Application of polyethersulfone membrane in enrichment of bacterium-containing specimens

Abstract

The invention belongs to the technical field of medical detection, and particularly relates to a method for treating a bacterium-containing sample by using a polyether sulfone (PES) membrane. In particular discloses application of a polyether sulfone membrane in bacterium-containing specimen treatment, the treatment application of the polyether sulfone membrane is impurity removal and / or thallus enrichment of the bacterium-containing specimen, when the application of the polyether sulfone membrane is impurity removal of the bacterium-containing specimen, the polyether sulfone membrane is selected from a large-aperture membrane, the aperture is selected from 0.8-5 [mu] m, and the aperture is selected from 0.8-5 [mu] m. The application of the polyethersulfone membrane is that when thalli of a bacterium-containing specimen are enriched, the polyethersulfone membrane is selected from small-aperture membranes, and the small aperture is selected from 0.22-0.8 [mu] m. When the system is used for treating bacterium-containing samples, the loss rate of thalli is low, the biological activity of the thalli is guaranteed, compared with an existing bacterium-containing sample treatment system, the treatment steps are simplified, the steps of repeated centrifuging, washing and the like of the samples are omitted, meanwhile, the dyeing and biological detection effects are further improved due to the fact that the loss rate of the thalli is reduced, and the detection efficiency is improved. The positive detection rate is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection, and in particular relates to a method for treating bacteria-containing samples using a polyethersulfone membrane (PES). Background Art

[0002] Bacteria are one of the important microorganisms that cause human diseases. Pathogenic bacteria often cause infections in multiple parts of the body, including the skin, eyes, ears, nose, throat, mouth, lungs, chest, brain, abdomen, urinary and female reproductive systems, and cardiovascular systems. There are more than a hundred common pathogenic bacteria. Due to the low content of bacteria in nature and in the infected site, tissues, and body fluids, it is extremely difficult for people to discover and detect bacteria. For bacterial infections, purification and enrichment of bacteria in limited clinical specimens will greatly improve the positive rate of clinical detection, timely discover and confirm the type of bacteria infected by the patient and its drug sensitivity, so as to provide appropriate treatment plans and achieve the purpose of effectively treating bacterial infectious diseases.

[0003] At present, the simplest and most direct way to improve the detection rate of bacteria is to enrich the bacteria in the sample. However, the current methods for enriching bacteria are relatively limited, and the most common method is to obtain precipitation by centrifugation. However, since bacteria easily form aerosols, and centrifugation often causes impurities in the specimen to precipitate together with the bacteria to be tested, thereby interfering with the detection of bacteria. Therefore, the centrifugation method is generally not recommended. Therefore, people are often forced to directly test the bacteria in the specimen, resulting in a low positive rate for bacterial detection. Since the properties of the specimens to be tested are relatively complex, the digestion and other treatment effects of the early specimens are not ideal. Therefore, how to quickly and effectively remove impurities and enrich bacteria in the pre-treatment specimens has always been a problem to be solved. The microporous membrane filtration method is a relatively mature method, which is often used to remove pathogenic microorganisms in clean solutions. The existing small membrane filtration method directly enriches bacteria on the filter membrane, and then places the membrane under a microscope for observation. In this case, the background effect caused by the existence of the filter membrane and the overlapping and blocking of bacteria caused by the thickness of the filter membrane will have an adverse effect on the microscopic examination effect. Therefore, it is considered to wash off the bacteria on the small membrane filtration method membrane, dissolve it in liquid, and then observe the smear, which will significantly improve the microscopic examination effect. With respect to the existing domestic and foreign eluent formulas, our experiments found that it can improve the elution rate compared with elution with pure 7H9 culture medium, but it does not exceed 50%. At the same time, combined with the enrichment rate calculation, the bacterial yield does not exceed 30% (enrichment rate x elution rate = bacterial yield). Therefore, choosing a membrane with suitable material and pore size for enrichment and a matching eluent for elution is the key to improving the bacterial yield. There are many types of microporous membranes. After filtering with microporous membranes, the bacteria are enriched on the membrane surface. However, due to the complex and irregular mesh pores of the microporous membrane, the enriched bacteria often cannot be effectively eluted, resulting in the loss of the actual available bacteria in the specimen. At the same time, considering the different formulas of eluents on the market, the experiment found that the effect was not ideal, and nearly half of the enriched bacteria were not eluted. Therefore, we considered using polyethersulfone membranes, using DMSO eluent to expand the upper micropores, and efficiently elute the bacteria from the microporous membrane to achieve the purpose of quickly and effectively removing impurities in the specimen and enriching the bacteria.

