CN105803044A - Starch degrading microorganism detection kit and method for detecting starch degrading microorganism - Google Patents
Starch degrading microorganism detection kit and method for detecting starch degrading microorganism Download PDFInfo
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Abstract
The invention discloses a starch degrading microorganism detection kit and a method for detecting a starch degrading microorganism. The starch degrading microorganism detection kit comprises a sample collecting tool, a disposable sterile medicinal ladle, a sterile collecting bag, a sample treatment reagent A, calibrated tracing reagents B1 and B2, an electronic transfer chain inhibitor C, a dying agent D and a disposable sterile operation tool set. The method for detecting the starch degrading microorganism by using the kit comprises the following steps of sample collecting, sample treating and cell separating, calibrating and tracing of the starch degrading microorganism, DAPI (4',6-diamidino-2-phenylindole) dying, microscopic observation, determination and statistic analysis of starch degrading bacteria abundance, and the like. According to the method disclosed by the invention, a starch degrading microorganism flora can be directly calibrated and monitored, so that the labor intensity is greatly reduced, the cost is lower, the operation process is simple and convenient, an operator needs not to be specifically trained, a detection site is not specially required, an experiment result is accurate, and relevant data can be rapidly provided for diagnosis personnel.
Description
Technical field:
The present invention relates to and relate to a kind of microbial detection reagent kit and a kind of method detecting microorganism, particularly a kind of starch degradation microbial detection reagent kit and use the method for starch degradation microorganism in this test kit detection human body.
Background technology
The a large amount of microorganisms perched in human body intestinal canal, are indispensable ingredients in organismic internal environment.The microorganism perched in adult's intestinal probably has more than 800 to plant, including antibacterial, Archimycetes, fungus, protozoon and a small amount of virus, total quantity is more than 10,000,000,000,000, it is approximately more than 10 times of body cell and sexual cell sum, owing to the microorganism of numerous kinds and quantity is perched in people's intestinal, enteric microorganism interferes significantly on human biology's feature by network of personal connections between host-microorganism and microorganism-microorganism.Intestinal microflora is " microorganism organ " (microbialorgan) of human body, different types of intestinal microflora associates with one another, and and host cell between constantly carry out the cellularity of communication for information, consumption, storage and redistribution energy, physiologically regulate and control important chemical substance convert and maintained by self replication and repair self.Meanwhile, reside in the microorganism in people's intestinal and play the important function such as energy metabolism regulation and control, xenobiotic metabolism, intestinal epithelial cell reparation, Intestinal Mucosal Immunity activation, host's behavior manipulation.
Enteric microorganism is directly related with human condition.As the microorganism with human body symbiosis, the interaction of enteric microorganism and human body decides the health degree of human body, when only enteric microorganism and human body are in a dynamic balance state, and the health status of guarantee human body.Relation between enteric microorganism and human diseases is extensively confirmed, and the disease that the mankind such as enteric microorganism and chronic metabolic diseases (such as obesity, diabetes etc.), digestive system disease (such as inflammatory enteritis, colorectal carcinoma, non-alcoholic fatty liver disease etc.), cardiovascular disease become increasingly conspicuous has important relation.
Human body intestinal canal microbial bacteria group structure is not unalterable, and it is as the growth of host, experienced by from newborn stage being continually changing to the old stage, and in this evolution, it is subject to the impact of the many factors such as the age of human body, genotype, local environment, food and antibiotic, therefore understand and control the impact on intestinal microflora structure of these factors, so that it may making the health status that human body maintenance is good.The factor affecting people's intestinal microflora structure is many-sided, due to the restriction of research method and means, and studying or general these factors at present.The modern life exists the factor that can upset human body intestinal canal microorganism species balance too much, such as infant is used too early antibiotic, destroying intestinal microflora balance and set up process, after making infant grow up, the risk of afflicted with inflammation enteritis, asthma, obesity etc. is greatly improved.Therefore, it is necessary to invent a kind of method monitoring human body intestinal microflora balance.
