CN109988810A - A kind of fast-bacteria-detection method and its application - Google Patents
A kind of fast-bacteria-detection method and its application Download PDFInfo
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Abstract
The invention discloses a kind of fast-bacteria-detection methods based on the catalysis reaction of Bacteria suppression glucose oxidase, it is related to microbial rapid detection field, it is characterized in that, the detection is by obtaining starch-iodide paper, and be attached in substrate, glucose solution is added on starch-iodide paper and object to be detected mixes, glucose oxidase solution is then added and is incubated for 10-20min, finally according to colour developing situation and color change, judge in object to be detected with the presence or absence of bacterium.Rapid and convenient of the present invention, significant effect, idea is novel, low in cost, it can be achieved that the detection of indifference broad spectrum of bacteria, suitable for large-scale promotion and production.
Description
Technical field
The present invention relates to microorganism detection fields, more particularly to a kind of be catalyzed based on Bacteria suppression glucose oxidase to react
Fast-bacteria-detection method and its application.
Background technique
To individual, food industry, for hospital diagnosis with for society, germ contamination is the global public health prestige got worse
The side of body.Claim according to the World Health Organization, due to cholera, the bacteria-related diseases such as infectious diarrhea and septicemia, there are about 1,700,000 people every year
It is dead.Germ contamination is not only the main problem of developing country and the main problem of developed country.By the big of bacteria effect
Majority are baby, children, immunologic hypofunction person and the elderly, especially in the underdeveloped remote districts of medical condition.Greatly
Most bacterial infection diseases are by Escherichia coli, staphylococcus aureus, Listeria Monocytogenes, and mouse typhus is husky
Door Salmonella, pulmonary tuberculosis, streptococcus, caused by S. pullonum and Bacillus cercus.Especially for Escherichia coli and
Staphylococcus aureus, they are two kinds of most common infective bacterials, can cause serious disease, including diarrhea, anaemia, urine
Road feel dye, Skin and soft tissue infection, orbital cell ulitis, necrotizing fasciitis and kidney failure.
Although antibiotic can be used for treating most of bacterium infections, they constantly accelerate the appearance and biography of antibiotic-resistant bacteria
It broadcasts.Nowadays, many antibiotic are all invalid, but the discovery speed of antibiotics has declined to a great extent.In view of antibiotics development
Slowly, exploitation is quickly, accurately, convenient, and the method for detecting bacterium of early screening and low cost is clinical, food and environmental quality control
Field processed there is an urgent need to.
The whole world, which has begun, has carried out various effort to detection and Quantifying Bacteria, including plate culture, polymerase chain are anti-
It answers (PCR), enzyme linked immunosorbent assay (ELISA) (ELISA) and the inspection policies based on chemical sensor etc., preceding several method has it
Advantage, but they be all based on laboratory and cannot be tested at the scene, which has limited them in the practice of everyday life
In be used for bacterial analysis effectiveness.Improvement requirement leads to the development of chemical sensor, including colorimetric detection, fluorescence detection, electrification
Detection etc. is learned, the detection method for being based especially on colorimetric is more and more closed since its is easily operated and vision-based detection
Note.Wherein, due to the development of nanotechnology, nano material is widely used in colorimetric bacterium because of its unique physicochemical properties
Detection, but still remain defect.
The inspiration of the advantages of by colorimetric method, we have developed one kind based on the catalysis reaction of Bacteria suppression glucose oxidase
The simple and novel colorimetric method of (BIGR, hereinafter referred to as BIGR), for quickly detecting bacterium living.Method based on BIGR
It can be carried out on paper base material.Glucose metabolism in our research and utilization bacterium simultaneously realizes broad spectrum of bacteria detection, total score
The time is analysed in 20 minutes, each microcell only needs the sample and reagent less than 10 microlitres.The successful inspection of bacterium in mouse ascites
Survey also indicates that this method has the potentiality of the fast vision detection pathogen in clinical diagnosis, to detect the wound with Quantifying Bacteria
New and breakthrough provides a new thinking.
Summary of the invention
In view of the above drawbacks of the prior art, the present invention provides a kind of conveniently fast-bacteria-detection method and
It is applied.
To achieve the above object, it is fast to provide a kind of bacterium based on the catalysis reaction of Bacteria suppression glucose oxidase by the present invention
Fast detection method, which is characterized in that described detection method includes the following steps:
Step 1: obtaining starch-iodide paper, and be attached in substrate；
Step 2: glucose solution and object to be detected being added on starch-iodide paper, mixes；
Step 3: glucose oxidase (GOX) solution is added and is incubated for, mixes, is incubated for 1-60min；
Step 4: according to colour developing situation and color change, judging in object to be detected with the presence or absence of bacterium.