[0004] In the prior art, patent CN112574863A discloses a bacteria detection method based on double-layer membrane filtration. The detection method of the present invention comprises the following steps: first, the sample to be tested is filtered through a filter containing a double-layer filter membrane composed of filter membranes of different pore sizes, so that the bacteria are enriched on the surface of the filter membrane, and then staining and bacteria detection are performed on the double-layer filter membrane. Patent CN112577794A discloses a preparation method for enriching bacteria by using shadowless glue to adhere to the membrane and its application. This method enriches the bacteria on the surface of the filter membrane by filtering, stains the filter membrane after filtration, and then uses shadowless glue to adhere the stained bacteria on the filter membrane, effectively avoiding the problem that the filter membrane cannot be washed with the staining solution, resulting in the inability to observe bacteria. Although this type of method is simple, the staining effect is not ideal; or the filter membrane is difficult to decolorize after staining, affecting the microscopic examination effect. Patent CN112574938B discloses a sputum treatment solution for enriching bacteria through membrane filtration and its application. The sputum treatment solution is composed of the following components: 0.5-4% sodium hydroxide, 0.1-1% dithiothreitol and 6-12% sodium hypochlorite. Since the sputum treatment solution contains a strong alkaline solution, Mycobacterium tuberculosis in the sputum can be completely inactivated, thereby improving the safety of operators. However, only a single bacterium can be detected.

[0005] Polyethersulfone membrane (PES) is a polymer material with excellent comprehensive performance, good chemical stability, acid and alkali resistance, heat resistance and aging resistance, etc. It has been widely used in ultrafiltration, microfiltration, reverse osmosis, gas separation, alcohol / water separation, olefin / alkane separation, immobilized carrier, hemodialysis, etc. Especially in the field of hemodialysis, due to the strong hydrophobicity of the surface of polyethersulfone membrane material, it is easy to produce adsorption and deposition on the membrane surface, so that the membrane pores are blocked, causing membrane pollution, reducing the performance of the membrane and shortening the service life. Therefore, in the prior art, more are based on the defects of the membrane in application, and further improve the technology of preparing polyethersulfone membrane material. Summary of the invention

[0006] The present invention is based on the discovery that polyethersulfone (PES) filter membrane can remove most impurities in clinical liquid specimens and can enrich and elute bacteria including Mycobacterium tuberculosis with an extremely low bacterial cell loss rate. On this basis, the present invention is completed.

[0007] In a first aspect, the present invention provides an application of a polyethersulfone membrane in the treatment of bacteria-containing specimens.

[0008] Furthermore, the polyethersulfone membrane can be used for the treatment of bacteria-containing specimens for impurity removal and / or bacterial enrichment.

[0009] Furthermore, when the polyethersulfone membrane is used to remove impurities from bacteria-containing specimens, the polyethersulfone membrane is selected from large-pore membranes, and the pore size is selected from 0.8-5 μm, preferably 1.2-4 μm, preferably 1.2-3 μm, and preferably 1.5-2 μm.

[0010] Furthermore, when the polyethersulfone membrane is used for enrichment of bacteria in bacterial specimens, the polyethersulfone membrane is selected from small pore size membranes, and the small pore size is selected from 0.22-0.8 μm, preferably 0.22-0.6 μm, and preferably 0.22-0.45 μm.

[0011] Furthermore, the specimens include but are not limited to original clinical specimens, culture specimens, and specimens processed by a processing method.

[0012] Furthermore, the bacteria-containing specimens include but are not limited to sputum specimens, pleural effusion, ascites, pericardial effusion, cerebrospinal fluid and other body fluid specimens to be tested.

[0013] In one embodiment, after the bacteria-containing specimen is treated with polyethersulfone membrane bacteria enrichment, the bacteria enriched on the polyethersulfone membrane are further eluted with an eluent, and the eluent includes but is not limited to 7H9 culture medium, glycerol, Tween80 solution and DMSO solution.

[0014] Furthermore, the volume concentration of the eluent is 1%-20%, preferably 5%-15, preferably 5%-10%, preferably 1%-5%.

[0015] Furthermore, the preferred volume concentration is 1%-20% DMSO solution.

[0016] In a second aspect, the present invention provides a filtering and / or enrichment system for bacteria-containing specimens, the system comprising a pretreatment module, a filtration module, a bacteria enrichment module and an elution module; wherein the sample pretreatment module processes the collected specimen to obtain a homogenized specimen sample; the filtration module uses a large-pore filter membrane and / or filter to filter impurities in the homogenized specimen sample; the enrichment module uses an enrichment device with a small pore size to enrich the bacteria in the specimen after filtering the impurities, and the bacteria are enriched on the front side of the enrichment device, and the filtration module, bacteria enrichment module and elution module of the filtration and / or enrichment system are all membranes made of polyethersulfone (PES); the elution module uses an elution solution to perform reverse elution from the enrichment device to elute the enriched bacteria.

[0017] Furthermore, the eluent includes but is not limited to 7H9 culture medium, glycerol, Tween80 solution and DMSO solution.