Human body intestinal canal microorganism plays an important role in the nutrient metabolism of human body, after food enters intestinal, each quasi-microorganism has been involved in this metabolic process, some functional microbials take part in the degraded of complicated macromolecular substances (starch, protein and fat), their metabolite to the stable of healthy human body intestinal microbial population and balance, the nutrition of human body, physiology and immunologic function, disease, aging, carcinogenic etc. all have significant effect.Human body intestinal canal microbiota and host's starch metabolism relation are brand-new research fields, and the precedence that the dietary polysaccharides that host is taken in by which microorganism concrete and the starch degradation in host's energy metabolism about, starch degradation microorganism actually the utilizes mechanism etc. making approach and microbiota and the relation of energy metabolism, microbiota control host's starch metabolism mutually how, between various microorganism all needs to be studied.
Existing research shows, health and alimentation are all had corresponding impact by the difference of the energy absorption that body energy takes in the difference between consumption, microbiota causes, and cause obese people microbiota to seem to help host more effectively to absorb energy.Recent study infers that the antibacterial of human body intestinal canal is likely to relevant to obesity, this is because evidence suggests that the people of obesity is different with the composition of partially thin people's intestinal microflora, result of study shows, the microbiota of obese type mice is likely to more efficiently to obtain energy from daily ration residue than the microbiota of bias tyre mice, thus pointing out human body to be likely to have similar effect.
Starch in mankind's conventional food be the amylose (amylose) by 25% and 75% amylopectin (amylopectin) constitute.Starch is mainly hydrolyzed by pancreatic amylase (amylase) in small intestinal, thereby it is assumed that starch degradation flora plays important role on formation obese people.The basic function of intestinal microflora is that the nutrient participating in human body converts, being produced as, with short-chain fatty acid, the main path that the carbohydrate metabolism at center is enteric microorganism ecosystem energy metabolism in intestinal, wherein the metabolism of starch converts at human nutrition thing and plays an important role in energy metabolism.The response of the starch degradation flora factor to external world understood and follow the trail of in human body intestinal canal, thereby through controlling these factors, the intestinal microflora state that maintenance is good, finally realize human health important in inhibiting.Communicate with each organ because the upper and lower two ends of people's digestive tract are outwards open, internal.Therefore intestinal contents often carries out material, energy or communication for information with extraneous and other organs, the microbe groups of the broad categories of symbiosis, enormous amount in intestinal, under normal circumstances that host health is useful, once there is careless mistake, alteration of intestinal flora in mucosa, its metabolite by mucosa absorption, then can cause the generation of human body diseases.Therefore detection and monitoring starch degradation microorganism species, it is possible to provide relevant diagnostic message, the approach of further control effect intestinal microflora and factor for people, make human body be in the state of health.
Up to now, the method for almost all of research human body intestinal canal starch degradation microorganism species all carries out with pure culture so that the stability that As time goes on these microbiologic populations are presented by people is short in understanding.JeremiahJ.Faith and colleague thereof have developed a kind of sequence measurement and accurately follow the trail of these microbial strains, they find that those eat special diet and the microbial strain of people lost weight there occurs change, it was shown that the change of intestinal microflora can as the mark of host health and function.But, current all of research means and method are all isolated strains on the basis of pure culture, adopt the method for molecular microbial ecology to study special bacterial strain further.And to (being namely need not by isolation of pure culture bacterial strain) under (insitu) state in position, detect and monitor the effect in enteral nutrition thing conversion process of the intestinal starch degradation microorganism species and mechanism is but understood seldom, particularly in position under (insitu) state, the method demarcating special intestinal starch degradation microorganism species not yet develops.
Human body is as independent individuality, and based on the reason of the aspects such as dietary habit, health and gene, the composition of everyone intestinal microflora is different.nullStarch degradation microorganism species is one of most important functions flora in human body intestinal canal microorganism，Owing to lacking the original position information of the microorganism species of the relevant participation intestinal starch metabolism of system，The understanding of enteric microorganism effect in human body starch metabolism process is studied mainly through pure culture，But，In human intestinal microorganisms group, only minority (< 20%) can by pure culture,And differ reflection microorganism surely in the effect of complicated ecosystem original position and function by the pure culture microbial physiology Biochemical Information that obtains of research，In addition，The method of pure culture requires over isolated strains，And each strain antibacterial is carried out physiology，Biochemical Research，Length consuming time、Relatively costly、Operating process is complicated，Particularly operator need the special training of acceptance just can complete the task (proving which antibacterial belongs to starch degradation flora) to antibacterial Function Identification.
