Enrichment method of bacterium-containing specimen and application thereof

Abstract

The invention belongs to the technical field of medical detection, particularly relates to a bacterium-containing specimen enrichment method and application thereof, and particularly discloses a bacterium-containing specimen enrichment and elution method which comprises the following steps: S1, pretreating a bacterium-containing specimen; s2, filtering the pretreated sample obtained in the step S1 through a filter membrane; s3, carrying out membrane enrichment on the filtered sample through a filter membrane; and S4, carrying out reverse elution on the thalli enriched on the front surface of the membrane by using an eluent. According to the method, complex steps in conventional specimen treatment can be omitted, the thallus loss rate is extremely low, and the concentration of the obtained bacterial liquid is high; and the method also has the advantages of simplicity, convenience and rapidness in operation, higher biological safety, low cost, environmental friendliness and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection, and in particular relates to a method for enriching bacteria-containing specimens and an application thereof. Background Art

[0002] The main difficulty in the discovery and diagnosis of bacterial infection is that the pathogens in the specimens are few in content and low in concentration, which makes it impossible to detect them sensitively by most common bacterial detection methods. Therefore, purification and enrichment of bacteria in limited clinical specimens has become an urgent problem to be solved. Solving this problem will greatly promote the timely discovery of pathogens, confirm the type of bacteria infected by patients and their drug sensitivity, and thus provide appropriate treatment plans to achieve the purpose of effective treatment.

[0003] At present, there is no effective method for enriching bacteria except the centrifugation method. However, the centrifugation method often causes bacteria to form aerosols and has low biosafety, and it is also easy to cause impurities in the specimen to precipitate together with the bacteria to be tested, which is not conducive to the detection of bacteria in the specimen. The microporous membrane filtration method is a relatively mature method and 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 shielding 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 the liquid, and then observe the smear, which will significantly improve the microscopic examination effect. For the existing domestic and foreign eluent formulas, experiments have found that it can increase 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 matching elution solution for elution is the key to improving the bacterial yield.

[0004] There are many types of microporous filter membranes, which can be divided into acid-resistant filter membranes [such as nylon (NY or PA) membranes] and alkali-resistant filter membranes [such as polyvinylidene fluoride membranes (PVDF)] according to the tolerance to pH. After filtering with a microporous filter membrane, the bacteria are enriched on the membrane surface. However, since the microporous filter membrane has a complex and irregular mesh pore channel, the enriched bacteria often cannot be effectively eluted, resulting in the loss of bacteria that can actually be obtained in the specimen. There are many different formulations of flushing liquids on the market, but the effects are not ideal, and in the face of microporous filter membranes of various materials, there are many types of flushing liquids that can be selected, and it is impossible to determine the optimal pairing scheme. Therefore, the present invention takes into account the acid-base tolerance of different microporous filter membranes, and can use an eluent with a certain pH to efficiently elute the bacteria from the microporous filter membrane to achieve the purpose of quickly and effectively removing impurities in the specimen and enriching the bacteria.

[0005] In the prior art, patent CN112574863A discloses a bacteria detection method based on double-layer membrane filtration. The detection method of the invention includes the following steps: first, the sample to be tested is filtered through a filter containing a double-layer filter membrane composed of filter membranes with 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 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, thereby avoiding the problem that the filter membrane cannot be washed off 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, which affects 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.

[0006] At present, according to the tolerance of membranes to acid and alkalinity, there are acid-resistant membranes [such as nylon (NY or PA) membranes], alkali-resistant membranes [such as polyvinylidene fluoride (PVDF) membranes], and membranes that are both acid-resistant and alkali-resistant [such as polytetrafluoroethylene (PTFE) membranes]. Among them, nylon (NY or PA) membranes are suitable for filtering aqueous solutions and most organic solutions, and are widely used in biological sample preparation and filtration where other membranes are not suitable or difficult to use. Nylon membranes are naturally hydrophilic, flexible, durable, and tear-resistant. They can be sterilized at high temperatures at 135°C. They are chemically stable and can tolerate a variety of organic and inorganic compounds such as alcohols, esters, oils, hydrocarbons, halogenated hydrocarbons, and organic oxides. However, their structure will be significantly damaged when infiltrated by acids with a concentration of >1N. Polyvinylidene fluoride (PVDF), also known as polyvinylidene fluoride, is a polymer filter membrane material. It has the heat resistance, chemical resistance and UV resistance of fluoropolymers. It has good corrosion resistance to most solvents, acids and salts. It is a filter membrane with very low protein adsorption among known filter membranes. Therefore, it is often used for sterilization and particle removal of liquids such as protein solutions, tissue culture media, antibiotics and ethanol. It can also be used for biological tests (such as receptor binding studies, TCA precipitation) and other applications, but it is not recommended to filter alkaline solutions >1N. Polytetrafluoroethylene (PTFE) is a thermoplastic with excellent corrosion resistance, insulation and high temperature resistance. It can be used in the range of -180℃ to +260℃. Polytetrafluoroethylene has low surface energy and low surface tension. It is a typical hydrophobic material with wide chemical compatibility. It can withstand strong solvents such as DMSO, THF, DMF, dichloromethane and chloroform. It is used for filtering most organic solutions, especially strong solvents that other filter membranes cannot withstand. After hydrophilic treatment, it can filter water-oil mixed solutions.

