Kit for dissociating microorganisms in microbial aggregates, dissociated microbial suspension and application

Abstract

The present invention proposes a kit for dissociating microorganisms in microbial aggregates, a dissociated microbial suspension and an application thereof. The microbial aggregates mainly include biofilms generated in environments such as sewage, pipes, oral cavity, surface of human implants, rock surface in rivers, hull surface, hot spring and deep-sea hot water outlet, rumen of ruminants, and bacterial floccules generated in activated sludge, etc. The kit belongs to the field of biotechnology and can solve the technical problems of the existing dilution coating plate method, such as long experimental cycle, difficulty in obtaining non-free strains, and damage to microorganisms by physical methods and chemical dispersants. The kit for dissociating microorganisms in microbial aggregates includes a filter and a reagent composed of glycoside hydrolase, protease and abrasive. After the microbial aggregate sample is dissociated by the kit, a dissociated microbial suspension is obtained, and the microbial dissociation efficiency is 70% to 99%, wherein the viable cell rate is above 90%. The present invention has the advantages of high microbial extraction efficiency, little damage to microorganisms, and short dissociation cycle. The dissociated microbial suspension can be directly used for subsequent cell detection, sorting, culture, and sequencing, which greatly improves the probability of obtaining live strains and low-abundance strains, and enriches the flora information obtained from samples.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a kit for dissociating microorganisms in microbial aggregates, a dissociated microbial suspension and applications. The microbial aggregates mainly include biofilms generated in environments such as sewage, pipes, oral cavity, surfaces of human implants, rock surfaces in rivers, hull surfaces, hot springs and deep-sea hot water outlets, and the rumen of ruminants, as well as bacterial flocs generated in activated sludge. Background Art

[0002] Microbial aggregates mainly include biofilms generated in environments such as sewage, pipes, oral cavity, human implant surfaces, rock surfaces in rivers, hull surfaces, hot springs and deep-sea hot water outlets, rumen of ruminants, and bacterial flocs generated in activated sludge. In the biofilm method, microorganisms attach to the surface of the carrier to form biofilms, which can adsorb organic matter in sewage and decompose it; during the treatment of activated sludge, microorganisms will form bacterial flocs, which help improve the sedimentation performance of sludge. In addition, biofilms and their derivative materials are also widely used in the medical field, not only playing a role in the prevention and treatment of biofilm-related infections, but also showing great potential in drug delivery and tissue engineering. Research on microorganisms that constitute microbial aggregates such as biofilms and bacterial flocs will help develop new environmental governance technologies, prevent and treat biofilm infections, and promote environmental protection and human health. Since the microbial single cells in microbial aggregates play their respective functions at different levels of the biofilm, in the process of studying microbial aggregates, it is necessary to focus on the microbial single cells that form microbial aggregates.

[0003] At present, the commonly used method for isolating single microbial cells is the traditional dilution plate method, which usually takes 24-36 hours, and most of the microorganisms are still wrapped in microbial aggregates. It has the disadvantages of long experimental cycle, low number of microbial species obtained on the plate, and inaccurate abundance. Other dissociation methods, including physical methods such as ultrasound and heat treatment, and chemical methods such as chemical dispersants, often cause damage to the bacteria when the efficiency of microbial dissociation is improved, resulting in a decrease in the vitality of the entire bacterial community. The microbial samples obtained by the above dissociation methods have the disadvantages of long experimental cycle, low dissociation efficiency, and damage to the bacteria. It is difficult to meet the research on bacterial activity, colony composition, and acquisition of low-abundance bacteria in the samples through subsequent cell detection, sorting, culture, and sequencing.

[0004] Therefore, it is necessary to provide a kit for dissociating microorganisms in microbial aggregates. Summary of the invention

[0005] The present invention aims at the technical problems that the dilution coating plate method used in the above-mentioned existing methods for dissociating microbial single cells in microbial aggregates has a long experimental cycle, non-free strains are difficult to obtain, and physical methods and chemical dispersants are harmful to microorganisms. The present invention provides a kit for dissociating microorganisms in microbial aggregates, a dissociated microbial suspension and an application. The kit is used for dissociation treatment, and the microbial dissociation efficiency is 70% to 99%, wherein the viable cell rate is greater than 90%, the extraction efficiency is high, and the damage to the microorganisms is small. The proportion of culturable viable bacteria in the obtained dissociated microbial suspension is increased by at least 5 times, which can meet the requirements of subsequent direct use for cell detection, sorting, cultivation and sequencing. It has the advantages of short microbial dissociation cycle, high viable cell ratio of dissociated microorganisms, and the extraction of non-free microorganisms. It can be applied to the dissociation of microorganisms in microbial aggregates such as biofilms generated in sewage, pipelines, oral cavity, surfaces of human implants, rock surfaces in rivers, hull surfaces, hot springs and deep-sea hot water outlets, and the rumen of ruminants, as well as bacterial flocs generated in activated sludge.