[0018] Furthermore, the concentration of the eluent is 1%-20%, preferably 5%-15%, preferably 5%-10%; preferably 1%-5%.

[0019] Furthermore, the reverse elution is to wash from the reverse side of the enrichment device using an eluent.

[0020] Furthermore, the enrichment device is a device having a membrane made of polyethersulfone (PES).

[0021] Furthermore, the pore size of the membrane made of polyethersulfone (PES) material is selected from 0.22-0.8 μm, preferably 0.22-0.6 μm, and preferably 0.22-0.45 μm.

[0022] Furthermore, the large-pore filter membrane in the filtration module is selected from a membrane made of polyethersulfone (PES); the large-pore diameter is selected from 0.8-5 μm, preferably from 1.2-4 μm, preferably from 1.2-3 μm, and preferably from 1.5-2 μm.

[0023] Furthermore, the filter is a filter device having a membrane made of polyethersulfone (PES) material, preferably a needle filter.

[0024] Furthermore, the shape of the polyethersulfone (PES) membrane includes but is not limited to a circle.

[0025] Furthermore, the membranes made of polyethersulfone (PES) are all hydrophilic.

[0026] Furthermore, the standardized treatment of the specimen is to adopt a certain treatment method to obtain direct sample filtration, and the treatment method includes but is not limited to alkaline digestion solution treatment.

[0027] In one embodiment, the system is a double filter filtration enrichment device, which consists of an upper filter and a lower filter, which are used in series, and the upper filter is located above the lower filter.

[0028] In a third aspect, the present invention provides a method for filtering and / or enriching a bacterial specimen, the method comprising the following steps:

[0029] S1. Pre-treat the bacterial specimens;

[0030] S2. Filter the pre-treated sample of S1 through a filter membrane;

[0031] S3. The filtered sample is passed through a filter membrane to enrich the bacteria on the membrane;

[0032] S4. Use the elution solution to reversely elute the bacteria enriched on the front side of the membrane.

[0033] Furthermore, the specimens described in S1 include but are not limited to original clinical specimens, culture specimens, and specimens processed by the processing method.

[0034] Furthermore, as described in S1, the bacterial specimens include but are not limited to sputum specimens, pleural effusion, ascites, pericardial effusion and cerebrospinal fluid specimens.

[0035] Furthermore, the specimen pretreatment is to obtain a homogenized specimen sample by adopting a certain processing method.

[0036] Furthermore, as described in S2, the filtration is to filter the bacteria-containing test sample using a membrane made of large-pore polyethersulfone (PES) material to remove impurities in the test sample.

[0037] Furthermore, the bacterial enrichment as described in S3 is to enrich the bacteria on the membrane by passing the sample from which impurities have been removed through a membrane made of small-pore polyethersulfone (PES).

[0038] Furthermore, the macropore size is selected from 0.8-5 μm, preferably from 1.2-4 μm, preferably from 1.2-3 μm, preferably from 1.5-2 μm.

[0039] Furthermore, the small pore size is selected from 0.22-0.8 μm, preferably from 0.22-0.6 μm, preferably from 0.22-0.45 μm.

[0040] Furthermore, the reverse elution is to use the elution solution to wash from the reverse side of the membrane enriched with bacteria.

[0041] Furthermore, the eluent includes but is not limited to 7H9 culture medium, glycerol, Tween80 solution and DMSO solution.

[0042] Furthermore, the volume concentration of the eluent is 1%-20%, preferably 5%-15, preferably 5%-10%, preferably 1%-5%.

[0043] Beneficial Effects

[0044] When processing bacterial samples, the system of the present invention not only has a low bacterial loss rate but also ensures the biological activity of the bacteria. Compared with existing bacterial sample processing systems, the processing steps are simplified and steps such as repeated centrifugation and washing of samples are eliminated. At the same time, since the bacterial loss rate is reduced, the effects of staining and biological detection are further improved, and the positive detection rate is increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The mass of impurities filtered out of the sample through filter membranes of different pore sizes is the percentage of the original sample mass.

[0046] Figure 2The samples were filtered through filter membranes with different pore sizes and then counted by centrifugal microscopy (experimental group) and directly counted by centrifugal microscopy (control group) for comparison. DETAILED DESCRIPTION

[0047] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0048] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0049] The term "front side" as used herein refers to the side of the filter or filter membrane that directly contacts the sample and allows the sample to pass through, and the other side opposite to it is the back side.