Summary of the invention
nullThe invention provides a kind of starch degradation microbial detection reagent kit and the method for detection starch degradation microorganism，The molecular microbial ecology ways and means exempting to cultivate and BODIPY amylase (amylase) dyeing is adopted to demarcate and follow the trail of human body intestinal canal starch degradation microorganism species under home state，Its principle is: the organic macromolecule BODIPY with fluorescence is combined with starch，The fluorescence in BODIPY is made to be wrapped in the inside of conjugate，Do not observe under fluorescence microscope，It is positioned at the α-amylase between Cytoplasm wall and can be hydrolyzed BODIPY fluorescence starch，Discharge the short-movie section hydrolyzate with fluorescence，These hydrolyzate are deposited on around the microorganism wall of relevant enzyme vigor，The single cell with specific alpha-amylase activity that be fluorescently labeled be can be observed under fluorescence microscope.
Meanwhile, in order to get rid of the false positive in dyeing course, distinguishing and have hydrolytic enzyme activities and do not have hydrolytic enzyme activities but can absorb the cell (error flag) of hydrolyzate, the present invention adds various electron transmission chain inhibitor to ensure the reliability of result in cultivation.Its principle be microorganism species can be hydrolyzed or ferment add labelling chemicals (starch of BODIPY labelling), therefore can all cells of the markd metabolite of absorption band also all can be labeled, and the microorganism that can suppress adding electron transmission chain inhibitor absorbs the hydrolyzate with fluorescently-labeled starch, prevent the false positive owing to active absorption causes with the hydrolyzate of fluorescent marker, it is ensured that viewed be entirely starch degradation microorganism with fluorescently-labeled microorganism.
For reaching above-mentioned purpose, the invention provides a kind of starch degradation microbial detection reagent kit, for starch degradation microorganism in Quantitative detection human excrement and urine's sample, this detection kit includes the disposable sterilized medicine spoon of sample collecting instrument and sterile collection bag, also includes sample processing reagent A, demarcates tracer reagent B1, B2, electron transport chain inhibitor C, stain D, disposable sterilized operation tool group.
Preferably, described sample processing reagent A is sterilizing 800mlPBS buffer, pH=7.0-8.0.
Preferably, described demarcation tracer reagent B1, B2 respectively sterilizing 5-20mlTris-HCl buffer (10-15mmol/L, and the green fluorescence starch dye liquor of 2-20mlBODIPY (boron fluoride two pyroles fluorescent dye, boron-dipyrromethene) labelling pH7.0-8.0).
Preferably, described electron transport chain inhibitor C is 2-30ml sterilizing 10-100mmol/L Hydrazoic acid,sodium salt, 10-100mmol/L 1080 (Monsanto), 10-100mmol/L iodo-acetamide sodium mixed solution.
Preferably, described stain D is 0.5-10ml sterilizing 0.003-0.005mg/mlDAPI dye liquor.
Described disposable sterilized operation tool group includes serum bottle, aluminium foil, gelatin coating microscope slide at the bottom of glove, thermos flask, plastic stir rod, 50mL beaker, BioRad homogenate bag, 50ml centrifuge tube, 2mL centrifuge tube, 10mL width, each 1-40 of quantity.
Preferably, described starch degradation microorganism include participating in human body intestinal canal starch degradation, there is cyto-architectural antibacterial.