[0006] In order to achieve the above-mentioned purpose, the technical solution adopted by the present invention is: a kit for dissociating microorganisms in microbial aggregates, comprising a filter and a reagent composed of glycoside hydrolase, protease and abrasive. After the microbial aggregate sample is treated by the kit, a microbial suspension containing single cells is obtained, and the microbial dissociation efficiency is 70% to 99%, among which the single cell release rate reaches 80% to 99%, the viable cell rate is greater than 90%, and the proportion of culturable viable bacteria in the obtained dissociated microbial suspension is increased by at least 5 times.

[0007] In some embodiments, the pore size of the filter is 2 to 100 μm; the glycoside hydrolases include but are not limited to amylase, cellulase, hemicellulase, sucrase, lactase, maltase; the proteases include but are not limited to trypsin, plasmin, proteinase K, papain, cathepsin; the abrasives include but are not limited to garnet, corundum, and quartz sand.

[0008] In some of the embodiments, the action temperature of glycoside hydrolase is 25-65°C, the action pH is 4.5-9.0, and the action concentration is 0.5-500 mg / mL; the action temperature of protease is 25-65°C, the action pH is 4-12.5, and the action concentration is 5-2000 μg / mL; the particle size range of abrasive is 5-100 mesh, and the action concentration is 0.1-1 g / mL. This technical solution uses glycoside hydrolase and protease of the above concentrations to maximize the enzymatic effect, promote the lysis of microbial aggregates and release microorganisms, and improve the extraction efficiency; by limiting the particle size of the abrasive, the particles in the sample can be fully ground to expose the microorganisms.

[0009] In some embodiments, the dissociation treatment method comprises the following steps:

[0010] Take a sample of microbial aggregates and make it into a suspension. After the first centrifugation, discard the supernatant, add glycoside hydrolase, protease, and abrasive, and incubate after high-speed vortex shaking;

[0011] After the incubation, the supernatant was discarded after the second centrifugation to terminate the enzymatic reaction;

[0012] Sterile water was added to resuspend and the dissociated microbial suspension was obtained after filtration.

[0013] Various bacteria in activated sludge form mesh extracellular polymers by secreting extracellular polysaccharides (EPS) and specific proteins, which wrap a large number of cells to form bacterial micelles; the main components of microbial biofilms include polysaccharides, proteins, nucleic acids and lipid substances; the enzyme reagent of the present invention uses glycoside hydrolases in combination with proteases, which can decompose bacterial micelles and biofilms, expose microorganisms, improve the extraction efficiency of microbial single cells, and cause little damage to microorganisms.

[0014] In some embodiments, the first centrifugal treatment condition is 8000-14000 rpm, and the time is 1-120 minutes; the second centrifugal treatment condition is 8000-14000 rpm, and the time is 1-120 minutes.

[0015] In some embodiments, the high-speed vortex oscillation condition is set to a rotation speed of 1000-3000 rpm and a time of 10-60 minutes. The technical solution uses high-speed vortex oscillation to allow the abrasive to fully grind the microbial aggregates in the sample, expose the microorganisms, and improve the extraction efficiency.

[0016] In some embodiments, the incubation conditions are 25-65°C, pH 4.5-9.0, rotation speed 20-220 rpm, and incubation time 0.5-6 hours. Incubation under the above conditions can fully exert the enzymatic effects of glycoside hydrolases and proteases.

[0017] In some embodiments, in the filtering step, the suspension is filtered through a 40-50 μm filter, a 15-20 μm filter, and a 5-10 μm filter in sequence. In this technical solution of the present invention, the filter is a multi-stage filter, and the pore size and filtering level of the filter can be selected according to the specific requirements of the filtration. Through multi-stage filtration, the extraction and purification efficiency can be improved.

[0018] The present invention also provides a dissociated microbial suspension, which is obtained by processing the kit for dissociating microorganisms in microbial aggregates. The dissociated microbial suspension is obtained by dissociating a microbial aggregate sample through the kit. The microorganisms in the dissociated microbial suspension exist in the form of single cells, and the microbial dissociation efficiency is 70% to 99%, wherein the viable cell rate is greater than 90%, and the proportion of culturable viable bacteria in the dissociated microbial suspension is increased by at least 5 times.