[0050] In one embodiment of the present invention, the polyethersulfone (PES) membrane is a fat-soluble membrane that is intolerant to dimethyl sulfoxide (DMSO). The use of DMSO can moderately digest the inner surface of the membrane microporous channel, expand the channel pore size during the elution process, and make it easier for bacteria to be eluted; the DMSO and digested PES particles contained in the final concentrated bacterial solution are harmless to the bacteria and can ensure the original normal biological activity of the bacteria; that is, in combination with Example 3 and Example 4, the bacterial enrichment rate and the bacterial elution rate are comprehensively considered to calculate the bacterial yield (bacterial enrichment rate x bacterial elution rate = bacterial yield), and a PES membrane with a pore size of 0.45 μm is used with 20% DMSO as an eluent to enrich and concentrate the bacteria, and the maximum amount of bacteria is obtained.

[0051] Example 1 Large-pore filter membrane filters impurities in the sample

[0052] (1) Take a batch of 1.5 ml centrifuge tubes, weigh them one by one, mark them on the lids, and set aside.

[0053] (2) Take 2 ml of each patient's sputum specimen and place it in a 5 ml centrifuge tube. Add an equal volume of NALC-NaOH solution (NaOH concentration 2%) (if the sputum sample is too thick, increase the volume of NALC-NaOH appropriately). Vortex to mix and let it stand for 15 min to digest. After completion, take two 1.5 ml portions and place them in two weighed 1.5 ml centrifuge tubes, marking them as sample 1 and sample 2.

[0054] (3) Use a 20 ml syringe to draw out all the sample in centrifuge tube No. 1, remove the needle, install a large-pore filter membrane (this example uses PES membranes with different pore sizes, namely 5 μm, 2 μm, 1.2 μm, and 0.8 μm), and then filter the sample back into the original tube.

[0055] (4) Sample No. 1 and Sample No. 2 were centrifuged at 12000 r for 5 min, and the supernatant was discarded and the bottom precipitate was retained.

[0056] (5) Weigh sample No. 1 and sample No. 2 using an electronic balance.

[0057] The experimental results show that the percentage of the weight of the sample after filtering through different pore size filters is different. The weight of impurities filtered by the 5μm pore size filter membrane accounts for 12.12% of the total weight of the sample, the weight of impurities filtered by the 2μm pore size filter membrane accounts for 25.91% of the total weight of the sample, and the weight of impurities filtered by the 1.2μm pore size filter membrane accounts for 51.2% of the total weight of the sample. The 0.8μm pore size filter membrane encounters too much resistance in the process of filtering impurities and cannot complete the entire filtering operation (see Figure 1 ). Therefore, the 1.2μm pore size filter membrane is the best for filtering impurities.

[0058] Example 2 Bacterial loss when filtering samples with different pore size membranes

[0059] 1. Filter group (experimental group)

[0060] (1) Take 2 ml of sputum sample from a clinical tuberculosis-positive patient, add an equal volume of NALC-NaOH solution (NaOH concentration 2%) (if the sputum sample is too thick, the volume of NALC-NaOH can be appropriately increased), shake and mix, and then let it stand for digestion for 15 minutes.

[0061] (2) Use a 20 ml syringe to draw out all the digested specimens in the centrifuge tube, remove the needle, install a large-pore filter membrane (this example uses PES membranes with different pore sizes, 5 μm, 2 μm, and 1.2 μm), and then filter the sample back into the original tube.

[0062] (3) Centrifuge the original tube at 3000r / min for 15min, discard the supernatant, and apply the precipitate on a glass slide, spreading it evenly into an oval film. Mark the frosted glass side of the slide with a pencil.

[0063] (4) After the slides were dried on a slide oven, they were placed on a staining rack, and auramine O staining solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main component of which is auramine O) was added to cover the bacterial film for staining for 15 minutes. After completion, the slides were rinsed off with water. Then, an acidic alcohol solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main components of which are hydrochloric acid and ethanol) was added to cover the bacterial film for decolorization for 3 minutes. After completion, the slides were rinsed off with water. Finally, potassium permanganate solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main component of which is potassium permanganate) was used to cover the bacterial film for re-staining for 1 minute. After completion, the slides were rinsed off with water.

[0064] (5) Place the stained slides on a slide dryer to dry, and then place them under a fluorescence microscope for observation and counting.

[0065] 2. Centrifugal group (control group)

[0066] (1) Take 2 ml of sputum sample from a clinical tuberculosis-positive patient, add an equal volume of NALC-NaOH solution (NaOH concentration 2%) (if the sputum sample is too thick, the volume of NALC-NaOH can be appropriately increased), shake and mix, and then let it stand for digestion for 15 minutes.

[0067] (2) After digestion, centrifuge the sputum specimen tube at 3000r / min for 15 min, discard the supernatant, and smear the precipitate on a glass slide, spreading it evenly into an oval film. Mark the frosted glass side of the slide with a pencil.

