CN103487571A - Detection method of acidophilic thermophilic bacterium in fruit juice, and inducer and color development agent of detection method - Google Patents
Detection method of acidophilic thermophilic bacterium in fruit juice, and inducer and color development agent of detection method Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The invention relates to a detection method of an acidophilic thermophilic bacterium in fruit juice, and an inducer and a color development agent of the detection method. The method comprises induction cultivation and color development detection, wherein the induction cultivation is to perform the induction cultivation on a to-be-detected fruit juice sample by utilizing the inducer; and the color development detection is to perform a color development reaction on the to-be-detected fruit juice sample subjected to the induction cultivation by utilizing the color development agent, and if an amber compound appears in a reaction system after the color development reaction, the to-be-detected fruit juice sample contains the acidophilic thermophilic bacterium. The inducer comprises vanillic acid and a medium. The color development agent comprises DNA (Deoxyribose Nucleic Acid) enzyme with horse radish peroxidase catalysis activity and hydrogen peroxide. The rapid color comparison detection method based on the DNA enzyme achieves on-site rapid qualitative and quantitative detection on the bacterium by constructing a biosensing technique based on the DNA enzyme, and has higher sensitivity and specificity.
Description
Technical field
The present invention relates to harmful microbe detection technique in fruit juice, be specifically related to for detection of the derivant of having a liking for sour thermoduric bacteria in fruit juice, developer and application thereof.
Background technology
Have a liking for sour thermoduric bacteria and be a kind of have a liking for acid, thermophilic, aerobic bacillus, be fruit juice contaminated bacteria main in thermoduric bacteria, its gemma can stand the pasteurization in acidic juice processing and survive, when it when condition is suitable, but amount reproduction, cause fruit juice sense organ and quality deterioration again.This bacterium mainly pollutes inspissated juice and the fruit drinks such as cider, orange juice, produces the bad flavor material, causes fruit juice putrid and deteriorated, the economic loss that can cause producing.
At present, having a liking for sour thermoduric bacteria detection method in fruit juice mainly contains and utilizes microbe growth and reach nutrient culture media separated between different microorganisms and separate and identify, molecular biology for detection (as round pcr, RT-PCR method) according to the generations different from the RNA sequence of the DNA between different microorganisms, the horseradish peroxidase brought according to the Catalyzed Synthesis By Peroxidase chemical reaction (HRP) detection method, also reliable high-end instrument detects the infrared spectrum detection of isolation technics etc.
Existingly have a liking for sour thermoduric bacteria detection technique above-mentioned, applying more is that bacterium separates and cultivates and the method for biochemical identification, but the shortcomings such as the method exist to detect, and index is many, complicated operation, consuming time, testing result hysteresis, cause can not feeding back in time the pollution situation of fruit juice in the juice production process and take corresponding anti-measure.
In addition, the traditional instrument detection that dependence detects a class as infrared spectrum has the sensitive height of detection, the advantages such as testing result good stability, the professional technique operating personnel that but large-scale instrument and equipment is expensive, need to possess relevant knowledge, have that testing cost is high, testing result waits shortcoming more slowly, greatly limited the large-scale application of large-scale instrument and equipment in field quick detection.
Summary of the invention
One of purpose of the present invention is to provide the detection method of having a liking for sour thermoduric bacteria in a kind of fruit juice, and to solve, the detection rates that existing detection method exists is low, poor specificity and the high problem of cost.
For this reason, have a liking for the detection method of sour thermoduric bacteria in fruit juice provided by the invention, it is characterized in that, comprise the following steps:
Induce cultivation: utilize derivant to be induced cultivation to samples of juice to be detected; Described derivant comprises vanillic acid and nutrient culture media, and described nutrient culture media is 402 nutrient culture media, YSG nutrient culture media, BSSA nutrient culture media, BAT nutrient culture media or KShi nutrient culture media.
Color developing detection: utilize developer and the samples of juice to be detected after inducing cultivation to carry out chromogenic reaction, occur in the reaction system after chromogenic reaction illustrating amber compound in samples of juice to be detected to contain and have a liking for sour thermoduric bacteria; Described developer comprises DNA enzyme and hydrogen peroxide.
The consumption of described vanillic acid and nutrient culture media is: l milliliter samples of juice to be detected need be used vanillic acid and 100 milliliters of nutrient culture media of 0.1～0.17 gram.
Described DNA enzyme comprises the Acetate-acetate buffer solution that single-chain nucleic acid sequence 5'-GTGGGTAGGGCGGGTTGG-3', protohemin, potassium chloride or ammonium chloride, sodium chloride, pH are 5.