[0019] The present invention also provides the use of dissociated microbial suspensions in direct cell detection, sorting, culturing and sequencing. Through the dissociation treatment of the kit for dissociating microorganisms in microbial aggregates of the present invention, the microbial dissociation efficiency is 70% to 99%, and most microorganisms can be separated, including species with low abundance, avoiding insufficient acquisition and detection of microorganisms in samples; and the microbial suspension obtained after treatment has a low impurity content, a high bacterial concentration, and most of them are in a single cell state, and can be sorted by microfluidic sorting equipment, and more abundant bacterial flora information can be obtained directly through cell detection, sorting, culturing and sequencing.

[0020] Compared with the prior art, the advantages and positive effects of the present invention are: the kit for dissociating microorganisms in microbial aggregates of the present invention has the advantages of short single cell acquisition cycle, high extraction efficiency, little damage to microorganisms, and can also extract non-free microorganisms. After pretreatment by the kit for dissociating microorganisms in microbial aggregates of the present invention, the microbial dissociation efficiency is 70% to 99%, of which the viable cell rate is greater than 90%, and the proportion of culturable viable bacteria in the obtained dissociated microbial suspension is increased by at least 5 times, which meets the subsequent direct use in cell detection, sorting, culture and sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a bar graph showing the ratio of the number of colonies in the sample suspension before and after filtration of Example 6 of the present invention and Comparative Example 7. DETAILED DESCRIPTION

[0022] In order to be able to understand the characteristics and technical contents of the embodiments of the present invention in more detail, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the embodiments described below are only part of the embodiments of the present invention, rather than all the embodiments of the present invention. In the following technical description, for the convenience of explanation, a full understanding of the disclosed embodiments is provided through multiple details. However, one or more embodiments can still be implemented without these details. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection requested by the present invention.

[0023] The embodiment of the present invention provides a kit for dissociating microorganisms in microbial aggregates, a dissociated microbial suspension and an application. The kit for dissociating microorganisms in microbial aggregates of the present invention first promotes the lysis of microbial aggregates through the hydrolysis of glycoside hydrolases and proteases and the grinding of abrasives to release microorganisms, and then separates the microorganisms through gradient filtration. It can be used for the separation of single microbial cells in microbial aggregates in sewage, pipelines, and oral samples, improves the dispersion of biofilms, promotes the full release of bacterial cells and fungal spores, and maintains the vitality of bacterial cells. It has the advantages of short acquisition cycle, high extraction efficiency, little damage to microorganisms, and can also extract non-free microorganisms, and the obtained microorganisms exist in the form of single cells.

[0024] The embodiment of the present invention provides a kit for dissociating microorganisms in microbial aggregates, including a filter and a reagent composed of a glycoside hydrolase, a protease and an abrasive. After the microbial aggregate sample is dissociated by the kit for dissociating microorganisms in microbial aggregates, the proportion of culturable live bacteria in the obtained dissociated microbial suspension is increased by at least 5 times, and the microbial dissociation efficiency is 70% to 99%, which meets the direct application requirements of subsequent cell detection, sorting, culture and sequencing. In a preferred embodiment, the action concentration of the glycoside hydrolase is 0.5 to 500 mg / mL, the pH is 4.5 to 9.0, and the action concentration of the protease is 5 to 2000 μg / mL, and the pH is 4 to 12.5. Within the above concentration and pH range, the glycoside hydrolase and the protease can maximize the enzymatic effect, promote the lysis of the microbial aggregates and release the microorganisms, and improve the extraction efficiency.

[0025] The dissociation treatment method of the present invention is to obtain a microorganism-enriched precipitate by centrifuging a microorganism aggregate sample, add active enzymes and abrasives, shake, and perform static enzymolysis, obtain a suspension after centrifugation, and obtain a dissociated microorganism suspension by centrifugation after multi-stage filtration. The dissociation treatment method specifically includes the following steps:

[0026] 1-5 mL of the microbial aggregate sample to be treated is drawn into a centrifuge tube, and the sample is centrifuged for the first time at 8000-14000 rpm for 1-120 minutes, and the supernatant is discarded to obtain a sediment enriched with bacteria, and 0.5-50 mL of glycoside hydrolase with a volume of 0.5-500 mg / mL and a protease with a volume of 0.01-1 mL and a concentration of 1-200 mg / mL, as well as 0-50 mL of sterile water are added to the centrifuge tube, and the pH is adjusted to 4.5-9.0 to decompose polysaccharides and proteins, and 0.1-5 g of abrasive with a particle size of 5-100 mesh is added to the centrifuge tube, and high-speed vortex oscillation is performed, and the speed is set to 1000-3000 rpm. The treatment time is 10-60 minutes, so that the abrasive grinds the particles in the sample during the high-speed oscillation process to expose the microorganisms;