Further, the substrate in the step 1 can be but not limited to the scraps of paper, slide, sheet metal, the face of the substrate
Product is greater than the area of starch-iodide paper.Substrate excess area may be used as holding.
Further, the starch-iodide paper in the step 1 is obtained by puncher, and shape can be but not limited to
Round, rectangular, diamond shape or hexagon.Preferably, the starch-iodide paper is circle, diameter 0.6cm, size of foundation base 1cm
×1.5cm。
Further, the concentration of glucose solution is 0.1-2mol in the step 2, and volume is 1-10 μ L.Preferred concentration
For 2mol, volume is 3 μ L,
Further, incubation time is 10-20min in the step 3, and being incubated for place is the reaction chamber for keeping constant humidity
In.
Further, the reaction chamber in the step 3 further includes silica-gel desiccant, and the reaction chamber is sealing room, can be protected
Hold steady temperature and humidity.The silica-gel desiccant is 250g, and sealing chamber size is 13cm × 27cm × 20cm.
Further, glucose oxidase (GOX) solution concentration in the step 3 is 1.8-90U/mL, the body of addition
Product is 1-10 μ L.
Further, the step 4 further comprises recording color change by scanner, and measure the ash in color hole
Angle value carries out quantitative analysis.
Further, the quantitative analysis in the step 4 is carried out by establishing the standard curve of various concentration bacterium.It is described
Standard curve is established by the way that the bacterium of various concentration is added into reaction system.Preferably, in step 2 and step 3 glucose and
The concentration of GOX is respectively 1mol/L and 9U/L, and preferably incubation time is 10min in the step 3.
Further, the fast-bacteria-detection method based on chromogenic reaction can be used for one or more bacteria combinations
Quick detection.
Further, fast-bacteria-detection method of the invention has different Monitoring lower-cuts to different bacteriums, is proposed
Method LOD for Escherichia coli be 7.48 × 103CFU/mL is 3.1 × 10 for staphylococcus aureus3CFU/mL。
The present invention also provides a kind of fast-bacteria-detection methods based on the catalysis reaction of Bacteria suppression glucose oxidase
Kit, which is characterized in that the kit is detected using method as described above.The kit includes that glucose is molten
Liquid, glucose oxidase solution, starch-iodide paper etc., the kit can be used for the quick detection of clinical sample, combined standard
Curve and scanner can quantify bacterium clinical sample.
The present invention provides a kind of broad spectrum of bacteria based on paper base quickly to detect, and there is the fast vision in clinical diagnosis to examine
Survey the effect of pathogen.Present invention process is simple, cheap, widely used, novel concept, is suitble to Large scale processes production.
Detailed description of the invention
Fig. 1 is the schematic diagram of the fast-bacteria-detection method based on BIGR chromogenic reaction；
Fig. 2 is the manufacture schematic diagram of paper base test equipment；
Fig. 3 is bacterium to H2O2Aoxidize iodide and the influence with iodine and Starch formation compound；
Fig. 4 is mouse ascites experiment fast-bacteria-detection process and result；
Fig. 5 reacts glucose after ten minutes and the optimization matrix and average gray value of GOX on test paper；
Fig. 6 is the colour developing vision diagram of variety classes bacterium and gray value diagram；
The Escherichia coli and staphylococcus aureus of Fig. 7 various concentration and the relationship of gray value ratio；
Fig. 8 is colour developing and the quantitative result of different bacterium combination.
Specific embodiment
Multiple preferred embodiments of the invention are introduced below with reference to Figure of description, keep its technology contents more clear and just
In understanding.The present invention can be emerged from by many various forms of embodiments, and protection scope of the present invention not only limits
The embodiment that Yu Wenzhong is mentioned.
Embodiment 1
Bacteria Culture and purifying
Use Escherichia coli (ATCC25922), staphylococcus aureus (ATCC29213), enterococcus faecalis (ATCC33186)
With streptococcus mutans (UA159) and S. pullonum (S. pullonum), all experiments are inoculated in Luria-
In Bertani (LB) culture medium (10% peptone, 5% yeast extract and 10% sodium chloride).LB liquid, which is inoculated with single bacterium colony, to be permitted
Perhaps bacterial growth stays overnight (24 hours, 37 DEG C, vibrate with 150rpm).Then, bacterium is centrifuged 10 minutes and is spent with 3600rpm
Ion water washing is three times.Remaining bacterial precipitation is suspended in the deionized water of various concentration by the supernatant for discarding these samples
In.Meanwhile in order to obtain dead bacterium, bacterial suspension is diluted with water to required concentration, then at autoclave (150 DEG C)
Middle sterilizing 30 minutes further to detect.