Described hydrogen peroxide consumption is: the 4mM hydrogen peroxide: 50 μ L,
The component of described DNA enzyme is:
0.3 μ M single-chain nucleic acid sequence 5'-GTGGGTAGGGCGGGTTGG-3':50 μ L;
1 μ M protohemin: 50 μ L;
20mM Klorvess Liquid: 100 μ L;
150mM sodium chloride solution: 100 μ L;
The Acetate-acetate buffer solution that pH is 5: 600 μ L.
The described condition of cultivation of inducing is: 20～60 ℃, cultivate 13～15 hours.
The condition of described color developing detection is: 20～30 ℃, 15～45 minutes.
Pol in described samples of juice to be detected is 8～12 ° of Brix.
Further, the detection method of having a liking for sour thermoduric bacteria in fruit juice provided by the invention comprises the following steps:
(1) make and have a liking for the typical curve between sour thermoduric bacteria concentration and uv absorption intensity:
(2) measure the content of having a liking for sour thermoduric bacteria in samples of juice to be detected: comprise and utilize above-mentioned derivant to be induced cultivation to samples of juice to be detected, utilize above-mentioned developer and the samples of juice to be detected after inducing cultivation to carry out chromogenic reaction, detect the ultraviolet absorption value of coloring reaction system, with, read from typical curve the content of having a liking for sour thermoduric bacteria in samples of juice to be detected.
It is a kind of for detection of detecting the derivant of having a liking for sour thermoduric bacteria in fruit juice that another purpose of the present invention is to provide, and this derivant is a kind of in above-mentioned several derivant.
It is a kind of for detection of detecting the developer of having a liking for sour thermoduric bacteria in fruit juice that one of another purpose of the present invention is to provide, and what this developer was above-mentioned several developers is a kind of.
Compared with prior art, beneficial effect of the present invention is as follows:
The quick colorimetric detection method based on DNA enzyme (DNAzyme) that the present invention proposes, by building the technology of the bio-sensing based on DNA enzyme (DNAzyme), realize that the quantitative and qualitative analysis detection fast of this bacterium scene is had to higher sensitivity and specificity.
The accompanying drawing explanation
The metabolic chart of guaiacol when Fig. 1 is different bacteria concentration sample induced reaction;
Fig. 2 is the chromogenic reaction result that in embodiment 2, different bacteria concentrations are corresponding;
The absorbance collection of illustrative plates that Fig. 3 is the coloring reaction system that in embodiment 2, different bacteria concentration bacterium are corresponding;
The typical curve that Fig. 4 is embodiment 2.
Embodiment
DNA enzyme of the present invention refers to can be under acid condition, and catalyzing hydrogen peroxide oxidation guaiacol produces a kind of enzyme of amber compound four poly-guaiacol.
The present invention single-chain nucleic acid sequence 5'-GTGGGTAGGGCGGGTTGG-3' used can form G tetrad structure, and under the potassium chloride existence condition, this G tetrad can form DNA enzyme (DNAzyme) in conjunction with protohemin.This enzyme can the catalysis hydrogen peroxide and guaiacol between chemical reaction.
Nutrient culture media used in the present invention specifically can select 402 nutrient culture media, YSG nutrient culture media, BSSA nutrient culture media, BAT nutrient culture media, KShi nutrient culture media etc. to can be used for cultivating the nutrient culture media of having a liking for sour thermoduric bacteria cultivation.Wherein solid medium is for bacterium counting, and fluid nutrient medium is for the cultivation metabolism of bacterium.Wherein the formula of 402 nutrient culture media is as follows: CALCIUM CHLORIDE DIHYDRATE 0.25g, epsom salt 0.50g, ammonium sulfate 0.20g, potassium dihydrogen phosphate 3.00g, yeast extract 2.00g, glucose 5.00g; White vitriol 0.10g, tetrahydrate manganese chloride 0.03g, boric acid 0.30g, six water cobalt chloride 0.20g, copper chloride dihydrate 0.01g, Nickel dichloride hexahydrate 0.02g, sodium molybdate 0.03g; Sterilized water 1000mL;
Known in being added with the nutrient culture media of vanillic acid, have a liking for sour thermoduric bacteria and can utilize vanillic acid to carry out self metabolic activity, produce guaiacol.
Under acid condition, hydrogen peroxide oxidation guaiacol under the catalytic action of DNAzyme produces the poly-guaiacol of amber compound four.The poly-guaiacol of this amber compound four can detect by an unaided eye, specifically by observing amber with blank pipe (blank does not cause finally not having guaiacol to generate for adding vanillic acid).And the poly-guaiacol of this amber compound four is that the 420nm place has stronger UV, visible light optical absorption peak at wavelength, and the different uv absorption intensity of its content is also different.