[0068] (3) Place the slide on a slide oven to dry, then place it on a staining rack, add auramine O staining solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main component is auramine O) to cover the bacterial film for staining for 15 minutes, and then rinse it off with water; then add acid alcohol solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main components are hydrochloric acid and ethanol) to cover the bacterial film for decolorization for 3 minutes, and then rinse it off with water; finally, use potassium permanganate solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main component is potassium permanganate) to cover the bacterial film for re-staining for 1 minute, and then rinse it off with water.

[0069] (4) Place the stained slides on a slide dryer to dry, and then place them under a fluorescence microscope for observation and counting.

[0070] (5) Calculation of bacterial loss rate:

[0071]

[0072] Comparison of samples filtered through membranes with different pore sizes and then centrifuged and counted (experimental group) and samples directly centrifuged and counted (control group) Figure 2As shown, the samples were counted under a centrifugal microscopy after passing through filter membranes with different pore sizes, and were also counted under a microscopy after direct centrifugation. The results were that the average number of bacteria counted under a centrifugal microscopy after filtration through a 5μm pore size filter membrane was 58.1, the average number of bacteria counted under a centrifugal microscopy after filtration through a 2μm pore size filter membrane was 54.8, the average number of bacteria counted under a centrifugal microscopy after filtration through a 1.2μm pore size filter membrane was 51.1, and the average number of bacteria counted directly under a centrifugal microscopy was 60, indicating that filter membranes with pore sizes of 5μm, 2μm, and 1.2μm will produce a small amount of bacterial loss when filtering impurities, but the maximum bacterial loss rate does not exceed 15%.

[0073] Combining Example 1 and Example 1, considering both the impurity filtration effect and the bacterial loss, it is best to use a 1.2 μm pore size filter membrane as a large pore size filter membrane.

[0074] Example 3 Effect of small pore size filtration membranes of different materials on enrichment of bacteria in samples

[0075] 1. Filtration method (experimental group)

[0076] (1) Take 2 ml of sputum sample from a clinical tuberculosis-positive patient, add an equal volume of NALC-NaOH solution (NaOH concentration 2%) (if the sputum sample is too thick, the volume of NALC-NaOH can be appropriately increased), shake and mix, and then let it stand for digestion for 15 minutes.

[0077] (2) Use a 20 ml syringe to drain all the digested specimens in the centrifuge tube, remove the needle, and install a large-pore filter membrane (in this example, 1.2μm pore size PES After filtering through a membrane, the sample was returned to the original tube.

[0078] (3) Use a 20 ml syringe to draw out all the filtered samples in the centrifuge tube, remove the needle, install a small-pore filter membrane (this example uses filter membranes of different materials and pore sizes, the materials are PTFE and PES, and the pore sizes are 0.22 μm, 0.45 μm, and 0.8 μm, respectively), filter the sample back into the original tube, and mark it as a waste liquid tube.

[0079] (4) Centrifuge the waste liquid tube at 3000r / min for 15min, discard the supernatant, and apply the precipitate on a glass slide, spreading it evenly into an oval film. Mark the frosted glass side of the slide with a pencil.

[0080] (5) Place the slide on a slide oven to dry, then place it on a staining rack, add auramine O staining solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main component is auramine O) to cover the bacterial film for staining for 15 minutes, and then rinse it off with water; then add acid alcohol solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main components are hydrochloric acid and ethanol) to cover the bacterial film for decolorization for 3 minutes, and then rinse it off with water; finally, use potassium permanganate solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main component is potassium permanganate) to cover the bacterial film for re-staining for 1 minute, and then rinse it off with water.

[0081] (6) Place the stained slides on a slide dryer to dry, and then place them under a fluorescence microscope for observation and counting.

[0082] 2. Centrifugal group (control group)

[0083] (1) Take 2 ml of sputum sample from a clinical tuberculosis-positive patient, add an equal volume of NALC-NaOH solution (NaOH concentration 2%) (if the sputum sample is too thick, the volume of NALC-NaOH can be appropriately increased), shake and mix, and then let it stand for digestion for 15 minutes.

[0084] (2) After digestion, centrifuge the sample at 3000r / min for 15 min, discard the supernatant, and apply the precipitate on a glass slide, spreading it evenly into an oval film. Mark the frosted glass side of the slide with a pencil.

[0085] (3) Place the slide on a slide oven to dry, then place it on a staining rack, add auramine O staining solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main component is auramine O) to cover the bacterial film for staining for 15 minutes, and then rinse it off with water; then add acid alcohol solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main components are hydrochloric acid and ethanol) to cover the bacterial film for decolorization for 3 minutes, and then rinse it off with water; finally, use potassium permanganate solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main component is potassium permanganate) to cover the bacterial film for re-staining for 1 minute, and then rinse it off with water.

[0086] (4) Place the stained slides on a slide dryer to dry, and then place them under a fluorescence microscope for observation and counting.