[0027] After high-speed vortex shaking, the centrifuge tube is placed at 25-65° C. and 20-220 rpm for incubation for 0.5-6 hours to allow the glycoside hydrolase and protease to fully function, and then centrifuged for a second time at 8000-14000 rpm for 1-120 minutes, and the supernatant is discarded to terminate the enzymatic reaction;

[0028] The precipitate was resuspended in 5-20 mL of sterile water to obtain suspension A, which was diluted and filtered through a 40-50 μm filter, a 15-20 μm filter, and a 5-10 μm filter to obtain suspension B;

[0029] After the suspension B is recovered, it is centrifuged for a third time at 8000-14000 rpm for 1-120 minutes, the supernatant is discarded, and the obtained precipitate is washed twice with 1-15 mL of sterile water, and then the bacteria at the bottom of the centrifuge tube are resuspended with an appropriate amount of sterile water to obtain a purified dissociated microbial suspension.

[0030] Another aspect of the present invention provides a dissociated microbial suspension obtained by dissociation treatment of the above-mentioned kit for dissociating microorganisms in microbial aggregates, wherein the microorganisms in the dissociated microbial suspension exist in the form of single cells, and the microbial dissociation efficiency is 70% to 99%.

[0031] The present invention also provides the use of the dissociated microbial suspension in direct cell detection, sorting, cultivation and sequencing.

[0032] In order to more clearly and in detail introduce the microbial single cell dissociation pretreatment method and pretreatment kit provided in the embodiments of the present invention, they will be described below in conjunction with specific embodiments.

[0033] Example 1

[0034] Prepare normal saline: weigh 0.43 g NaCl and dissolve it in 500 mL sterile water. After fully dissolved, sterilize it in a 121°C autoclave for 20 minutes to obtain normal saline;

[0035] Preparation of 100 mg / mL α-amylase: weigh 1000 mg of α-amylase powder and dissolve it in 10 mL of sterile water to obtain 100 mg / mL α-amylase;

[0036] Prepare 10 mg / mL papain: weigh 100 mg of papain powder and dissolve it in 10 mL of sterile water to obtain 10 mg / mL papain;

[0037] Take 1 mL of sewage sample in a centrifuge tube, centrifuge at 14000 rpm for 10 minutes, remove the supernatant, and resuspend the precipitate with saline to obtain a suspension;

[0038] Add 2 mL of 100 mg / mL α-amylase, 0.1 mL of 10 mg / mL papain, and 1 g of quartz sand to the suspension, adjust the pH to 7.0, vortex at high speed for 30 minutes, and then incubate at 37°C, 180 rpm for 3 hours;

[0039] After the incubation, the mixture was centrifuged at 14000 rpm for 10 minutes, the supernatant was removed, and the precipitate was resuspended with sterile water to obtain a suspension;

[0040] After 40 μm, 20 μm, and 10 μm filters are sequentially connected in series, the suspension is filtered through the above filters in sequence to obtain a filtrate.

[0041] Example 2

[0042] Prepare normal saline: weigh 0.43 g NaCl and dissolve it in 500 mL sterile water. After fully dissolved, sterilize it in a 121°C autoclave for 20 minutes to obtain normal saline;

[0043] Prepare 0.5 mg / mL α-amylase: weigh 5 mg of α-amylase powder and dissolve it in 10 mL of sterile water to obtain 0.5 mg / mL α-amylase;

[0044] Prepare 10 mg / mL papain: weigh 100 mg of papain powder and dissolve it in 10 mL of sterile water to obtain 10 mg / mL papain;

[0045] Take 1 mL of sewage sample in a centrifuge tube, centrifuge at 14000 rpm for 10 minutes, remove the supernatant, and resuspend the precipitate with saline to obtain a suspension;

[0046] 2 mL of 0.5 mg / mL α-amylase, 0.05 mL of 10 mg / mL papain, and 0.02 g of quartz sand were added to the suspension, the pH was adjusted to 7.0, high-speed vortexing was performed for 30 minutes, and then incubated at 37°C, 180 rpm for 6 hours;

[0047] After the incubation, the mixture was centrifuged at 14000 rpm for 10 minutes, the supernatant was removed, and the precipitate was resuspended with sterile water to obtain a suspension;

[0048] After 40 μm, 20 μm, and 10 μm filters are sequentially connected in series, the suspension is filtered through the above filters in sequence to obtain a filtrate.