[0007] In the present invention, for bacteria-containing specimens, due to the limited amount of specimens and the small amount and low concentration of pathogens in the specimens, most common bacteria detection methods currently cannot detect them sensitively. At the same time, in the process of membrane elution after filtration, due to the different eluents, the elution efficiency of pathogens cannot be guaranteed, and it cannot be guaranteed that the bacteria will not be inactivated by the eluent, thereby affecting the enrichment efficiency of pathogens. Therefore, it is urgent to develop a new pathogen enrichment method for bacteria-containing specimens. Summary of the invention

[0008] The present invention has discovered a method for efficiently filtering and removing most of the impurities in a specimen and enriching bacteria based on a filter membrane, and eluting the enriched bacteria by utilizing the acid and alkali resistance of the filter membrane. Based on this, the present invention has been completed.

[0009] In a first aspect, the present invention provides a method for enriching and eluting a bacterial specimen, the method comprising the following steps:

[0010] S1. Pre-treat the bacterial specimens;

[0011] S2. Filter the pre-treated sample of S1 through a filter membrane;

[0012] S3. The filtered sample is enriched by a filter membrane;

[0013] S4. Use the elution solution to reversely elute the bacteria enriched on the front side of the membrane.

[0014] Furthermore, the specimens described in S1 include but are not limited to original clinical specimens, culture specimens, and specimens processed by the processing method.

[0015] 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.

[0016] Furthermore, the specimen pretreatment is to obtain a homogenized specimen sample by a certain treatment method for filtration, and the treatment method includes but is not limited to acid digestion treatment and alkaline digestion solution treatment.

[0017] Furthermore, as described in S2, the filtration is to filter the bacteria-containing test specimen using a large-pore acid-resistant or alkali-resistant filter membrane to remove impurities in the test specimen.

[0018] Furthermore, the 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 small-pore alkali-resistant or acid-resistant filtration membrane.

[0019] 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.

[0020] 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.

[0021] Furthermore, the acid-resistant membrane includes but is not limited to polytetrafluoroethylene (PTFE) membrane and polypropylene (PP) membrane.

[0022] Furthermore, the alkali-resistant membrane includes but is not limited to polytetrafluoroethylene (PTFE) membrane, polypropylene (PP) membrane and polyethersulfone (PES) membrane.

[0023] Furthermore, the acid-intolerant filter membrane includes but is not limited to nylon (NY or PA) membrane and mixed cellulose (MCE) membrane.

[0024] Furthermore, the alkali-inresistant filter membrane includes but is not limited to polyvinylidene fluoride (PVDF) membrane and mixed cellulose (MCE) membrane.

[0025] Furthermore, the eluent described in S4 includes an acidic or alkaline eluent.

[0026] Further, the acidity of the acidic eluent is expressed in terms of H + The concentration is measured at 0.01-2 mol / L.

[0027] Furthermore, the alkalinity of the alkaline eluent is expressed as OH - The concentration is measured at 0.01-2 mol / L.

[0028] Furthermore, the acidic eluent includes but is not limited to HCL solution and H2SO4 solution.

[0029] Furthermore, the alkaline eluent includes but is not limited to KOH solution and NaOH solution.

[0030] Furthermore, the eluent includes but is not limited to eluent 1, eluent 2, 7H9 culture medium, HCL solution, NaOH solution and Tween80 solution, the eluent 1 contains 0.05M KH2PO4, 1M NaCl, 0.1% Tween 80, and 0.1% sodium hexametaphosphate, and the eluent 2 contains 0.02% Tween 80 and 0.02% sodium hexametaphosphate.

[0031] Furthermore, the volume of the eluent is 100-2500 μL, and the retention time of the eluent on the membrane is 5-25 s.