Embodiment 2
The Catalytic color reaction of glucose and GOX
The glucose (2mol, 1mol, 0.5mol, 0.1mol) and GOX (90U/ of various concentration are prepared with PBS (pH=7.4)
ML, 18U/mL, 9U/mL, 1.8U/mL).The round starch-iodide paper that diameter is 0.6cm is obtained using puncher, then will
It is attached on substrate (1cm × 1.5cm).Then successively 3 μ L glucose solution and 3 μ LGOX are added on round paper.Test paper is put
Enter the humidity (room temperature) in the sealing room (13cm × 27cm × 20cm) with 250g silica-gel desiccant to keep constant.Reaction 10
After minute, color change is recorded.Fig. 2 shows the manufacturing process of paper base test equipment, will strike out iodide-starch paper
Diameter is the circle of 0.6cm and is attached on substrate, wherein 1 indicates round starch-iodide paper, 2 indicate that substrate adheres to starch-
The region of iodide paper, 3 indicate gripping area.
Embodiment 3
Bacteria Detection based on chromogenic reaction
Preparation 1 × 109CFU/mL bacterium, the light corresponded to the pass at the 600nm of Microplate Reader measurement are close
Degree 1.Bacterium is diluted to various concentration: 1 × 108,1×107,1×106,1×105With 1 × 104CFU/mL (in PBS).?
1mol/L glucose and 0.05mg/mL GOX are used in subsequent experiment.3 μ L glucoses are added into paper first, are then added 3
μ L bacterial solution.After these solution are completely soaked, 3 μ LGOX are added and react it in the reaction chamber 20 minutes.Then, lead to
It over-scans instrument and records color change, and measure the gray value in color hole to carry out quantitative analysis.Fig. 1 is BIGR diagram, GOX catalysis
Dioxygen oxidation generates H2O2.The iodide and H being previously loaded on paper2O2Reaction generates iodine.Then compound with iodine and Starch formation
Object generates apparent navy blue on test paper.In the presence of bacterium, due to bacterium consumption of glucose living Catalytic color reaction by
To inhibition, and light color is left on test paper.Naked eyes can be easily observed color change.
In order to verify bacterium meeting this concept of consumption of glucose during metabolism, by 30 μ L bacteriums (5 × 108CFU/
ML) it is added to (final bacterial concentration: 5 × 10 in 270 μ L22.6mmol/L glucose6CFU/mL).It cultivates after twenty minutes, grape
Sugared concentration is significantly lower than initial value.In order to determine whether bacterium will affect H2O2Reaction between iodide, by 3 μ L30%
H2O2With 3 μ LddH2O is added on a test paper, and 3 μ L30%H are added2O2, by 3 μ L bacteriums (5 × 108CFU/mL it) is added another
In one bacterium.As shown in figure 3, the color of two round paper is similar, show bacterium to H2O2Aoxidize iodide and with iodine and starch
The influence for forming compound is negligible.Therefore, when bacterium is loaded on test paper, due to lacking glucose, GOX catalysis
Reaction is suppressed, and leads to the light color on test paper.Bacterium is more, and color is more shallow.By the gray value of analysis test paper, we can be measured
Change the concentration of bacterium.
Embodiment 4
Mouse ascites experiment
6 male C57 mouse (~8 weeks and~25g) have been used in this study.At isoflurane gas anesthetic
Reason.Contain 5 × 10 to 3 male mice injections8The 4mL dialyzate of CFU/mL staphylococcus aureus is to its enteral.Other 3
Male mice injects the pure dialyzate of 4mL.After 2 hours, mouse ascites sample is taken out for detecting.Directly analyze ascites sample, nothing
It need to be further purified.It is demonstrated experimentally that the method proposed is applied successfully to detect the ascites sample from infecting mouse.Specifically
Process and result are shown in Fig. 4, by 5 × 108CFU/mL staphylococcus aureus is mixed into peritoneal dialysis solution to maintain mouse abdomen
Chamber infection model, control are PBS, and error bar indicates three duplicate experiments.
Embodiment 5
Optimize glucose and GOX
In order to and sensitive ratio colour response is obtained when bacterial incubations, we optimize the amount of GOX and glucose.Such as Fig. 5
It is shown, the GOX and glucose of various concentration are prepared with PBS, and successively 0.3 μ L glucose solution and GOX are added on test paper.Reaction
After ten minutes, the color change on test paper is recorded.In Fig. 5, it is evident that with the increase of glucose or GOX concentration, blue is more
It is deep.This visual explanation is also graphically illustrated by calculating average gray value rate.Comprehensively consider, we select 9U/mL
GOX and 1mol/L glucose becomes as our test condition because it provided maximum color within 10 minutes time
Change.