[0049] Example 3

[0050] Prepare normal saline: weigh 0.43 g NaCl and dissolve it in 500 mL sterile water. After fully dissolved, sterilize it in a 121°C autoclave for 20 minutes to obtain normal saline;

[0051] Preparation of 500 mg / mL α-amylase: weigh 5000 mg of α-amylase powder and dissolve it in 10 mL of sterile water to obtain 500 mg / mL α-amylase;

[0052] Prepare 10 mg / mL papain: weigh 100 mg of papain powder and dissolve it in 10 mL of sterile water to obtain 10 mg / mL papain;

[0053] Take 1 mL of sewage sample in a centrifuge tube, centrifuge at 14000 rpm for 10 minutes, remove the supernatant, and resuspend the precipitate with saline to obtain a suspension;

[0054] Add 2 mL of 500 mg / mL α-amylase, 0.5 mL of 10 mg / mL papain, and 2 g of quartz sand to the suspension, adjust the pH to 7.0, vortex at high speed for 30 minutes, and then incubate at 37°C, 180 rpm for 0.5 hour;

[0055] After the incubation, the mixture was centrifuged at 14000 rpm for 10 minutes, the supernatant was removed, and the precipitate was resuspended with sterile water to obtain a suspension;

[0056] After 40 μm, 20 μm, and 10 μm filters are sequentially connected in series, the suspension is filtered through the above filters in sequence to obtain a filtrate.

[0057] Example 4

[0058] Prepare normal saline: weigh 0.43 g NaCl and dissolve it in 500 mL sterile water. After fully dissolved, sterilize it in a 121°C autoclave for 20 minutes to obtain normal saline.

[0059] Preparation of 100 mg / mL α-amylase: weigh 1000 mg of α-amylase powder and dissolve it in 10 mL of sterile water to obtain 100 mg / mL α-amylase;

[0060] Prepare 10 mg / mL papain: weigh 100 mg of papain powder and dissolve it in 10 mL of sterile water to obtain 10 mg / mL papain;

[0061] Take 1 mL of sewage sample in a centrifuge tube, centrifuge at 14000 rpm for 10 minutes, remove the supernatant, and resuspend the precipitate with saline to obtain a suspension;

[0062] Add 2 mL of 100 mg / mL α-amylase, 0.1 mL of 10 mg / mL papain, and 1 g of quartz sand to the suspension, adjust the pH to 4.5, vortex at high speed for 30 minutes, and then incubate at 37°C, 180 rpm for 3 hours;

[0063] After the incubation, the mixture was centrifuged at 14000 rpm for 10 minutes, the supernatant was removed, and the precipitate was resuspended with sterile water to obtain a suspension;

[0064] After 40 μm, 20 μm, and 10 μm filters are sequentially connected in series, the suspension is filtered through the above filters in sequence to obtain a filtrate.

[0065] Example 5

[0066] Prepare normal saline: weigh 0.43 g NaCl and dissolve it in 500 mL sterile water. After fully dissolved, sterilize it in a 121°C autoclave for 20 minutes to obtain normal saline;

[0067] Preparation of 100 mg / mL α-amylase: weigh 1000 mg of α-amylase powder and dissolve it in 10 mL of sterile water to obtain 100 mg / mL α-amylase;

[0068] Prepare 10 mg / mL papain: weigh 100 mg of papain powder and dissolve it in 10 mL of sterile water to obtain 10 mg / mL papain;

[0069] Take 1 mL of sewage sample in a centrifuge tube, centrifuge at 14000 rpm for 10 minutes, remove the supernatant, and resuspend the precipitate with saline to obtain a suspension;

[0070] 2 mL of 100 mg / mL α-amylase, 0.1 mL of 10 mg / mL papain, and 1 g of quartz sand were added to the suspension, the pH was adjusted to 9.0, high-speed vortexing was performed for 30 minutes, and then incubated at 37°C, 180 rpm for 3 hours;

[0071] After the incubation, the mixture was centrifuged at 14000 rpm for 10 minutes, the supernatant was removed, and the precipitate was resuspended with sterile water to obtain a suspension;

[0072] After 40 μm, 20 μm, and 10 μm filters are sequentially connected in series, the suspension is filtered through the above filters in sequence to obtain a filtrate.