[0032] In one embodiment of the present invention, alkali-resistant bacteria such as Mycobacterium tuberculosis are selected to digest the specimen with NaOH solution to homogenize the specimen, an alkali-resistant large-pore filter membrane such as a PTFE membrane is used to filter impurities, an acid-intolerant small-pore filter membrane such as a nylon membrane is used to enrich the bacteria on the front side of the membrane, and a specific acidic eluent such as an HCL solution is used for backwashing. The acidic eluent digests the inner surface of each micropore to a certain extent, increases the pore size of the micropores, and allows the enriched bacteria to be efficiently eluted. Finally, the eluted bacterial solution is promptly subjected to acid-base neutralization to maintain the long-term activity of the bacteria.

[0033] In another embodiment of the present invention, acid-resistant bacteria such as Mycobacterium tuberculosis are treated with HCL solution to digest the specimen to make the specimen homogenized, an acid-resistant large-pore filter membrane such as a PTFE membrane is used to filter impurities, an alkali-intolerant small-pore filter membrane such as a PVDF membrane is used to enrich the bacteria on the front side of the membrane, and a specific alkaline eluent such as a NaOH solution is used for backwashing. The alkaline eluent digests the inner surface of each micropore to a certain extent, increases the pore size of the micropores, and allows the enriched bacteria to be efficiently eluted. Finally, the eluted bacterial solution is promptly subjected to acid-base neutralization to maintain the long-term activity of the bacteria.

[0034] 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 is used to pretreat the collected specimens to obtain acidic or alkaline specimens that can be further detected; the filtration module is used to filter impurities in the solution specimens through acid-resistant or alkali-resistant filter membranes and / or filters for the pretreated specimens; the enrichment module is used to enrich the bacteria in the filtered specimens using an alkali-resistant or acid-resistant and small-pore filter membrane, and the bacteria are enriched on the front side of the filter membrane; the elution module is used to perform reverse elution using an elution solution from the enrichment module to elute the bacteria from the filter membrane.

[0035] Furthermore, the eluent includes an acidic or alkaline eluent.

[0036] Further, the acidity of the acidic eluent is expressed in terms of H + The concentration is measured at 0.01-2 mol / L.

[0037] Furthermore, the alkalinity of the alkaline eluent is expressed as OH - The concentration is measured at 0.01-2 mol / L.

[0038] Furthermore, the acidic eluent includes but is not limited to HCL solution and H2SO4 solution.

[0039] Furthermore, the alkaline eluent includes but is not limited to KOH solution and NaOH solution.

[0040] Furthermore, the eluent includes but is not limited to eluent 1, eluent 2, 7H9 culture medium, HCL solution, NaOH solution and Tween80 solution, the eluent 1 contains 0.05M KH2PO4, 1M NaCl, 0.1% Tween 80 and 0.1% sodium hexametaphosphate, and the eluent 2 contains 0.02% Tween 80 and 0.02% sodium hexametaphosphate.

[0041] Furthermore, the volume of the eluent is 100-2500 μL, and the retention time of the eluent on the membrane is 5-25 s.

[0042] 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.

[0043] 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.

[0044] Furthermore, the acid-resistant membrane includes but is not limited to polytetrafluoroethylene (PTFE) membrane and polypropylene (PP) membrane.

[0045] Furthermore, the alkali-resistant membrane includes but is not limited to polytetrafluoroethylene (PTFE) membrane, polypropylene (PP) membrane and polyethersulfone (PES) membrane.

[0046] Furthermore, the acid-intolerant filter membrane includes but is not limited to nylon (NY or PA) membrane and mixed cellulose (MCE) membrane.

[0047] Furthermore, the alkali-inresistant filter membrane includes but is not limited to polyvinylidene fluoride (PVDF) membrane and mixed cellulose (MCE) membrane.

[0048] Furthermore, the filter may be a double filter device, wherein the double filter filtration and enrichment device is composed of an upper filter and a lower filter, which are used in combination in series, and the upper filter is located above the lower filter.

[0049] Furthermore, the dual-filter filtration and enrichment device can not only perform the impurity removal and bacteria collection operations of systemic tuberculosis clinical samples based on the device as a whole, but can also be separated and used separately for the impurity removal and bacteria enrichment operation steps.