[0087] (5) Calculation of bacterial enrichment rate:

[0088]

[0089] As shown in Table 1abc, when filtering samples with filter membranes of different materials and pore sizes, different degrees of bacterial loss will occur. Therefore, the amount of bacteria that can be enriched by the same sample through filter membranes of different materials and pore sizes is different. Among them, the PES membrane with a pore size of 0.45μm has the best enrichment effect, and the bacterial enrichment rate can reach 79.43%.

[0090] Table 1a Bacteria count in a single field of view under a 40x objective lens in the experimental group (individuals)

[0091]

[0092] Table 1b Bacteria count in a single field of view under a 40x objective lens in the centrifugation group (units)

[0093]

[0094] Table 1c Enrichment effect of bacteria in samples after filtration based on filter membranes of different materials and pore sizes

[0095]

[0096] Example 4: Different eluents are used to elute the enriched cells using small-pore filtration membranes made of different materials

[0097] 1. Filtration method (elution group and waste liquid group):

[0098] (1) Take 2 ml of sputum sample from a clinical tuberculosis-positive patient, add an equal volume of NALC-NaOH solution (NaOH concentration 2%) (if the sputum sample is too thick, the volume of NALC-NaOH can be appropriately increased), shake and mix, and then let it stand for digestion for 15 minutes.

[0099] (2) Use a 20 ml syringe to draw out all the digested specimens in the centrifuge tube, remove the needle, install a large-pore filter membrane (in this example, a 1.2 μm pore size PES membrane), and filter the sample back into the original tube.

[0100] (3) Use a 20 ml syringe to draw out all the filtered samples in the centrifuge tube, remove the needle, install a small-pore filter membrane (this example uses 0.45 μm pore filter membranes made of different materials, PTFE and PES), filter the sample back into the original tube, and mark it as a waste liquid tube.

[0101] (4) Remove the small-pore filter membrane and install it in reverse on a 20 ml syringe filled with eluent (this example uses eluents of different formulas). Slowly push two drops into a 1.5 ml centrifuge tube, add 0.1-0.2 g of carboxymethyl cellulose, mix well, and mark for later use.

[0102] (5) Take 1 drop of the push solution from the 1.5 ml centrifuge tube and drop it on the slide. Spread it evenly into an oval film and mark the frosted glass side of the slide with a pencil. (Elution group)

[0103] (6) Centrifuge the waste liquid tube at 3000r for 15min, discard the supernatant, take the precipitate and spread it on a glass slide, evenly spreading it into an oval film, and mark the frosted glass side of the slide with a pencil. (Waste liquid group)

[0104] (7) After the slides were dried on a slide oven, they were placed on a staining rack, and auramine O staining solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main component of which is auramine O) was added to cover the bacterial film for staining for 15 minutes. After completion, the slides were rinsed off with water. Then, an acidic alcohol solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main components of which are hydrochloric acid and ethanol) was added to cover the bacterial film for decolorization for 3 minutes. After completion, the slides were rinsed off with water. Finally, potassium permanganate solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main component of which is potassium permanganate) was used to cover the bacterial film for re-staining for 1 minute. After completion, the slides were rinsed off with water.

[0105] (8) Place the stained slides on a slide dryer to dry, and then place them under a fluorescence microscope for observation and counting.

[0106] 2. Centrifugation method (centrifugation group):

[0107] (1) Take 2 ml of sputum sample from a clinical tuberculosis-positive patient, add an equal volume of NALC-NaOH solution (NaOH concentration 2%) (if the sputum sample is too thick, the volume of NALC-NaOH can be appropriately increased), shake and mix, and then let it stand for digestion for 15 minutes.

[0108] (2) After digestion, centrifuge the sample at 3000r / min for 15 min, discard the supernatant, and apply the precipitate on a glass slide, spreading it evenly into an oval film. Mark the frosted glass side of the slide with a pencil.

[0109] (3) Place the slide on a slide oven to dry, then place it on a staining rack, add auramine O staining solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main component is auramine O) to cover the bacterial film for staining for 15 minutes, and then rinse it off with water; then add acid alcohol solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main components are hydrochloric acid and ethanol) to cover the bacterial film for decolorization for 3 minutes, and then rinse it off with water; finally, use potassium permanganate solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main component is potassium permanganate) to cover the bacterial film for re-staining for 1 minute, and then rinse it off with water.

[0110] (4) Place the stained slides on a slide dryer to dry, and then place them under a fluorescence microscope for observation and counting.