[0073] Example 6

[0074] Preparation of 10 mg / mL α-amylase: weigh 100 mg of α-amylase powder and dissolve it in 10 mL of sterile water to obtain 10 mg / mL α-amylase;

[0075] Prepare 10 mg / mL papain: weigh 100 mg of papain powder and dissolve it in 10 mL of sterile water to obtain 10 mg / mL papain;

[0076] Take a biofilm sample and make it into a suspension;

[0077] Take 1 mL of the sample suspension in a 15 mL centrifuge tube, centrifuge at 14000 rpm for 10 minutes, discard the supernatant and obtain the precipitate;

[0078] Add 2 mL of 10 mg / mL α-amylase solution, 0.5 mL of 10 mg / mL papain, and 0.1 g of quartz sand to the precipitate, adjust the pH to 6.5, vortex at high speed for 30 minutes, and then incubate at 37°C, 180 rpm for 3 hours;

[0079] After the incubation, centrifuge at 14000 rpm for 10 minutes, discard the supernatant, resuspend the precipitate with sterile water, connect 40 μm, 20 μm, and 10 μm filters in series, and filter the suspension through the above filters in sequence to obtain the filtrate.

[0080] Comparative Example 1

[0081] Take 1 mL of sewage sample in a centrifuge tube, centrifuge at 14000 rpm for 10 minutes, remove the supernatant, and resuspend the precipitate with sterile water to obtain a suspension;

[0082] After 40 μm, 20 μm, and 10 μm filters are sequentially connected in series, the suspension is filtered through the above filters in sequence to obtain a filtrate.

[0083] Comparative Example 2

[0084] Prepare normal saline: weigh 0.43 g NaCl and dissolve it in 500 mL sterile water. After fully dissolved, sterilize it in a 121°C autoclave for 20 minutes to obtain normal saline;

[0085] Preparation of 100 mg / mL α-amylase: weigh 1000 mg of α-amylase powder and dissolve it in 10 mL of sterile water to obtain 100 mg / mL α-amylase;

[0086] Prepare 10 mg / mL papain: weigh 100 mg of papain powder and dissolve it in 10 mL of sterile water to obtain 10 mg / mL papain;

[0087] Take 1 mL of sewage sample in a centrifuge tube, centrifuge at 14000 rpm for 10 minutes, remove the supernatant, and resuspend the precipitate with saline to obtain a suspension;

[0088] 2 mL of 100 mg / mL α-amylase and 0.1 mL of 10 mg / mL papain were added to the suspension, the pH was adjusted to 7.0, ultrasonicated in an ice-water bath at 200 W for 3 min, and then incubated at 37 °C and 180 rpm for 3 h;

[0089] After the incubation, the mixture was centrifuged at 14000 rpm for 10 minutes, the supernatant was removed, and the precipitate was resuspended with sterile water to obtain a suspension;

[0090] After 40 μm, 20 μm, and 10 μm filters are sequentially connected in series, the suspension is filtered through the above filters in sequence to obtain a filtrate.

[0091] Comparative Example 3

[0092] Prepare alkaline physiological saline-Tween buffer: weigh 0.43 g NaCl and dissolve it in 500 mL sterile water. After it is fully dissolved, add 250 μL Tween 20, adjust the pH to about 9.14 with NaOH, and then sterilize it in a high pressure sterilizer at 121°C for 20 minutes to obtain alkaline physiological saline-Tween buffer;

[0093] Take 1 mL of sewage sample in a centrifuge tube, centrifuge at 14000 rpm for 10 minutes, remove the supernatant, and resuspend the precipitate in alkaline saline-Tween buffer to obtain a suspension;

[0094] 0.1 g of quartz sand was added to the suspension, vortexed at high speed for 30 minutes, centrifuged at 14000 rpm for 10 minutes, the supernatant was removed, and the precipitate was resuspended with sterile water to obtain a suspension;

[0095] After 40 μm, 20 μm, and 10 μm filters are sequentially connected in series, the suspension is filtered through the above filters in sequence to obtain a filtrate.

[0096] Comparative Example 4

[0097] Prepare alkaline physiological saline-Tween buffer: weigh 0.43 g NaCl and dissolve it in 500 mL sterile water. After it is fully dissolved, add 250 μL Tween 20, adjust the pH to about 9.14 with NaOH, and then sterilize it in a high pressure sterilizer at 121°C for 20 minutes to obtain alkaline physiological saline-Tween buffer;

[0098] Preparation of 100 mg / mL α-amylase: weigh 1000 mg of α-amylase powder and dissolve it in 10 mL of sterile water to obtain 100 mg / mL α-amylase;

[0099] Prepare 10 mg / mL papain: weigh 100 mg of papain powder and dissolve it in 10 mL of sterile water to obtain 10 mg / mL papain;

[0100] Take 1 mL of sewage sample in a centrifuge tube, centrifuge at 14000 rpm for 10 minutes, remove the supernatant, and resuspend the precipitate in alkaline saline-Tween buffer to obtain a suspension;

[0101] Add 0.1 g of quartz sand to the suspension, sonicate in an ice-water bath at 200 W for 3 min, and remove the supernatant by centrifugation;

[0102] Then, 2 mL of 100 mg / mL α-amylase and 0.1 mL of 10 mg / mL papain were added, and the mixture was incubated at 37°C and 180 rpm for 3 h.