[0050] In one embodiment of the present invention, alkali-resistant bacteria such as Mycobacterium tuberculosis are selected to digest the specimen with NaOH solution to homogenize the specimen, an alkali-resistant large-pore filter membrane such as a PTFE membrane is used to filter impurities, an acid-intolerant small-pore filter membrane such as a nylon membrane is used to enrich the bacteria on the front side of the membrane, and a specific acidic eluent such as an HCL solution is used for backwashing. The acidic eluent digests the inner surface of each micropore to a certain extent, increases the pore size of the micropores, and allows the enriched bacteria to be efficiently eluted. Finally, the eluted bacterial solution is promptly subjected to acid-base neutralization to maintain the long-term activity of the bacteria.

[0051] In another embodiment of the present invention, acid-resistant bacteria such as Mycobacterium tuberculosis are treated with HCL solution to digest the specimen to make the specimen homogenized, an acid-resistant large-pore filter membrane such as a PTFE membrane is used to filter impurities, an alkali-intolerant small-pore filter membrane such as a PVDF membrane is used to enrich the bacteria on the front side of the membrane, and a specific alkaline eluent such as a NaOH solution is used for backwashing. The alkaline eluent digests the inner surface of each micropore to a certain extent, increases the pore size of the micropores, and allows the enriched bacteria to be efficiently eluted. Finally, the eluted bacterial solution is promptly subjected to acid-base neutralization to maintain the long-term activity of the bacteria.

[0052] In a third aspect, the present invention provides a system for filtering and / or enriching bacteria-containing specimens, and its application in filtering or enriching bacteria in bacteria-containing specimens.

[0053] The system includes a pre-treatment module, a filtration module, a bacterial enrichment module and an elution module; wherein the sample pre-treatment module is used to pre-treat the collected specimens to obtain acidic or alkaline specimens that can be further detected; the filtration module is used to filter impurities in the solution specimens through acid-resistant or alkali-resistant filter membranes and / or filters for the pre-treated specimens; the enrichment module is used to enrich the bacteria in the filtered specimens using a filter membrane that is not alkali-resistant or acid-resistant and has a small pore size, and the bacteria are enriched on the front side of the filter membrane; the elution module is used to perform reverse elution using an elution solution from the enrichment module to elute the bacteria from the filter membrane.

[0054] Furthermore, the eluent includes an acidic or alkaline eluent.

[0055] Further, the acidity of the acidic eluent is expressed in terms of H + The concentration is measured at 0.01-2 mol / L.

[0056] Furthermore, the alkalinity of the alkaline eluent is expressed as OH - The concentration is measured at 0.01-2 mol / L.

[0057] Furthermore, the acidic eluent includes but is not limited to HCL solution and H2SO4 solution.

[0058] Furthermore, the alkaline eluent includes but is not limited to KOH solution and NaOH solution.

[0059] Furthermore, the eluent includes but is not limited to eluent 1, eluent 2, 7H9 culture medium, HCL solution, NaOH solution and Tween80 solution, the eluent 1 contains 0.05M KH2PO4, 1M NaCl, 0.1% Tween 80 and 0.1% sodium hexametaphosphate, and the eluent 2 contains 0.02% Tween 80 and 0.02% sodium hexametaphosphate.

[0060] Furthermore, the volume of the eluent is 100-2500 μL, and the retention time of the eluent on the membrane is 5-25 s.

[0061] 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.

[0062] Furthermore, the small pore size is selected from 0.22-0.8 μm, preferably 0.22-0.6 μm, preferably 0.22-0.45 μm.

[0063] Furthermore, the acid-resistant membrane includes but is not limited to polytetrafluoroethylene (PTFE) membrane and polypropylene (PP) membrane.

[0064] Furthermore, the alkali-resistant membrane includes but is not limited to polytetrafluoroethylene (PTFE) membrane, polypropylene (PP) membrane and polyethersulfone (PES) membrane.

[0065] Furthermore, the acid-intolerant filter membrane includes, but is not limited to, a polytetrafluoroethylene (PTFE) membrane, a polypropylene (PP) membrane, and a polyethersulfone (PES) membrane.

[0066] Furthermore, the alkali-inresistant filter membrane includes but is not limited to polyvinylidene fluoride (PVDF) membrane and mixed cellulose (MCE) membrane.

[0067] Furthermore, the specimens include but are not limited to original clinical specimens, culture specimens, and specimens processed by a processing method.

[0068] Furthermore, the bacteria-containing specimens include but are not limited to sputum specimens, pleural effusion, ascites, pericardial effusion and cerebrospinal fluid specimens.