Embodiment 6
Detection and quantitative broad spectrum of bacteria
By 3 μ L bacteriums (6 × 108CFU/mL it) is added on test paper, 3 μ L glucoses and 3 μ LGOX is then added.Bacterium
Ultimate density is 2 × 108CFU/mL.As shown in fig. 6, the sample containing different bacterium leaves obviously on test paper due to BIGR
Light color.All these test files are recorded, are then passed throughIt is analyzed to calculate average gray value, to make figure
Solve explanation.Vision, which is read and illustrated, shows that this method can be used for detecting extensive bacterium.Vision is read and is illustrated
Show that this method can be used for detecting extensive bacterium.In order to study the sensitivity of proposed method, we are further tested
The Escherichia coli and staphylococcus aureus (10 of various concentration4–108CFU/mL).As shown in fig. 7, gray value rate is with bacterial concentration
Proportional increase.Detection limit (LOD): LOD=3S/M is calculated using following formula, wherein S indicates the standard deviation value of blank sample, M
It is range of linearity internal standard slope of a curve.According to the formula, the LOD of the method proposed for Escherichia coli be 7.48 ×
103CFU/mL is 3.1 × 10 for staphylococcus aureus3CFU/mL。
7, the detection of mixed cell and quantitative
In clinical practice, multiple pathogen is usually existed simultaneously in infection site.Therefore, regardless of different chains, amount
It is actually meaningful to change total bacterial concentration.For this ability for the method that inspection institute proposes, six groups of mixed cells are prepared,
Every group of mixture comprising five kinds of bacterium chains, the Escherichia coli including various concentration and different proportion, staphylococcus aureus, excrement
Enterococcus, streptococcus mutans, S. pullonum.All six groups of total concentrations are fixed as 5 × 108CFU/mL.Such as Fig. 8 institute
Show, obviously gently than control group (1mL PBS), but apparent difference is not observed in six groups of color between them.Gray value
The qualitative assessment that rate provides also demonstrates us and quantifies the ideal performance (Fig. 8) of the method for mixed cell.
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be within the scope of protection determined by the claims.
Claims (10)
1. a kind of fast-bacteria-detection method based on the catalysis reaction of Bacteria suppression glucose oxidase, which is characterized in that described
Detection method includes the following steps:
Step 1: obtaining starch-iodide paper, and be attached in substrate；
Step 2: glucose solution and object to be detected being added on starch-iodide paper, mixes；
Step 3: glucose oxidase solution is added and is incubated for, mixes, is incubated for 1-60min；
Step 4: according to colour developing situation and color change, judging in object to be detected with the presence or absence of bacterium.
2. the fast-bacteria-detection method as described in claim 1 based on the catalysis reaction of Bacteria suppression glucose oxidase,
It is characterized in that, the substrate in the step 1 can be but not limited to the scraps of paper, slide, sheet metal, and the area of the substrate, which is greater than, to form sediment
Powder-iodide paper area.
3. the fast-bacteria-detection method as described in claim 1 based on the catalysis reaction of Bacteria suppression glucose oxidase,
It is characterized in that, the starch-iodide paper in the step 1 is obtained by puncher, and shape can be but not limited to round, side
Shape, diamond shape or hexagon.
4. the fast-bacteria-detection method as described in claim 1 based on the catalysis reaction of Bacteria suppression glucose oxidase,
It is characterized in that, the concentration of glucose solution is 0.1-2mol in the step 2, and volume is 1-10 μ L.
5. the fast-bacteria-detection method based on chromogenic reaction as described in claim 1, which is characterized in that in the step 3
Incubation time is 10-20min, and being incubated for place is to keep constant in the reaction chamber of humidity.
6. the fast-bacteria-detection method as described in claim 1 based on the catalysis reaction of Bacteria suppression glucose oxidase,
It is characterized in that, the reaction chamber in the step 3 further includes silica-gel desiccant.
7. the fast-bacteria-detection method as described in claim 1 based on the catalysis reaction of Bacteria suppression glucose oxidase,
It is characterized in that, the glucose oxidase solution concentration in the step 3 is 1.8-90U/mL, and the volume of addition is 1-10 μ L.
8. the fast-bacteria-detection method as described in claim 1 based on the catalysis reaction of Bacteria suppression glucose oxidase,
It is characterized in that, the step 4 further comprises recording color change by scanner, and the gray value for measuring color hole carries out
Quantitative analysis.
9. the fast-bacteria-detection method as described in claim 1 based on the catalysis reaction of Bacteria suppression glucose oxidase,
It is characterized in that, the quantitative analysis in the step 4 is carried out by establishing the standard curve of various concentration bacterium.
10. a kind of kit of the fast-bacteria-detection method based on the catalysis reaction of Bacteria suppression glucose oxidase, feature
It is, the kit uses claim 1-9 any one the method such as to carry out fast-bacteria-detection.
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Cited By (1)
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| CN111635929A (en) * | 2020-06-05 | 2020-09-08 | 上海交通大学 | Double-enzyme amplification system and bacterial activity detection method based on same |
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