不同材质的微生物捕捉滤膜在屏障环境设施中的应用效果初探

doi:10.12300/j.issn.1674-5817.2024.004

摘要/Abstract

摘要：

目的通过探究不同材质滤膜的微生物捕捉性能，评估其对屏障环境实验设施中啮齿类动物健康状况的监测效果。方法以嗜肺巴斯德杆菌和金黄色葡萄球菌为代表菌株，在实验室条件下模拟滤膜捕捉微生物的过程，以捕捉粉尘质量、最低检测限度及荧光定量PCR检测的Ct值差异综合评估5种具有吸附、透气性能的自选材质滤膜（M1～M5）和商品化捕捉滤膜（T1）的微生物捕捉效果。将性能最优的自选材质滤膜置于屏障设施内笼架的通风管道中，用相应笼架的哨兵鼠作为对照组，以表皮葡萄球菌和大肠埃希菌为指示菌，计算阳性检出率和符合率，探讨其作为微生物捕捉滤膜在屏障设施内监测实验动物健康状况的可行性。结果从捕捉粉尘质量看，自选材质滤膜M3（滤径为0.1 μm的无纺布可作为材质）的捕捉效果仅次于T1，捕捉量达0.126 g；对于金黄色葡萄球菌，除M4外，其余材质的滤膜最低检测限度均为10² CFU/g；对于嗜肺巴斯德杆菌，所有滤膜的最低检测限度皆为10² CFU/g；但M3的荧光定量PCR扩增结果Ct值显著小于其他材质，故5种自选材质滤膜中M3的捕捉效果最好。滤膜检测验证实验中，表皮葡萄球菌在哨兵鼠粪便和M3中的阳性检出率分别为50.00%（6/12）和58.33%（7/12），符合率为92%；大肠埃希菌在哨兵鼠粪便和M3中阳性检出率均为50.00%（6/12），符合率为100%。结论 5种自选材质滤膜中，M3捕捉效果最佳；在屏障环境设施内，M3对于表皮葡萄球菌的监测效果优于哨兵鼠。因此，滤径为0.1 μm的无纺布可作为微生物捕捉滤膜材质，为笼架排风粉尘PCR监测用微生物捕捉滤膜的材质选择和应用提供借鉴经验。

关键词: 微生物捕捉滤膜, 屏障环境设施, 笼架排风粉尘, 荧光定量PCR法, 哨兵鼠

Abstract:

Objective To evaluate the effectiveness of filter membranes made from different materials in monitoring the health status of rodents in barrier environment facilities by investigating their microbial capture performance. Methods Pasteurella pneumotropica (Pp) and Staphylococcus aureus (Sa) were used as representative strains to simulate the process of microbial capture by filter membranes under laboratory conditions. The microbial capture effectiveness of five self-selected filter membranes (M1, M2, M3, M4, and M5) with adsorption and breathability properties and a commercial filter membrane (T1) were comprehensively evaluated based on captured dust mass, minimum detection limit, and differences in Ct values obtained through fluorescence quantitative PCR detection. The best-performing self-selected filter membrane was placed in the ventilation ducts of cage racks within the barrier facility, with sentinel mice in corresponding cage racks as the control group. Staphylococcus epidermidis and Escherichia coli were used as indicator bacteria to calculate the positive detection rate and coincidence rate, thereby exploring the feasibility of using microbial capture filter membranes to monitor the health status of experimental animals in barrier facilities. Results In terms of the captured dust mass, the self-selected filter membrane M3 (non-woven filter membrane with a diameter of 0.1 um)；showed a capture effectiveness second only to T1, with a capture mass of 0.126 g. For Sa, all filter membranes except M4 had a minimum detection limit of 102 CFU/g. For Pp, the minimum detection limit for all filter membranes was 10² CFU/g. However, the Ct value of the quantitative fluorescence PCR amplification results for M3 was significantly lower than that of other materials, indicating that M3 had the best capture performance among the five self-selected materials. In the filter detection verification experiment, the positive detection rate of Staphylococcus epidermidis in sentinel mouse feces and M3 was 50.00% (6/12) and 58.33% (7/12), respectively, with a coincidence rate of 92%. The positive detection rate of Escherichia coli in both sentinel mouse feces and M3 was 50.00% (6/12), with a coincidence rate of 100%. Conclusion Among the 5 self-selected filter membranes, M3 exhibits the best capturing performance. Within the barrier environment facilities, M3 outperforms sentinel mice in monitoring Staphylococcus epidermidis. Therefore, non-woven filter membrane with a diameter of 0.1 um； can be used as the material for microbial capture filter membranes, providing valuable insights for the selection and application of microbial capture filter membranes used in PCR monitoring of cage exhaust air dust.

Key words: Microbial capture filter membrane, Barrier environment facilities, Cage exhaust air dust, Fluorescence quantitative PCR

中图分类号:

Q95-33

孔垂琴,田苗苗,蔡利东,等. 不同材质的微生物捕捉滤膜在屏障环境设施中的应用效果初探[J]. 实验动物与比较医学, 2024, 44(4): 436-444. DOI: 10.12300/j.issn.1674-5817.2024.004.