Present invention also offers and a kind of use the method for starch degradation microorganism in this test kit detection human body, i.e. starch degradation microorganism in detection human excrement and urine, comprise the following steps:
(1) sample collecting: use disposable sterilized medicine spoon and sterile collection bag to gather fresh human excrement and urine, carry out sample treatment under 18-40 DEG C of constant temperature in 20min；
(2) sample treatment and cell separation: add sample processing reagent A in sample and mix；By the liquid at high speed shelves homogenate of mixing, filter, filtrate is centrifuged and removes supernatant, and precipitate is resuspended in sample processing reagent A stand-by；In the experimentation waiting next step, cell suspending liquid can be stored in the refrigerator of 4 DEG C；
(3) demarcation of starch degradation microorganism and spike: be centrifuged by cell suspending liquid, removes supernatant, adds and demarcates tracer reagent B1, B2, adds electron transport chain inhibitor C, and lid is built, and the tight lucifuge of rapid Aluminium Foil Package；Room temperature shake 30-50min, speed is maintained at 200-300rpm；Draw 10-20 μ L reactant liquor under gnotobasis to be spread evenly across on gelatin coating microscope slide, and lucifuge is air-dry in gnotobasis；
(4) DAPI dyeing: taking 100-200ul stain D, to be covered on air-dry microscope slide uniformly spreadable, is placed in the aseptic operation box of dark dyeing 10-20min；Stain D is gone, and carefully rinses 2-3 with distilled water all over (each 1min)；Lucifuge is also air-dry in aseptic operation box；
(5) microscope is observed: be placed in by dried microscope slide on fluorescence microscopy endoscope objective lens platform, 100 × object lens under carry out observing and taking pictures, each sample at least gathers 30 sets (every sets of data picture includes green fluorescence starch dyeing picture and a DAPI dyes picture) data picture；
(6) determination of starch degradation bacteria abundance and statistical analysis: use image analysis software ImageJ to analyze the cell number (microbial count) of the green fluorescence starch stained positive starch degradation cell number in same set of digital picture and DAPI stained positive respectively, green fluorescence starch staining cell adopts the manual count method counting provided in ImageJ, and blue-fluorescence DAPI staining cell adopts automatic count method:
Cell number/total cellular score × 100% of the abundance of starch degradation antibacterial=green fluorescence starch dyeing
T inspection and variance analysis all complete in excel.
Preferably, it is provided that a kind of method making gelatin smear, it is characterised in that: comprise the following steps:
(1) take 7 carry sheet glass shelf, microscope slide is filled in carry sheet glass shelf；3.5L distilled water adds 4-5 and drips detergent, put into carry sheet glass shelf, boil 10min；Taking out carry sheet glass shelf, dry in the air 3min；With distilled water, detergent is rinsed well, the microscope slide state of being kept upright is dried；
(2) adding the ten sulfate dihydrate potassium chromium of 2.5g and the gelatin of 0.44g in 3.5L distilled water, heating, to about 70 DEG C, puts into carry sheet glass shelf, constant temperature 10min after gelatin dissolves substantially；Take out carry sheet glass shelf, microscope slide is tiltedly stood on test tube rack limit and be drying to obtain.
nullThe invention provides a kind of method of starch degradation microorganism in starch degradation microbial detection reagent kit and detection human body，The molecular microbial ecology ways and means exempting to cultivate and BODIPY amylase (amylase) dyeing is adopted to demarcate and follow the trail of human body intestinal canal starch degradation microorganism species under home state，Stability not only for definition and diagnosis human body starch degradation microorganism species has great importance，And be the effect in further appreciating that the utilization in human nutrition thing metabolism of the intestinal starch degradation microorganism species and converting and function，Obtain the original position information of the Nomenclature Composition and Structure of Complexes of original position intestinal starch degradation microorganism species，Understand the ecological relation in enteral nutrition thing utilizes and converts between starch degradation microorganism species and human body，Lay the foundation for exploring the intestinal starch degradation microorganism species mechanism of action in body metabolism disease occurs further.
The present invention provides method and former culture-dependent method to compare, it is characterized as being and can directly demarcate and monitor starch degradation microorganism species, shorten reduction labor intensity significantly, less costly, operating process is easy, and the training that operator need not be special, to the place detected without special requirement, experimental result is accurate, can be quickly provided to the data that diagnostic personnel is relevant.Meanwhile, the design of test kit also greatly simplify operating process, reduction operation error and the environment impact on result, makes experimental result relatively reliable.
Accompanying drawing explanation
Fig. 1 uses the method flow diagram of starch degradation microorganism in this test kit detection human body；
Fig. 2 is fresh human body fecal microorganism group Green fluorescence starch stained positive starch degradation microorganism microphotograph example；
Fig. 3 is the total microbial cell microphotograph example in DAPI dyeing fresh human body fecal microorganism group.
Detailed description of the invention
Below by way of specific instantiation, embodiments of the present invention being described, those skilled in the art the content disclosed by this specification can understand other advantages and effect of the present invention easily.The present invention can also be carried out by additionally different detailed description of the invention or apply, and the every details in this specification based on different viewpoints and application, can also carry out various modification or change under the spirit without departing from the present invention.
Before further describing the specific embodiment of the invention, it should be appreciated that protection scope of the present invention is not limited to following specific specific embodiments；It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments, rather than in order to limit the scope of the invention；In specification and claims of the present invention, unless additionally explicitly pointed out in literary composition, singulative " ", " one " and " this " include plural form.