Thus, can carry out chromogenic reaction by containing after the samples of juice of having a liking for sour thermoduric bacteria is cultivated under proper condition, then judge whether reaction system produces amber compound and realize having a liking for the quantitative detection of sour thermoduric bacteria.
The inventor, based on to above-mentioned mechanism, utilizes the DNAzyme technology to carry out fast detecting to the sour thermoduric bacteria of having a liking in fruit juice.
At first, the inventor considers how just to induce the sour thermoduric bacteria of having a liking in fruit juice to produce guaiacol, has designed the vanillic acid of Different adding amount as derivant, impels the sour thermoduric bacteria of having a liking in fruit juice can produce guaiacol and detected by the DNA enzyme in cultivation.The inventor finds when the vanillic acid addition is less than 0.1 gram (in every 100 milliliters of nutrient culture media), having a liking for sour thermoduric bacteria energy growth and breeding does not still have guaiacol to produce, and show that the vanillic acid that is less than 0.1 gram (in every 100 milliliters of nutrient culture media) can not be used as the derivant addition; When the vanillic acid addition is greater than 0.17 gram (in every 100 milliliters of nutrient culture media), having a liking for sour thermoduric bacteria can not can not produce guaiacol by growth and breeding, show that the vanillic acid that is greater than 0.17 gram (in every 100 milliliters of nutrient culture media) can not be used as the derivant addition; , have a liking for sour thermoduric bacteria energy normal growth and produce guaiacol when 0.1～0.17 gram (in every 100 milliliters of nutrient culture media) when the vanillic acid addition, can detect for the DNA enzyme.
In order further to determine specifically to induce, have a liking for the induced reaction time that sour thermoduric bacteria produces guaiacol, sterile working will be had a liking for sour thermoduric bacteria (DSM3922, be preserved in German DSMZ) be inoculated in liquid 402 nutrient culture media (in the 100mL nutrient culture media containing 0.17 gram vanillic acid as derivant), under 45 ℃ of shaken cultivation conditions, cultivate, in incubation at interval of 2h, the 1mL fluid nutrient medium is got in sterile working, the centrifugal 5min of 5000rpm, get supernatant, be determined at 270nm place ultraviolet absorption peak, make the dynamic monitoring figure of incubation time and guaiacol generation.As shown in Figure 1,0.17 gram vanillic acid as the derivant condition under, by cultivation being had a liking for to the whole dynamic process of sour thermoduric bacteria generation guaiacol, monitored, the present invention finds, after having a liking for sour thermoduric bacteria cultivation 14h, the sour thermoduric bacteria of having a liking for of different initial bacteria concentrations produces the obviously guaiacol of different amounts, and the guaiacol of the difference amount that this time point (14h) produces is enough to be judged by chromogenic reaction, for this reason, can carry out the qualitative and quantitative detection that difference is had a liking for sour thermoduric bacteria bacteria concentration according to coloration method provided by the invention.On the other hand, can guarantee to realize having a liking for the fast detecting of sour thermoduric bacteria within a short period of time, cultivation provided by the invention detects after having a liking for sour thermoduric bacteria 14h, is conducive to realize the demand of this fast detecting.Therefore, the present invention selects 13～15h as monitoring point.
The present invention utilizes developer to form the theoretical foundation of DNA enzyme, and (under appropraite condition, the DNA base sequence of G enrichment can the quadrantal structure of spontaneous formation G, and under the condition that has potassium ion, potassium ion contributes to stablize this G tetrad structure.Once add protohemin, its will be stable with potassium ion the combination of G tetrad, the final quadrantal DNA enzyme of protohemin-G-that forms), the concentration by regulation and control reaction buffer pH value (as Acetate-acetate buffer solution), protohemin and G enrichment sequence affects DNAzyme formation and catalytic reaction.Thereby select suitable DNAzyme catalysis regulation and control guaiacol-hydrogen peroxide reaction system, to utilize the rear ultraviolet absorption value that produces amber compound of reaction, the oxidation reaction effect of judgement DNAzyme catalysis guaiacol-hydrogen peroxide reaction system under different condition.
Below the specific embodiment that the inventor provides, so that technical scheme of the present invention is further explained to explanation.
Embodiment 1:
This embodiment is to having a liking for the qualitative detection of sour thermoduric bacteria in samples of juice.
The samples of juice to be detected of this embodiment is 70 ° of Brix concentrated apple juices for enough buying the pol of just having processed in fruit juice enterprise, and diluting this fruit juice to pol with sterilized water is 8-12 ° of Brix.
Derivant is: 100 milliliters of 402 nutrient culture media+0.17g vanillic acids.
