度(21±5)℃、相对湿度为(85±5)％的环境中预处理至少4h；
(2)将金黄色葡萄球菌CMCC(B)26003接种在适量的胰酪大豆胨液体培养基中，在37±2℃振荡培养(24±2)h，然后用1.5％的蛋白胨将上述培养物稀释至成5×105CFU/mL浓度的菌悬液；
(3)将步骤(2)得到的金黄色葡萄球菌菌悬液雾化，喷出的细菌气溶胶平均颗粒直径(MPS)，应为3.0±0.3μm；细菌气溶胶分布的几何标准差应不超过1.5,以1mL/min的流量，将通过管路气溶胶垂直喷送到固定在采样器上的口罩上，喷送1min，再停止喷雾，喷送无菌空气1min，气溶胶经过口罩进入冲击式液体采集器中，每个采集器用20mL无菌蛋白胨水收集穿透过样品的气溶胶，将通过采样器的气体流速控制在28.3L/min的条件下，共进行三次平行试验；
(4)无菌操作将采集器中的蛋白胨水用电子细菌计数器计数，分别得出加有口罩样品的蛋白胨水中的细菌数量，即细菌穿透值，以及不加口罩的蛋白胨水中的细菌数量，即阳性对照值；
(5)按公式计算结果：
Figure PCTCN2022138248-appb-000001
式中：
T：细菌穿透值
C：阳性对照值平均值。
与现有技术相比，本发明的有益效果如下：
1、利用本方法可以省去细菌培养的时间，将实验时间由过去的48小时以上缩短为几个小时，极大的节省了时间成本。
2、本方法可以减少依靠安德森采样器所引起的系统误差，增加实验数据的可靠性。
3、本方法可以减轻试验人员的工作负担，简化工作流程和操作，提升检验结果的可靠性，增强数据的真实性。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
1、所用试剂：胰酪大豆胨琼脂培养基(TSA)；胰酪大豆胨液体培养基(TSB)；蛋白胨水；金黄色葡萄球菌CMCC(B)26003。
2、样品预处理：试验前将样品放置在温度(21±5)℃、相对湿度为(85±5)％的环境中预处理至少4h。
3、试验用细菌悬液制备：将金黄色葡萄球菌CMCC(B)26003接种在适量的胰酪大豆胨液体培养基中，在37±2℃振荡培养(24±2)h，然后用1.5％的蛋白胨将上述培养物稀释至成5×10 5CFU/mL浓度的菌悬液。
4、将金黄色葡萄球菌菌悬液雾化，喷出的细菌气溶胶平均颗粒直径(MPS)，应为3.0±0.3μm；细菌气溶胶分布的几何标准差应不超过1.5,以1mL/min的流量，将通过管路气溶胶垂直喷送到固定在采样器上的口罩上，喷送1min，再停止喷雾，喷送无菌空气1min。气溶胶经过口罩进入冲击式液体采集器中，每个采集器用20mL无菌蛋白胨水收集穿透过样品的气溶胶，将通过采样器的气体流速控制在28.3L/min的条件下。共进行三次平行试验。
5、试验结束后，无菌操作将采集器中的蛋白胨水用电子细菌计数器计数，分别得出加有口罩样品的蛋白胨水中的细菌数量，即细菌穿透值，以及不加口罩的蛋白胨水中的细菌数量，即阳性对照值。
6、按公式计算结果：
Figure PCTCN2022138248-appb-000002
式中：
T：细菌穿透值
C：阳性对照值平均值
实施例：
取3只口罩，试验前将样品放在21℃、相对湿度85％的环境中预处理4h。
将金黄色葡萄球菌CMCC(B)26003接种在50mL的胰酪大豆胨液体培养基中，37℃振荡培养24h，然后用1.5％的蛋白胨将上述培养物稀释至成5 ×10 5CFU/mL浓度的菌悬液。
试验系统中不放入口罩，连接冲击式液体采集器，将通过采样器的气体流速控制在28.3L/min，向喷雾器输送细菌悬液的时间设定为1min，空气压力和采样器运行时间设定为2min，运行三次，平衡试验系统。
不放入样品，启动试验系统，将细菌气溶胶收集到已灭菌、含有20mL无菌蛋白胨水的冲击式液体采集器中，作为阳性质控，将采集器中的蛋白胨水用细菌计数器计数细菌个体数量，确定气溶胶流量，应为(2200±500)CFU，跟据情况调整菌液浓度及气体流速。
阳性质控测试完成后，放入新的已灭菌、含有20mL无菌蛋白胨水的冲击式液体采集器，将试验样品夹在采样器上端，被测试面向上。按照上述程序进行采样。在一批试验样品测试完成后，再测试一次阳性质控。总共进行三次试验。
在一批试验样品测试完成后，然后收集2min气溶胶室中的空气样品，作为阴性质控，在此过程中，不能向喷雾器中输送细菌悬液。
试验结束后，无菌操作将采集器中的蛋白胨水用细菌计数器计数，分别得出加有口罩样品的蛋白胨水中的细菌数量，即细菌穿透值，以及不加口罩的蛋白胨水中的细菌数量，即阳性对照值。
利用公式计算各个口罩的细菌过滤效率。
本发明方法检测该批口罩，细菌过滤效率为99.8％，原方法该批次口罩的细菌过滤效率99.9％，检测结果无显著差异。
细菌计数器可使用细胞流体计数器或使用细菌电子计数器，或其他可用于对液体中单个细菌进行计数的仪器设备。
需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、 “包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。

Claims (5)

  1. 一种用于检测口罩细菌过滤效率的方法，其特征在于，包括以下步骤：
    (1)将样品放置在温度16-26℃、相对湿度为80-90％的环境中预处理至少4h；
    (2)将金黄色葡萄球菌CMCC(B)26003接种在胰酪大豆胨液体培养基中，在35-39℃振荡培养22-26h，然后用1.5％的蛋白胨将上述培养物稀释至成5×10 5CFU/mL浓度的菌悬液；
    (3)将步骤(2)得到的金黄色葡萄球菌菌悬液雾化成气溶胶喷出，以1mL/min的流量，将通过管路气溶胶垂直喷送到固定在采样器上的口罩上，喷送1min，再停止喷雾，喷送无菌空气1min，气溶胶经过口罩进入冲击式液体采集器中，每个采集器用20mL无菌蛋白胨水收集穿透过样品的气溶胶，将通过采样器的气体流速控制在28.3L/min的条件下；
    (4)无菌操作将采集器中的蛋白胨水用电子细菌计数器计数，分别得出加有口罩样品的蛋白胨水中的细菌数量，即细菌穿透值，以及不加口罩的蛋白胨水中的细菌数量，即阳性对照值；
    (5)按公式计算结果：
    Figure PCTCN2022138248-appb-100001
    式中：
    T：细菌穿透值
    C：阳性对照值平均值。
  2. 根据权利要求1所述的一种用于检测口罩细菌过滤效率的方法，其特征在于，所述步骤(2)中，细菌气溶胶平均颗粒直径(MPS)为3.0±0.3μm，细菌气溶胶分布的几何标准差应不超过1.5。
  3. 根据权利要求1所述的一种用于检测口罩细菌过滤效率的方法，其特征在于，所述步骤(2)需进行三次平行试验。
  4. 根据权利要求1所述的一种用于检测口罩细菌过滤效率的方法，其特征在于，所述步骤(1)中，温度为21℃，湿度为85％。
  5. 根据权利要求1所述的一种用于检测口罩细菌过滤效率的方法，其特征在于，所述步骤(2)中，在37℃振荡培养24h。
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