When embodiment provides numerical range, it should be appreciated that unless the present invention is otherwise noted, between two end points and two end points of each numerical range, any one numerical value all can be selected for.Unless otherwise defined, the same meaning that all technology used in the present invention and scientific terminology and those skilled in the art of the present technique are generally understood that.Except the concrete grammar used in embodiment, equipment, material, record according to those skilled in the art's grasp to prior art and the present invention, it is also possible to use similar with the method described in the embodiment of the present invention, equipment, material or that be equal to any method of prior art, equipment and material to realize the present invention.
Unless otherwise indicated, the experimental technique that disclosed in this invention, detection method, preparation method all adopt the routine techniques of the conventional molecular biology of the art, biochemistry, technique of analytical chemistry and association area.
Starch degradation microbial detection reagent kit of the present invention, can be used for starch degradation microorganism in Quantitative detection human excrement and urine's sample, including the disposable sterilized medicine spoon of sample collecting instrument and sterile collection bag, described test kit also includes sample processing reagent A, demarcates tracer reagent B1, B2, electron transport chain inhibitor C, stain D, disposable sterilized operation tool group；Described sample processing reagent A is sterilizing 800ml1 × PBS, pH=7.0；The green fluorescence starch dye liquor of described demarcation tracer reagent B1, B2 respectively sterilizing 8ml1 × Tris-HCl buffer (10mmol/L, pH7.8) and 4mlBODIPY (boron fluoride two pyroles fluorescent dye, boron-dipyrromethene) labelling；Described electron transport chain inhibitor C is 12ml sterilizing 3mmol/L Hydrazoic acid,sodium salt, 2mmol/L 1080 (Monsanto), 4mmol/L iodo-acetamide sodium mixed solution；Described stain D is 1.6ml sterilizing 0.005mg/mlDAPI dye liquor；Described disposable sterilized operation tool group includes the microscope slide of serum bottle, aluminium foil, gelatin coating at the bottom of glove, thermos flask, plastic stir rod, 50mL beaker, BioRad homogenate bag, 50ml centrifuge tube, 2mL centrifuge tube, 10mL width, each 20 of quantity；What described starch degradation microorganism included participating in human body intestinal canal starch degradation has cyto-architectural antibacterial.
The method of starch degradation microorganism in detection human excrement and urine of the present invention, concrete steps:
(1) sample collecting: use disposable sterilized medicine spoon and sterile collection bag to gather the fresh human excrement and urine of 50g, carry out sample treatment under 37 DEG C of constant temperatures in 20min；
(2) sample treatment and cell separation: use aseptic plastic stirring rod to be mixed by fecal specimens, then weighs 10g fresh excreta with aseptic 50ml beaker, adds sample processing reagent A and mix；The liquid of mixing is transferred in aseptic BioRad homogenate bag, at BioRad homogenizer top gear homogenate 8min, is filtered by the mistake filter grid of homogenate bag side；By filtrate collection in aseptic 50ml centrifuge tube, 5000g is centrifuged 15min；Remove supernatant, and precipitate is resuspended in 5ml sample processing reagent A stand-by；In the experimentation waiting next step, cell suspending liquid can be stored in the refrigerator of 4 DEG C；
(3) demarcation of starch degradation microorganism and spike: being transferred to by cell suspending liquid in aseptic 2mleppendof centrifuge tube, 4500g is centrifuged 10min；Remove supernatant, add and demarcate tracer reagent B1, B2, mixing liquid is transferred in the serum bottle of the wide end of aseptic 10ml, add electron transport chain inhibitor C lid and build, and the tight lucifuge of rapid Aluminium Foil Package；The shake 30min that is fixed on rotary shaker by the serum bottle wrapped room temperature, speed is maintained at 220rpm；Serum bottle is transferred to aseptic operation box, opens serum bottle, draw 10 μ L and be spread evenly across on gelatin coating microscope slide, and in aseptic operation box air-dry 20min, whole process needs lucifuge；
(4) DAPI dyeing: take 80ul stain D with liquid-transfering gun and be covered on air-dry microscope slide central authorities, uniformly spreadable with rifle head, it is placed in the aseptic operation box of dark dyeing 20-30min；Stain D is gone, and carefully rinses 2-3 with distilled water all over (each 1min)；Lucifuge in aseptic operation box air-dry 20 minutes；
(5) microscope is observed: be placed in by dried microscope slide on fluorescence microscopy endoscope objective lens platform, 100 × object lens under carry out observing and taking pictures, each sample at least gathers 30 sets (every sets of data picture includes green fluorescence starch dyeing picture and a DAPI dyes picture) data picture；
(6) determination of starch degradation bacteria abundance and statistical analysis: use image analysis software ImageJ to analyze the cell number (microbial count) of the green fluorescence starch stained positive starch degradation cell number in same set of digital picture and DAPI stained positive respectively, green fluorescence starch staining cell adopts the manual count method counting provided in ImageJ, and DAPI staining cell adopts automatic count method:
Cell number/total cellular score × 100% of the abundance of starch degradation antibacterial=green fluorescence starch dyeing
T inspection and variance analysis all complete in excel.