[0069] Beneficial Effects

[0070] The method of the present invention can avoid the complicated steps in conventional specimen processing, the bacterial cell loss rate is extremely low, and the obtained bacterial solution concentration is high; it also has the advantages of simple and rapid operation, higher biosafety, low cost, and environmental friendliness. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] 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.

[0072] Figure 2 The 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

[0073] 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.

[0074] 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.

[0075] In one embodiment of the present invention, alkali-resistant bacteria such as Mycobacterium tuberculosis are selected to digest and treat specimens with alkaline solutions such as NaOH solutions to homogenize the specimens, and impurities are filtered out using alkali-resistant large-pore filter membranes such as PTFE membranes. A small-pore filter membrane that is not acid-resistant such as a nylon membrane is used to enrich the bacteria on the front side of the membrane, and a specific acidic eluent such as an HCL solution is used for backwashing. The acidic eluent digests the inner surface of each micropore to a certain extent, increases the pore size of the micropores, and allows the enriched bacteria to be efficiently eluted. Finally, the eluted bacterial solution is promptly subjected to acid-base neutralization to maintain the long-term activity of the bacteria.

[0076] In another embodiment of the present invention, acid-resistant bacteria such as Mycobacterium tuberculosis are treated with an acidic solution such as HCL solution to digest the specimen, so that the specimen is homogenized, an acid-resistant large-pore filter membrane such as a PTFE membrane is used to filter impurities, an alkali-intolerant small-pore filter membrane such as a PVDF membrane is used to enrich the bacteria on the front side of the membrane, and a specific alkaline eluent such as a NaOH solution is used for backwashing. The alkaline eluent digests the inner surface of each micropore to a certain extent, increases the pore size of the micropores, and allows the enriched bacteria to be efficiently eluted. Finally, the eluted bacterial solution is promptly subjected to acid-base neutralization to maintain the long-term activity of the bacteria.

[0077] In the present invention, the alkaline eluent is used for acid-resistant bacteria, and the acidic eluent is used for acid-resistant bacteria. Since the eluent is neutralized soon after flushing the filter membrane, it has no effect on the biological activity of the bacteria. This study uses Mycobacterium tuberculosis, which is both acid-resistant and alkali-resistant, so both acid-base eluents are applicable. Therefore, in the present invention, a nylon membrane with a pore size of 0.45 μm and 2N HCL are used as the eluent, and a PVDF membrane with a pore size of 0.45 μm and 2N NaOH are used as the eluent to concentrate and enrich the bacteria, and the maximum amount of bacteria is obtained.

[0078] The eluent involved in the present invention, itself and the tiny particles generated by part of the eluent digesting the inner surface of the micropores, have no effect on the growth and survival of bacteria during the detection period, and can still ensure that the bacteria maintain their original biological activity.

[0079] In the present invention, the selection of large-pore filter membrane needs to be based on the acidity and alkalinity of the specimen digestion fluid. Acidic treatment fluid requires a large-pore acid-resistant filter membrane, and alkaline digestion fluid requires a large-pore alkaline filter membrane. You can also directly choose a large-pore PTFE filter membrane that is both acid-resistant and alkali-resistant (this experiment uses a large-pore PTFE filter membrane). The specific selection also needs to be considered in combination with factors such as material cost and experimental effect.

[0080] The acid-resistant nylon membrane used in the present invention has a certain tolerance to alkali, but a low tolerance to digestive fluid with too high pH value. Therefore, it needs to be adjusted according to specific circumstances during use. After the specimen is treated with alkaline digestive fluid, an appropriate amount of acidic solution can be added to adjust it to weak alkalinity or neutrality.

[0081] The alkali-inresistant PVDF membrane used in the present invention has a certain tolerance to acid, but a low tolerance to digestive fluid with too low pH value. Therefore, it needs to be adjusted according to specific circumstances during use. After the specimen is treated with acidic digestive fluid, an appropriate amount of alkaline solution can be added to adjust it to weak acid or neutral.

[0082] 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.

[0083] Example 1 Filtering impurities in samples using filter membranes with different pore sizes

[0084] 1. Take a batch of 1.5 ml centrifuge tubes, weigh them one by one, and mark them on the lids for later use;

[0085] 2. Take 2 ml of each patient's sputum specimen in a 5 ml centrifuge tube, 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 15 minutes to digest. After completion, take two 1.5 ml portions and divide them into two weighed 1.5 ml centrifuge tubes, marked as sample 1 and sample 2;

[0086] 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 acid- and alkali-resistant PTFE membranes with different pore sizes, with pore sizes of 5μm, 2μm, 1.2μm, and 0.8μm), and then filter the sample back into the original tube.