[0111] (5) Calculation method of elution rate:

[0112]

[0113] Table 2a The number of bacteria in a single field of view under a 40x objective lens in the elution group (individuals)

[0114]

[0115]

[0116] Table 2d Effect of using different eluents to elute the bacteria enriched in filter membranes of different materials

[0117]

[0118] The effect of using different eluents to elute the bacteria enriched on the filter membranes of different materials is shown in Table 2abcd. The proportion of the number of bacteria after eluting the filter membranes of different materials that have been enriched by using different eluents to the total number of bacteria enriched on the membranes is different. The experiment used a variety of eluents, including 7H9 culture medium, glycerol (concentrations of 20%, 10%, 5%, 1%, diluted with pure water), Tween80 solution (concentrations of 20%, 10%, 5%, 1%, diluted with 7H9 culture medium), DMSO solution (concentrations of 20%, 10%, 5%, 1%, diluted with pure water); at the same time, filter membranes of different materials were used for comparison, including PTFE membrane and PES membrane, with pore sizes of 0.45μm. Among them, 20% Tween 80 with PTFE membrane has the best bacterial elution effect, and 20% DMSO with PES membrane ranks fourth in bacterial elution effect.

[0119] Combining Example 3 and Example 4, the bacterial enrichment rate and bacterial elution rate were considered comprehensively to calculate the bacterial yield (bacterial enrichment rate x bacterial elution rate = bacterial yield). A PES membrane with a pore size of 0.45 μm and 20% DMSO were used as the eluent to enrich and concentrate the bacteria, and the maximum amount of bacteria was obtained.

[0120] Example 5: Staining the enriched bacteria using two methods: staining on the membrane and staining after elution

[0121] 1. Membrane staining

[0122] (1) Take 2 ml of sputum sample from a clinical tuberculosis-positive patient, add an equal volume of NALC-NaOH solution (NaOH concentration 2%) (if the sputum sample is too thick, the volume of NALC-NaOH can be appropriately increased), shake and mix, and then let it stand for digestion for 15 minutes.

[0123] (2) Use a 20 ml syringe to draw out all the digested specimens in the centrifuge tube, remove the needle, install a large-pore filter membrane (in this example, a 1.2 μm pore size PES membrane), and filter the sample back into the original tube.

[0124] (3) Use a 20 ml syringe to draw out all the filtered samples in the centrifuge tube, remove the needle, install a small-pore filter membrane (this example uses 0.45 μm pore filter membranes made of different materials, PTFE and PES), filter the sample back into the original tube, and mark it as a waste liquid tube.

[0125] (4) Remove the small-pore filter membrane, connect it forward to a 20 ml syringe filled with auramine O dye, and slowly push the syringe to cover the membrane surface. Remove it and let it stand for staining for 15 minutes. After completion, connect it forward to a 20 ml syringe filled with acid alcohol solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main components are hydrochloric acid and ethanol), and slowly push the syringe to cover the membrane surface with decolorizing solution. Remove it and let it stand for decolorization for 3 minutes. After completion, connect it forward to a 20 ml syringe filled with potassium permanganate solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main component is potassium permanganate), and slowly push the syringe to cover the membrane surface with re-staining solution. Remove it and let it stand for re-staining for 1 minute. After completion, connect it forward to a 20 ml syringe filled with pure water and a section of air, and slowly push the syringe to rinse the re-staining solution with pure water, and then drain the water with air.

[0126] (5) Remove the small-pore filter membrane and install it in reverse on a 20 ml syringe filled with eluent (in this example, different eluents are used for different filter membranes: PTFE membrane is eluted with 7H9 culture medium, and PES membrane is eluted with 20% DMSO solution). Slowly push two drops into a 1.5 ml centrifuge tube, add 0.1-0.2 g of carboxymethyl cellulose, mix well, and mark for later use.

[0127] (6) Take one drop of the reverse push liquid from the 1.5 ml centrifuge tube and drop it on a glass slide. Spread it evenly into an oval film and mark the frosted glass side of the slide with a pencil.

[0128] (7) Place the stained slides on a slide dryer to dry, then place them under a fluorescence microscope for observation, counting, and photographing.

[0129] 2. Staining after elution

[0130] (1) Take 2 ml of sputum sample from a clinical tuberculosis-positive patient, add an equal volume of NALC-NaOH solution (NaOH concentration 2%) (if the sputum sample is too thick, the volume of NALC-NaOH can be appropriately increased), shake and mix, and then let it stand for digestion for 15 minutes.

[0131] (2) Use a 20 ml syringe to draw out all the digested specimens in the centrifuge tube, remove the needle, install a large-pore filter membrane (in this example, a 1.2 μm pore size PES membrane), and filter the sample back into the original tube.

[0132] (3) Use a 20 ml syringe to draw out all the filtered samples in the centrifuge tube, remove the needle, install a small-pore filter membrane (this example uses 0.45 μm pore filter membranes made of different materials, PTFE and PES), filter the sample back into the original tube, and mark it as a waste liquid tube.