Wherein, the preparation method of described gelatin smear comprises the following steps:
(1) take 7 carry sheet glass shelf, microscope slide is filled in carry sheet glass shelf；3.5L distilled water adds 4-5 and drips detergent, put into carry sheet glass shelf, boil 10min；Taking out carry sheet glass shelf, dry in the air 3min；With distilled water, detergent is rinsed well, the microscope slide state of being kept upright is dried；
(2) adding the ten sulfate dihydrate potassium chromium of 2.5g and the gelatin of 0.44g in 3.5L distilled water, heating, to about 70 DEG C, puts into carry sheet glass shelf, constant temperature 10min after gelatin dissolves substantially；Take out carry sheet glass shelf, microscope slide is tiltedly stood on test tube rack limit and be drying to obtain.
Due to the utilization of technique scheme, the present invention compared with prior art has the advantage that
The present invention adopts the molecular microbial ecology ways and means exempting to cultivate and BODIPY amylase (amylase) dyeing to demarcate and follow the trail of human body intestinal canal starch degradation microorganism species under home state, stability not only for definition and diagnosis human body starch degradation microorganism species has great importance, and be the effect in further appreciating that the utilization in human nutrition thing metabolism of the intestinal starch degradation microorganism species and converting and function, obtain the original position information of the Nomenclature Composition and Structure of Complexes of original position intestinal starch degradation microorganism species, understand the ecological relation in enteral nutrition thing utilizes and converts between starch degradation microorganism species and human body, lay the foundation for exploring the intestinal starch degradation microorganism species mechanism of action in body metabolism disease occurs further.
Patented method of the present invention and former culture-dependent method compare, it is characterized as being and can directly demarcate and monitor starch degradation microorganism species, shorten reduction labor intensity significantly, less costly, operating process is easy, and the training that operator need not be special, to the place detected without special requirement, experimental result is accurate, can be quickly provided to the data that diagnostic personnel is relevant.Meanwhile, the design of test kit also greatly simplify operating process, reduction operation error and the environment impact on result, makes experimental result relatively reliable.
Below it is only the concrete exemplary applications of the present invention, protection scope of the present invention is not constituted any limitation.The technical scheme that all employing equivalents or equivalence are replaced and formed, all falls within rights protection scope of the present invention.
Claims (9)
1. a starch degradation microbial detection reagent kit, for starch degradation microorganism in Quantitative detection human excrement and urine's sample, including the disposable sterilized medicine spoon of sample collecting instrument and sterile collection bag, it is characterised in that: also include sample processing reagent A, demarcate tracer reagent B1, B2, electron transport chain inhibitor C, stain D, disposable sterilized operation tool group.
2. starch degradation microbial detection reagent kit according to claim 1, it is characterised in that: described sample processing reagent A is 800ml sterilizing PBS, pH=7.0-8.0.
3. starch degradation microbial detection reagent kit according to claim 1, it is characterised in that: described demarcation tracer reagent B1 is 5-20ml sterilizing Tris-HCl buffer, concentration 10-15mmol/L, pH7.0-8.0；Demarcate the green fluorescence starch dye liquor that tracer reagent B2 is 2-20ml sterilizing boron fluoride two pyroles fluorescent dye BODIPY labelling.