[0087] 4. Centrifuge sample 1 and sample 2 at 12000r for 5 min, discard the supernatant and retain the bottom precipitate.

[0088] 5. Weigh sample 1 and sample 2 using an electronic balance.

[0089] Experimental results: The percentage of impurities in the sample filtered through the filter membranes with different pore sizes to the original sample mass is as follows: Figure 1 As shown, the percentage of weight loss of the sample after filtering through filter membranes of different pore sizes accounts for different percentages of the weight of the sample before filtering treatment, namely, the weight of impurities filtered out by 5μm pore size filter membrane accounts for 11.35% of the total weight of the sample, the weight of impurities filtered out by 2μm pore size filter membrane accounts for 26.91% of the total weight of the sample, the weight of impurities filtered out by 1.2μm pore size filter membrane accounts for 59.85% of the total weight of the sample, and the weight of impurities filtered out by 0.8μm pore size filter membrane accounts for 65.54% of the total weight of the sample.

[0090] Therefore, the 0.8μm pore size filter membrane has the best effect in filtering impurities, and the 1.2μm pore size filter membrane has the second best effect in filtering impurities.

[0091] Example 2 Bacterial loss when filtering samples with different pore size membranes

[0092] 1. Filter group (experimental group)

[0093] (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.

[0094] (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, acid- and alkali-resistant PTFE membranes with different pore sizes are used), and then filter the sample back into the original tube.

[0095] (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.

[0096] (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.

[0097] (5) Place the stained slides on a slide dryer to dry, and then place them under a fluorescence microscope for observation and counting.

[0098] 2. Centrifugal group (control group)

[0099] (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.

[0100] (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.

[0101] (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.

[0102] (4) Place the stained slides on a slide dryer to dry, and then place them under a fluorescence microscope for observation and counting.

[0103] (5) Calculation of bacterial loss rate:

[0104]

[0105] Experimental results:

[0106] like Figure 2 As shown, the samples were counted under a centrifugal microscope after passing through filter membranes of different pore sizes and directly after centrifugation. The results were that the average number of bacteria counted under a centrifugal microscope after filtration through a 5μm pore size filter membrane was 64.6, the average number of bacteria counted under a centrifugal microscope after filtration through a 2μm pore size filter membrane was 63.6, the average number of bacteria counted under a centrifugal microscope after filtration through a 1.2μm pore size filter membrane was 62.4, the average number of bacteria counted under a centrifugal microscope after filtration through a 0.8μm pore size filter membrane was 52, and the average number of bacteria counted directly under a centrifugal microscope was 65.2. This shows that filter membranes with pore sizes of 5μm, 2μm, and 1.2μm will hardly produce bacterial losses when filtering impurities, while a filter membrane with a pore size of 0.8μm will produce about 20.25% bacterial loss during filtration.

[0107] Based on the above results, and taking into account the degree of impurity filtration and bacterial loss, the 1.2 μm filter membrane was selected as the upper large-pore filter membrane, which is most suitable for filtering impurities in clinical samples.

[0108] Example 3 Effect of small pore size filtration membranes of different materials on enrichment of bacteria in samples

[0109] 1. Filtration method (experimental group):

[0110] (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.

[0111] (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 acid- and alkali-resistant PTFE membrane is used), and then filter the sample back into the original tube.

[0112] (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, PVDF, and nylon, 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.

[0113] (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.

[0114] (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.

[0115] (6) Place the stained slides on a slide dryer to dry, and then place them under a fluorescence microscope for observation and counting.

[0116] 2. Centrifugal group (control group):

[0117] (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.

[0118] (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.

[0119] (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.

[0120] (4) Place the stained slides on a slide dryer to dry, and then place them under a fluorescence microscope for observation and counting.

[0121] (5) Calculation of enrichment rate:

[0122]

[0123] Experimental results:

[0124] The enrichment effect of bacteria in the sample after filtration by filter membranes of different materials and different pore sizes is shown in Table 1abc. Filter membranes of different materials and different pore sizes will produce different degrees of bacterial loss when filtering and collecting bacteria from the sample. Therefore, the amount of bacteria that can be enriched by the same sample through filter membranes of different materials and different pore sizes is different. Among them, the PVDF membrane with a pore size of 0.45μm and the nylon membrane with a pore size of 0.45μm have the best enrichment effect, and the bacterial enrichment rates are 89.77% and 90.36%, respectively.