[0103] After the incubation, the mixture was centrifuged at 14000 rpm for 10 minutes, the supernatant was removed, and the precipitate was resuspended with sterile water to obtain a suspension;

[0104] After 40 μm, 20 μm, and 10 μm filters are sequentially connected in series, the suspension is filtered through the above filters in sequence to obtain a filtrate.

[0105] Comparative Example 5

[0106] Prepare normal saline: weigh 0.43 g NaCl and dissolve it in 500 mL sterile water. After fully dissolved, sterilize it in a 121°C autoclave for 20 minutes to obtain normal saline;

[0107] Prepare acetic acid-potassium acetate extract: take 41g potassium acetate and dilute to 500mL, add 29.5mL glacial acetic acid and dilute to 500mL, mix the two in a volume ratio of 9:1, and adjust the pH to 5-6;

[0108] Take 1 mL of sewage sample in a centrifuge tube, centrifuge at 140,000 rpm for 5 minutes, remove the supernatant, and resuspend the precipitate with sterile saline to obtain a suspension;

[0109] Add 0.02 g TiO2, 25 uL 3.6% CH2O solution, and 50 uL acetic acid-potassium acetate extract to the suspension, shake at 27°C, 180 rpm for 15 min, and place in a 60°C water bath for 20 min;

[0110] After cooling, centrifuge at 4°C, 14000 rpm for 10 min, remove the supernatant, and resuspend the precipitate with sterile water to obtain a suspension;

[0111] After 40 μm, 20 μm, and 10 μm filters are sequentially connected in series, the suspension is filtered through the above filters in sequence to obtain a filtrate.

[0112] Comparative Example 6

[0113] Take 1 mL of sewage sample in a centrifuge tube, centrifuge it at 14000 rpm for 10 minutes, remove the supernatant, and resuspend the precipitate with sterile water to obtain a suspension.

[0114] Comparative Example 7

[0115] Take biofilm samples and prepare suspensions;

[0116] Take 1 mL of the sample suspension in a centrifuge tube, centrifuge at 14000 rpm for 10 minutes, remove the supernatant, and resuspend the precipitate with sterile water to obtain a suspension;

[0117] After 40 μm, 20 μm, and 10 μm filters are sequentially connected in series, the suspension is passed through the above filters in sequence for gradient filtration to obtain a filtrate.

[0118] Colony count

[0119] The microbial suspensions obtained in Examples 1-5 and Comparative Examples 1-6 were respectively counted using the dilution plate method and the blood cell counting plate method, and the viable bacterial rates of Examples 1-5 and Comparative Examples 1-6 were calculated according to the formula: culturable viable bacterial rate = dilution plate method counting result / blood cell counting plate method counting result × 100%. The bacterial count measured in the original sewage sample was the total number of microorganisms contained in the sample. The dissociation efficiency of Example 1 and Comparative Examples 1-5 was calculated according to the formula: dissociation efficiency = treated bacterial count / original bacterial count × 100%. The colony count statistics and dissociation efficiency are shown in Table 1.

[0120] Table 1 Statistics of viable bacterial rate and dissociation efficiency in microbial suspensions obtained in Examples 1-5 and Comparative Examples 1-6

[0121]

[0122]

[0123] As can be seen from the above, Examples 1-5 adopt the technical scheme of the present invention, and the viable bacteria rate in the obtained microbial suspension is greater than 90%, and the dissociation efficiency is greater than 70%; Comparative Examples 1-6 adopt other treatment methods, specifically, Comparative Example 1 only adopts a filtration treatment method, Comparative Example 2 adopts an ultrasound, α-amylase and papain enzymolysis and filtration treatment method, Comparative Example 3 adopts an alkali treatment, quartz sand grinding and filtration treatment method, Comparative Example 4 adopts an alkali treatment, quartz sand ultrasound, α-amylase and papain enzymolysis and filtration treatment method, Comparative Example 5 adopts a chemical reagent, oscillation, heat treatment and filtration treatment method, Comparative Example 6 uses the original suspension of the sewage sample without treatment, the viable bacteria rate in the microbial suspension obtained in Comparative Examples 1-6 is less than that in Examples 1-5, and the dissociation efficiency is much less than that in Examples 1-5, indicating that the technical scheme of the present invention has high extraction efficiency and high viable bacteria rate.