KONG Chuiqin,TIAN Miaomiao,CAI Lidong,et al. Exploration on Application Effectiveness of Microbial Capture Filter Membranes from Different Materials in Barrier Environment Facilities[J]. Laboratory Animal and Comparative Medicine, 2024, 44(4): 436-444. DOI: 10.12300/j.issn.1674-5817.2024.004.

图/表 6

表1 不同材质的微生物捕捉滤膜信息

Table 1 Information of microbial capture filter membranes made from different materials

组别 Group	材质 Material	滤径/μm Filter diameter/μm	规格 Specification	厂家 Manufacturer
M1	玻纤滤材	0.1	H13高效	重庆再升科技股份有限公司
M2	PPK3熔喷布	0.1	90 g/m²	上海仁益制毡有限公司
M3	无纺布	0.1	80 g/m²	东莞市佳联达无纺布有限公司
M4	聚丙烯微孔滤膜	0.3	PP-T 0.22 μm	山东新华医疗器械股份有限公司
M5	脱脂纱布	0.2	7.5 cm×7.5 cm	徐州市徐卫卫生材料有限责任公司
T1	未知	未知	未知	泰尼百斯

图1 金黄色葡萄球菌（A）和嗜肺巴斯德杆菌（B）污染EAD模拟样品的混合效果注：Ct为循环阈值，CFU为菌落形成单位。

Figure 1 Mixing effect of exhaust air dust (EAD) simulated samples contaminated by Staphylococcus aureus (Sa) (A) and Pasteurella pneumotropica (Pp) (B)Note：Ct， cycle threshold； CFU， colony forming unit.

图2 不同材质滤膜的粉尘捕捉效果图注：图中M1、M2、M3、M4和M5为本次实验选用材质，T1为购买的成品。从观察结果来看，M3、M5和T1表面附着有大量粉尘，而M1、M2、M4表面附着粉尘相对较少；各滤膜材质附着粉尘前后的质量差从低到高依次为M4<M1<M2<M5<M3<T1。

Figure 2 Dust capture effectiveness of filter membranes made from different materialsNote：In the figure, M1, M2, M3, M4, and M5 were the materials selected for the experiment, and T1 was the purchased finished product. Based on visual observation, surfaces of M3, M5, and T1 had a large amount of dust attached, while surfaces of M1, M2, and M4 had relatively less dust attached. The mass differences of each filter membrane material, ranked from lowest to highest, were M4<M1<M2<M5<M3<T1.

表2 不同材质滤膜的粉尘捕捉效果评价

Table 2 Evaluation of dust capturing effectiveness of filter membranes made from different materials

材质种类 Material	材质大小/cm Size/cm	粉尘捕捉前滤膜质量/g m₁/g	粉尘捕捉后滤膜质量/g m₂/g	质量差/g m（m₁－m₂）/g
M1	5×3	0.196±0.003	0.266±0.000	0.070±0.049
M2	5×3	0.070±0.000	0.164±0.000	0.094±0.067
M3	5×3	0.390±0.000	0.516±0.000	0.126±0.089^*
M4	5×3	0.202±0.000	0.221±0.000	0.019±0.013
M5	5×3	0.203±0.000	0.286±0.000	0.084±0.059
T1	5×3	0.617±0.000	0.760±0.000	0.143±0.101^*

表3 不同材质滤膜捕捉金黄色葡萄球菌和嗜肺巴斯德杆菌和检测灵敏度和变异系数

Table 3 Detection sensitivity and coefficient of variation (CV) for Staphylococcus aureus (Sa) and Pasteurella pneumotropica (Pp) using filter membranes made from different materials

材质 Material Bacteria	细菌浓度/（CFU·g^-1） concentration/（CFU·g^-1）	金黄色葡萄球菌 Staphylococcus aureus (Sa)		嗜肺巴斯德杆菌 Pasteurella pneumotropica (Pp)
材质 Material Bacteria	细菌浓度/（CFU·g^-1） concentration/（CFU·g^-1）	循环阈值（ $x ¯$ ±s） Ct ( $x ¯$ ±s)	变异系数/% CV/%	循环阈值（ $x ¯$ ±s） Ct( $x ¯$ ±s)	变异系数/% CV/%
M1	10⁶	24.03±0.13	0.005	23.59±0.42	0.018
	10⁵	26.13±0.23	0.009	25.88±0.23	0.009
	10⁴	29.51±0.43	0.015	28.82±0.60	0.021
	10³	33.72±0.18	0.005	33.84±0.39	0.012
	10²	36.52±0.27	0.007	36.15±0.28	0.008
M2	10⁶	23.42±1.06	0.045	19.50±0.17	0.009
	10⁵	26.50±0.47	0.018	22.15±0.22	0.010
	10⁴	28.22±0.13	0.005	25.67±0.33	0.013
	10³	32.30±0.28	0.009	30.33±0.34	0.011
	10²	35.28±0.42	0.012	35.97±0.25	0.007
M3	10⁶	24.74±0.17	0.007	19.78±0.43	0.022
	10⁵	26.08±0.00	0.000	21.46±0.10	0.005
	10⁴	29.60±0.23	0.008	25.25±0.28	0.011
	10³	35.01±0.03	0.001	27.39±0.19	0.007
	10²	35.14±0.28^*	0.008	32.91±0.80^*	0.024
M4	10⁶	28.40±0.05	0.002	24.96±0.43	0.017
	10⁵	29.59±0.02	0.001	28.39±0.34	0.012
	10⁴	35.70±0.62	0.017	30.83±0.51	0.017
	10³	36.24±0.21	0.006	33.64±0.45	0.013
	10²	/	/	36.56±0.00	0.012
M5	10⁶	23.31±0.41	0.018	18.04±0.70	0.039
	10⁵	25.96±0.16	0.006	21.76±0.49	0.023
	10⁴	28.85±0.25	0.009	25.08±0.23	0.009
	10³	33.23±0.36	0.011	28.11±0.46	0.016
	10²	35.56±0.45	0.013	32.94±0.40	0.012
T1	10⁶	22.92±0.31	0.014	16.88±0.43	0.025
	10⁵	25.36±0.23	0.009	21.62±0.34	0.016
	10⁴	28.93±0.13	0.004	23.68±0.44	0.019
	10³	31.98±0.37	0.012	27.46±0.75	0.027
	10²	33.14±0.00^*	0.024	32.10±0.28^*	0.009