4. starch degradation microbial detection reagent kit according to claim 1, it is characterized in that: described electron transport chain inhibitor C is 2-30ml sterilizing mixed solution, this sterilizing mixed solution is made up of 10-100mmol/L Hydrazoic acid,sodium salt, 10-100mmol/L 1080 (Monsanto) and 10-100mmol/L iodo-acetamide sodium.
5. starch degradation microbial detection reagent kit according to claim 1, it is characterised in that: described stain D is the DAPI dye liquor of 0.5-10ml sterilizing, and concentration is 0.003-0.005mg/ml.
6. starch degradation microbial detection reagent kit according to claim 1, it is characterized in that: described disposable sterilized operation tool group includes serum bottle, aluminium foil, gelatin coating microscope slide at the bottom of glove, thermos flask, plastic stir rod, 50mL beaker, BioRad homogenate bag, 50ml centrifuge tube, 2mL centrifuge tube, 10mL width, each 1-40 of quantity.
7. starch degradation microbial detection reagent kit according to claim 1, it is characterised in that: what described starch degradation microorganism included participating in human body intestinal canal starch degradation has cyto-architectural antibacterial, Archimycetes, fungus and protozoon.
8. one kind uses the method for test kit detection starch degradation microorganism described in claim 1, it is characterised in that: comprise the following steps:
Sample collecting: use disposable sterilized medicine spoon and sterile collection bag to gather fresh human excrement and urine, carry out sample treatment under 18-40 DEG C of constant temperature in 20min；
Sample treatment and cell separation: add sample processing reagent A in sample and mix；By the liquid at high speed shelves homogenate of mixing, filter, filtrate is centrifuged and removes supernatant, and precipitate is resuspended in sample processing reagent A stand-by；In the experimentation waiting next step, cell suspending liquid can be stored in the refrigerator of 4 DEG C；
The demarcation of starch degradation microorganism and spike: be centrifuged by cell suspending liquid, remove supernatant, adds and demarcates tracer reagent B1, B2, adds electron transport chain inhibitor C, and lid is built, and the tight lucifuge of rapid Aluminium Foil Package；Room temperature shake 30-50min, speed is maintained at 200-300rpm；Draw 10-20 μ L reactant liquor under gnotobasis to be spread evenly across on gelatin coating microscope slide, and lucifuge is air-dry in gnotobasis；
DAPI dye: take 100-200ul stain D and be covered on air-dry gelatin coating microscope slide uniformly spreadable, be placed in dark aseptic operation box in dyeing 10-20min；Stain D is gone, and carefully rinses 2-3 time with distilled water, each 1min；Lucifuge is also air-dry in aseptic operation box；
Microscope is observed: be placed on fluorescence microscopy endoscope objective lens platform by dried gelatin coating microscope slide, 100 × object lens under carry out observing and taking pictures, each sample at least gathers 30 sets of data pictures, and every sets of data picture includes a green fluorescence starch dyeing picture and a DAPI dyeing picture；
The determination of starch degradation bacteria abundance and statistical analysis: use image analysis software ImageJ to analyze the cell number of the green fluorescence starch stained positive starch degradation cell number in same set of digital picture and DAPI stained positive respectively, green fluorescence starch staining cell adopts the manual count method counting provided in ImageJ, and DAPI staining cell adopts automatic count method:
Cell number/total cellular score × 100% of the abundance of starch degradation antibacterial=green fluorescence starch dyeing
T inspection and variance analysis all complete in excel.
9. method according to claim 8, it is characterised in that: the process step of described gelatin coating gelatin smear is as follows:
Take 7 carry sheet glass shelf, gelatin coating microscope slide is filled in carry sheet glass shelf；3.5L distilled water adds 4-5 and drips detergent, put into carry sheet glass shelf, boil 10min；Taking out carry sheet glass shelf, dry in the air 3min；With distilled water, detergent is rinsed well, the gelatin coating microscope slide state of being kept upright is dried；
Adding the ten sulfate dihydrate potassium chromium of 2.5g and the gelatin of 0.44g in 3.5L distilled water, heating, to 70 DEG C, puts into carry sheet glass shelf, constant temperature 10min after gelatin dissolves substantially；Take out carry sheet glass shelf, gelatin coating microscope slide is tiltedly stood on test tube rack limit and be drying to obtain.
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| CN108204962A (en) * | 2017-12-14 | 2018-06-26 | 夏云 | A kind of method of quick detection broiler chicken caecum protein hydrolytic bacteria |
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