[0125] Table 1a Bacteria count in a single field of view under a 40x objective lens in the experimental group (individuals)

[0126]

[0127] Table 1b Bacteria count in a single field of view under a 40x objective lens in the centrifugation group (units)

[0128]

[0129] Table 1c Enrichment effect of bacteria in samples after filtration with membranes of different materials and pore sizes

[0130]

[0131] Example 4: Different eluents are used to elute the enriched cells using small-pore filtration membranes made of different materials

[0132] 1. Filtration method (elution group and waste liquid group):

[0133] (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.

[0134] (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 acid- and alkali-resistant PTFE membrane is used), and then filter the sample back into the original tube.

[0135] (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, namely PTFE, PVDF, and nylon), filter the samples back into the original tube, and mark it as a waste liquid tube.

[0136] (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.

[0137] (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)

[0138] (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)

[0139] (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.

[0140] (8) Place the stained slides on a slide dryer to dry, and then place them under a fluorescence microscope for observation and counting.

[0141] 2. Centrifugation method (centrifugation group):

[0142] (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.

[0143] (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.

[0144] (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.

[0145] (4) Place the stained slides on a slide dryer to dry, and then place them under a fluorescence microscope for observation and counting.

[0146] (5) Elution rate:

[0147]

[0148] Comparison of results:

[0149] The effect of using different eluents to elute the bacteria enriched on the filter membranes of different materials is shown in Table 2abc. 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 eluent 1 (composition 0.05M KH2PO4, 1M NaCl, 0.1% Tween 80, 0.1% sodium hexametaphosphate), eluent 2 (composed of 0.02% Tween80, 0.02% sodium hexametaphosphate), 7H9 culture medium, HCL solution (the concentrations were 2N, 1N, 0.5N, and 0.25N, respectively, diluted with pure water), NaOH solution (the concentrations were 2N, 1N, 0.5N, and 0.25N, respectively, diluted with pure water), Tween80 solution (the concentrations were 20%, 10%, 5%, and 1%, respectively, diluted with 7H9 culture medium); at the same time, filter membranes of different materials were used for comparison, including PTFE membrane, PVDF membrane, and nylon membrane, with a pore size of 0.45μm.

[0150] The experimental results show that 2N HCL combined with nylon membrane has the best bacteria elution effect, while 2N NaOH combined with PVDF membrane has the second best bacteria elution effect.

[0151] Table 2a The number of bacteria in a single field of view under a 40x objective lens in the elution group (individuals)

[0152]

[0153]

[0154] Table 2b Bacteria count in a single field of view under a 40x objective lens in the waste liquid group (individuals)

[0155]

[0156] Table 2c Bacteria count in a single field of view under a 40x objective lens in the centrifugation group (units)

[0157]

[0158] Table 2d The elution rate of bacteria enriched in filter membranes of different materials by different eluents (%)

[0159]

[0160] In combination with Example 3 and Example 4, the bacterial enrichment rate and the bacterial elution rate were considered comprehensively, and the bacterial yield (bacterial enrichment rate x bacterial elution rate = bacterial yield) was calculated. A nylon membrane with a pore size of 0.45 μm and 2N HCL was used as an eluent, and a PVDF membrane with a pore size of 0.45 μm and 2N NaOH was used as an eluent to concentrate and enrich the bacteria, and the maximum amount of bacteria was obtained.

[0161] Example 5: Staining the enriched bacteria by staining on the membrane and staining after elution

[0162] 1. Membrane staining

[0163] (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.

[0164] (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 acid- and alkali-resistant PTFE membrane is used), and then filter the sample back into the original tube.

[0165] (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, namely PTFE, PVDF, and nylon), filter the sample back into the original tube, and mark it as a waste liquid tube.

[0166] (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.

[0167] (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, PVDF membrane is eluted with 2NNaOH solution, and nylon membrane is eluted with 2N HCL solution). Slowly push two drops into a 1.5 ml centrifuge tube, add 0.1-0.2 g carboxymethyl cellulose, mix well, and mark for later use.

[0168] (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.

[0169] (7) Place the stained slides on a slide dryer to dry, then place them under a fluorescence microscope for observation, counting, and photographing.

[0170] 2. Staining after elution

[0171] (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.

[0172] (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 acid- and alkali-resistant PTFE membrane is used), and then filter the sample back into the original tube.

[0173] (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, namely PTFE, PVDF, and nylon), filter the sample back into the original tube, and mark it as a waste liquid tube.