表3 不同材质滤膜捕捉金黄色葡萄球菌和嗜肺巴斯德杆菌和检测灵敏度和变异系数

Table 3 Detection sensitivity and coefficient of variation (CV) for Staphylococcus aureus (Sa) and Pasteurella pneumotropica (Pp) using filter membranes made from different materials

材质 Material Bacteria	细菌浓度/（CFU·g^-1） concentration/（CFU·g^-1）	金黄色葡萄球菌 Staphylococcus aureus (Sa)		嗜肺巴斯德杆菌 Pasteurella pneumotropica (Pp)
材质 Material Bacteria	细菌浓度/（CFU·g^-1） concentration/（CFU·g^-1）	循环阈值（ $x ¯$ ±s） Ct ( $x ¯$ ±s)	变异系数/% CV/%	循环阈值（ $x ¯$ ±s） Ct( $x ¯$ ±s)	变异系数/% CV/%
M1	10⁶	24.03±0.13	0.005	23.59±0.42	0.018
	10⁵	26.13±0.23	0.009	25.88±0.23	0.009
	10⁴	29.51±0.43	0.015	28.82±0.60	0.021
	10³	33.72±0.18	0.005	33.84±0.39	0.012
	10²	36.52±0.27	0.007	36.15±0.28	0.008
M2	10⁶	23.42±1.06	0.045	19.50±0.17	0.009
	10⁵	26.50±0.47	0.018	22.15±0.22	0.010
	10⁴	28.22±0.13	0.005	25.67±0.33	0.013
	10³	32.30±0.28	0.009	30.33±0.34	0.011
	10²	35.28±0.42	0.012	35.97±0.25	0.007
M3	10⁶	24.74±0.17	0.007	19.78±0.43	0.022
	10⁵	26.08±0.00	0.000	21.46±0.10	0.005
	10⁴	29.60±0.23	0.008	25.25±0.28	0.011
	10³	35.01±0.03	0.001	27.39±0.19	0.007
	10²	35.14±0.28^*	0.008	32.91±0.80^*	0.024
M4	10⁶	28.40±0.05	0.002	24.96±0.43	0.017
	10⁵	29.59±0.02	0.001	28.39±0.34	0.012
	10⁴	35.70±0.62	0.017	30.83±0.51	0.017
	10³	36.24±0.21	0.006	33.64±0.45	0.013
	10²	/	/	36.56±0.00	0.012
M5	10⁶	23.31±0.41	0.018	18.04±0.70	0.039
	10⁵	25.96±0.16	0.006	21.76±0.49	0.023
	10⁴	28.85±0.25	0.009	25.08±0.23	0.009
	10³	33.23±0.36	0.011	28.11±0.46	0.016
	10²	35.56±0.45	0.013	32.94±0.40	0.012
T1	10⁶	22.92±0.31	0.014	16.88±0.43	0.025
	10⁵	25.36±0.23	0.009	21.62±0.34	0.016
	10⁴	28.93±0.13	0.004	23.68±0.44	0.019
	10³	31.98±0.37	0.012	27.46±0.75	0.027
	10²	33.14±0.00^*	0.024	32.10±0.28^*	0.009

表4 滤膜M3的检测验证结果

Table 4 Detection validation results for filter membrane M3

检测项目 Testing item	M3滤膜 M3 filter	哨兵鼠粪便样品 Sentinel mouse faeces			符合率/% Coincidence rate/%
检测项目 Testing item	M3滤膜 M3 filter	阳性 Positive	阴性 Negative	汇总 Summary	符合率/% Coincidence rate/%
表皮葡萄球菌 Staphylococcus epidermidis	阳性	6	1	7	92
	阴性	0	5	5
	汇总	6	6	12
大肠埃希菌 Escherichia coli	阳性	6	0	6	100
	阴性	0	6	6
	汇总	6	6	12

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