[0124] The sample suspensions of Example 6 and Comparative Example 7 before and after filtration were respectively spread on LB plate culture medium for counting by dilution plate method. The number of colonies in Example 6 and Comparative Example 7 before and after filtration was compared with that in the attached figure. Figure 1 As shown, error bars are added in the figure with standard deviation, Figure 1It can be seen that, regardless of whether or not filtered, the ratio of the number of colonies in Example 6 to that in Comparative Example 7 is greater than 1, that is, the microorganisms dissociated from the biofilm samples treated with the kit of the present invention in Example 6 are higher than those from the untreated samples in Comparative Example 7; moreover, after filtration through the filter, the number of microorganisms dissociated in Example 6 can reach more than 100 times that of Comparative Example 7, indicating that the technical solution of the present invention has high extraction efficiency and can extract non-free microorganisms from the biofilm.

[0125] Dissociated microbial suspensions can be used directly for cell sorting

[0126] The microbial suspensions obtained in Example 1 and Comparative Example 4 were respectively subjected to single cell sorting by a single cell sorter, 5,000 single cells were sorted out from each, and the sorted single cells were subjected to single cell sequencing. The sequencing results showed that the single cells sorted in Example 1 contained at least 20 different microorganisms, while only 15 were obtained in Comparative Example 4. This indicates that the extraction efficiency of the technical solution of the present invention is high, and strains with lower abundance can be extracted.

[0127] Finally, it should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only the preferred feasible embodiments of the present invention, and the above-mentioned embodiments are only used to illustrate the technical scheme of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, modifications, evolutions and improvements made by ordinary technicians in this field to the technical scheme of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A kit for dissociating microorganisms in microbial aggregates, characterized in that: The kit comprises a filter and reagents composed of glycoside hydrolase, protease and abrasive. After the microbial aggregate sample is dissociated by the kit, a microbial suspension containing single cells is obtained. The microbial dissociation efficiency is 70% to 99%, wherein the viable cell rate is greater than 90%.

2. The kit for dissociating microorganisms in microbial aggregates according to claim 1, characterized in that The pore size of the filter is 2 to 100 μm; glycoside hydrolases include but are not limited to amylase, cellulase, hemicellulase, sucrase, lactase, and maltase; proteases include but are not limited to trypsin, plasmin, proteinase K, papain, and cathepsin; abrasives include but are not limited to garnet, corundum, and quartz sand.

3. The kit for dissociating microorganisms in microbial aggregates according to claim 1, characterized in that The action temperature of glycoside hydrolase is 25-65°C, the action pH is 4.5-9.0, and the action concentration is 0.5-500 mg / mL; the action temperature of protease is 25-65°C, the action pH is 4-12.5, and the action concentration is 5-2000 μg / mL; the particle size range of abrasive is 5-100 mesh, and the action concentration is 0.05-1 g / mL.

4. The kit for dissociating microorganisms in microbial aggregates according to any one of claims 1 to 3, characterized in that: The dissociation treatment method includes the following steps: Take a sample of microbial aggregates and make it into a suspension. After the first centrifugation, discard the supernatant, add glycoside hydrolase, protease, and abrasive, and incubate after high-speed vortex shaking; After the incubation, the supernatant was discarded after the second centrifugation to terminate the enzymatic reaction; Sterile water was added to resuspend and the dissociated microbial suspension was obtained after filtration.

5. The kit for dissociating microorganisms in microbial aggregates according to claim 4, characterized in that The first centrifugal treatment condition is 8000-14000 rpm, and the time is 1-120 minutes; the second centrifugal treatment condition is 8000-14000 rpm, and the time is 1-120 minutes.

6. The kit for dissociating microorganisms in microbial aggregates according to claim 4, characterized in that The conditions for high-speed vortex oscillation are set at a rotation speed of 1000 to 3000 rpm and a time of 10 to 60 minutes.

7. The kit for dissociating microorganisms in microbial aggregates according to claim 4, characterized in that The incubation conditions are as follows: temperature 25-65°C, pH 4.5-9.0, rotation speed 20-220 rpm, and incubation time 0.5-6 hours.

8. The kit for dissociating microorganisms in microbial aggregates according to claim 4, characterized in that: In the filtering step, the suspension is filtered through a 40-50 μm filter, a 15-20 μm filter, and a 5-10 μm filter in sequence.

9. A dissociated microbial suspension obtained by treating with the kit for dissociating microorganisms in microbial aggregates according to any one of claims 1 to 8, characterized in that: The dissociated microorganism suspension is obtained by dissociating a microorganism aggregate sample through the kit, and the microorganism dissociation efficiency is 70% to 99%, wherein the viable cell rate is greater than 90%.

10. Use of the dissociated microbial suspension according to claim 9 in direct cell detection, sorting, cultivation and sequencing.

Images