[0174] (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, PVDF membrane is eluted with 2NNaOH solution, and nylon membrane is eluted with 2N HCL solution). Slowly push two drops into a 1.5 ml centrifuge tube, add 0.1-0.2 g carboxymethyl cellulose, mix well, and mark for later use.

[0175] (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.

[0176] (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.

[0177] (7) Place the stained slides on a slide dryer to dry, then place them under a fluorescence microscope for observation, counting, and photographing.

[0178] Experimental results:

[0179] The effects of using different staining methods to stain the bacteria enriched by filter membranes of different materials are shown in Table 3. The experiment used two methods, staining on the membrane and staining after elution, to perform fluorescent staining on the bacteria enriched by PTFE membrane, PVDF membrane, and nylon membrane with a pore size of 0.45μm, and count the bacteria in a single field of view and measure the fluorescence intensity. Among them, whether it is fluorescence intensity or bacterial count, the best effect is to use staining after elution.

[0180] Table 3 Effects of different staining methods on the enriched bacteria on filter membranes of different materials

[0181]

Claims

1. A method for enriching and eluting 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 enriched by a filter membrane; S4. Use the elution solution to reversely elute the bacteria enriched on the front side of the membrane.

2. The enrichment and elution method according to claim 1, characterized in that: The specimens described in step S1 include but are not limited to original clinical specimens, culture specimens, and specimens processed by the processing method.

3. The enrichment and elution method according to claim 1, characterized in that: The filtration described in S2 is to filter the bacteria-containing test sample using a large-pore acid-resistant or alkali-resistant filter membrane to remove impurities in the test sample, and the large-pore diameter is selected from 0.8-5 μm.

4. The enrichment and elution method according to claim 1, characterized in that: The 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 small-pore alkali-resistant or acid-resistant filtration membrane, wherein the small pore size is selected from 0.22-0.8 μm.

5. The enrichment and elution method according to claim 1, characterized in that: The acid-resistant membrane includes but is not limited to polytetrafluoroethylene (PTFE) membrane and polypropylene (PP) membrane; the alkali-resistant membrane includes but is not limited to polytetrafluoroethylene (PTFE) membrane, polypropylene (PP) membrane and polyethersulfone (PES) membrane, the acid-insensitive filter membrane includes but is not limited to nylon (NY or PA) membrane and mixed cellulose (MCE) membrane; the alkali-insensitive filter membrane includes but is not limited to polyvinylidene fluoride (PVDF) membrane and mixed cellulose (MCE) membrane.

6. The enrichment and elution method according to claim 1, characterized in that: The eluent as described in S4 includes an acidic or alkaline eluent, wherein the acidity of the acidic eluent is expressed in terms of H + The concentration is 0.01-2 mol / L; the alkalinity of the alkaline eluent is OH - The concentration is measured at 0.01-2 mol / L.

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 is used to pre-treat the collected specimens to obtain acidic or alkaline specimens that can be further tested; the filtration module is used to filter impurities in the solution specimens through acid-resistant or alkaline filter membranes and / or filters; the enrichment module is used to enrich the bacteria in the filtered specimens using an alkali-resistant or acid-resistant small-pore filter membrane, and the bacteria are enriched on the front side of the filter membrane; the elution module is used to perform reverse elution using an eluent from the enrichment module to elute the bacteria from the filter membrane; the eluent includes an acidic or alkaline eluent.

8. The filtration and / or enrichment system according to claim 7, characterized in that The acidity of the acidic eluent is expressed in terms of H + The concentration is 0.01-2 mol / L; the alkalinity of the alkaline eluent is OH - The concentration is measured at 0.01-2 mol / L.

9. The filtration and / or enrichment system according to claim 7, characterized in that: The large pore size is selected from 0.8-5 μm; the small pore size is selected from 0.22-0.8 μm.

10. Application of a bacterial specimen filtration and / or enrichment system in bacterial specimen filtration or enrichment, the system as claimed in claim 7 comprising a pre-treatment module, a filtration module, a bacterial enrichment module and an elution module; wherein: The sample pre-treatment module is used to pre-treat the collected specimens to obtain acidic or alkaline specimens that can be further tested; the filtration module is used to filter impurities in the solution specimens through acid-resistant or alkali-resistant filter membranes and / or filters; the enrichment module is used to enrich the bacteria in the filtered specimens using an alkali-resistant or acid-resistant small-pore filter membrane, and the bacteria are enriched on the front side of the filter membrane; the elution module is used to perform reverse elution using an elution solution from the enrichment module to elute the bacteria from the filter membrane.

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