[0133] (4) Remove the small-pore filter membrane and install it in reverse on a 20 ml syringe filled with eluent (in this example, different eluents are used for different filter membranes: PTFE membrane is eluted with 7H9 culture medium, and PES membrane is eluted with 20% DMSO solution). Slowly push two drops into a 1.5 ml centrifuge tube, add 0.1-0.2 g of carboxymethyl cellulose, mix well, and mark for later use.

[0134] (5) Take one drop of the reverse push liquid from the 1.5 ml centrifuge tube and drop it on a glass slide. Spread it evenly into an oval film and mark the frosted glass side of the slide with a pencil.

[0135] (6) Place the slide on a slide oven to dry, then place it on a staining rack, add auramine O staining solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main component is auramine O) to cover the bacterial film for staining for 15 minutes, and then rinse it off with water; then add acid alcohol solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main components are hydrochloric acid and ethanol) to cover the bacterial film for decolorization for 3 minutes, and then rinse it off with water; finally, use potassium permanganate solution (purchased from Qingdao Lanche Biotechnology Co., Ltd., the main component is potassium permanganate) to cover the bacterial film for re-staining for 1 minute, and then rinse it off with water.

[0136] (7) Place the stained slides on a slide dryer to dry, then place them under a fluorescence microscope for observation, counting, and photographing.

[0137] Table 3 Effects of different staining methods on the enriched bacteria on filter membranes of different materials

[0138]

[0139] Comparison of results:

[0140] The effects of using different staining methods to stain the bacteria enriched in filter membranes of different materials are shown in Table 3. The experiment adopted two methods, on-membrane staining and post-elution staining, to perform fluorescent staining on the bacteria enriched in PTFE membrane and PES membrane with a pore size of 0.45 μm, respectively, and counted the bacteria in a single field of view and measured the fluorescence intensity. Among them, the best staining effect was achieved after elution in terms of both fluorescence intensity and bacterial count.

Claims

1. An application of a polyethersulfone membrane in the treatment of bacteria-containing specimens, wherein the treatment application of the polyethersulfone membrane is to remove impurities and / or enrich bacteria in bacteria-containing specimens. When the polyethersulfone membrane is used to remove impurities in bacteria-containing specimens, the polyethersulfone membrane is selected from large-pore membranes. When the polyethersulfone membrane is used to enrich bacteria in bacteria-containing specimens, the polyethersulfone membrane is selected from small-pore membranes. After the bacteria are enriched, an eluent is used for reverse elution to elute the enriched bacteria.

2. The use according to claim 1, characterized in that The pore size of the macroporous membrane is selected from 0.8-5 μm.

3. The use according to claim 1, characterized in that The pore size of the small-pore membrane is selected from 0.22-0.8 μm.

4. The use according to claim 1, characterized in that The specimens include but are not limited to original clinical specimens, culture specimens, and specimens processed by processing methods.

5. The use according to claim 1, characterized in that The bacteria-containing specimens include, but are not limited to, sputum specimens, pleural effusion, ascites, pericardial effusion, cerebrospinal fluid and other body fluid specimens to be tested.

6. The use according to claim 1, characterized in that The eluent includes, but is not limited to, 7H9 culture medium, glycerol, Tween80 solution and DMSO solution.

7. A filtration and / or enrichment system for bacterial specimens, the system comprising a pre-treatment module, a filtration module, a bacterial enrichment module and an elution module; wherein: The sample pre-treatment module processes the collected specimens to obtain homogenized specimen samples; the filtration module uses a large-pore filter membrane and / or filter to filter impurities in the homogenized specimen samples; the enrichment module uses a small-pore enrichment device to enrich the bacteria in the specimen after filtering the impurities, and the bacteria are enriched on the front side of the enrichment device. The filtration module, bacteria enrichment module and elution module of the filtration and / or enrichment system are all membranes made of polyethersulfone (PES); the elution module uses an eluent to perform reverse elution from the enrichment device to elute the enriched bacteria.

8. A system for filtering and / or enriching bacteria-containing specimens as claimed in claim 7, characterized in that: The small pore size of the membrane made of polyethersulfone (PES) material is selected from 0.22-0.8 μm, and the large pore size filter membrane in the filtration module is selected from a membrane made of polyethersulfone (PES); the large pore size is selected from 0.8-5 μm.

9. A method for filtering and / or enriching a bacterial specimen, the method comprising the following steps: S1. Pre-treat the bacterial specimens; S2. Filter the pre-treated sample of S1 through a filter membrane; S3. The filtered sample is passed through a filter membrane to enrich the bacteria on the membrane; S4. Use the eluent to reversely elute the bacteria enriched on the front side of the membrane; Reverse elution is to use the elution solution to wash the reverse side of the membrane where the bacteria are enriched.

10. The method for filtering and / or enriching bacteria-containing specimens as described in claim 9, wherein the eluent includes but is not limited to 7H9 culture medium, glycerol, Tween80 solution and DMSO solution; the volume concentration is 1%-20%.

Images