Induce cultivation: 1 milliliter of samples of juice to be detected is added in derivant under 45 ℃ of conditions, after shaking table shaken cultivation 14h, with aseptic transfer pipet, gets inoculum 1mL, and the centrifugal 5min of 5000rpm, get supernatant.
Developer is: the hydrogen peroxide of the 4mM of DNAzyme and 50 μ L.Wherein DNAzyme adds the 0.3 μ M single-chain nucleic acid sequence (5'-GTGGGTAGGGCGGGTTGG-3') of 50 μ L (buying in giving birth to work bioengineering (Shanghai) incorporated company), the 1 μ M protohemin of 50 μ L, the 20mM Klorvess Liquid of 100 μ L, the 150mM sodium chloride solution of 100 μ L in the 25mM Acetate-acetate buffer solution (pH5.0) of 600 μ L to, hatch 30min, form DNAzyme.
Chromogenic reaction: the supernatant that 50 μ L obtain reacts 15min with developer.By visual inspection reaction system change color, by contrasting with the blank pipe, finding in reaction system has amber compound to generate, and illustrates in fruit juice to contain and has a liking for sour thermoduric bacteria afterwards.
Embodiment 2:
This embodiment is to having a liking for the quantitative detection of sour thermoduric bacteria in samples of juice.
The samples of juice to be detected of this embodiment, derivant, developer are identical with embodiment 1.
The typical curve between sour thermoduric bacteria concentration and uv absorption intensity is had a liking in making:
(1) prepare the test strain sample
Sterile working, will have a liking for sour thermoduric bacteria (DSM3922 is preserved in German DSMZ) inoculation in triangular flask, and under 45 ℃ of conditions, upper shaking table shaken cultivation 24h, as the test strain sample.
(2) prepare initial bacteria suspension
Sterile working, by the centrifugal 5min of test strain sample 5000rpm obtained in (1) step, after abandoning supernatant, mix thalline with the 10mL sterilized water, as bacteria suspension.
Sterile working, pipette bacteria suspension 1mL, puts into the test tube that the 9mL sterilized water is housed, and makes bacterium liquid fully vibrate and mix, and microbial cell is disperseed, standing 20～30s, 10-
1dilution; Use again the 1mL aseptic straw, draw 10
-1dilution 1mL, move into and to be equipped with in the test tube of 9mL sterilized water, and pressure-vaccum 3 times, allow bacterium liquid mix, 10
-2dilution; Change again an aseptic straw, draw 10
-2dilution 1mL, move into and to be equipped with in the test tube of 9mL sterilized water, and also pressure-vaccum is 3 times, 10
-3dilution; By that analogy, serial dilution, make 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, 10
-9etc. a series of dilution bacterium liquid.Draw respectively 10 with three 1ml aseptic straws again
-4, 10
-5, and 10
-6each 1ml of dilution bacteria suspension, check the number and to put into the aseptic plate of the number of finishing, each plate is put 0.2ml.Finally, flat-plate inverted is placed in to 45 ℃ of constant incubators and cultivates 24h, calculating bacteria suspension concentration is 10
8cfu/mL, 10
-8dilution bacterium liquid is initial bacteria suspension.
(3) different bacteria concentration sample preparations
Sterile working pipettes initial bacteria suspension 1mL, puts into the test tube that the 9mL sterilized water is housed, and makes bacterium liquid fully vibrate and mix, and microbial cell is disperseed, standing 20～30s, 10
-1dilution; Use again the 1mL aseptic straw, draw 10
-1dilution 1mL, move into and to be equipped with in the test tube of 9mL sterilized water, and pressure-vaccum 3 times, allow bacterium liquid mix, 10
-2dilution; Change again an aseptic straw, draw 10
-2dilution 1mL, move into and to be equipped with in the test tube of 9mL sterilized water, and also pressure-vaccum is 3 times, 10
-3dilution; By that analogy, serial dilution, make 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, 10
-9etc. a series of dilution bacterium liquid.Obtain 10
5cfu/mL, 10
4cfu/mL, 10
3cfu/mL, 10
2the cfu/mL bacteria concentration.The number of finishing respectively, standby.
(4) induced reaction
Draw respectively 10 with the 1mL aseptic straw
5cfu/mL, 10
4cfu/mL, 10
3cfu/mL, 10
2each 1mL of a series of dilution such as cfu/mL bacterium liquid, add to respectively in the triangular flask that the equivalent derivant is housed, and under 45 ℃ of conditions, carries out this bacterium shaking table and cultivate, and cultivates 14h.
(5) chromogenic reaction
Get the have a liking for sour thermoduric bacteria centrifuged supernatant of 50 μ L after induced reaction and add in developer, hatch 45min, observe the change color of different bacteria concentration samples.As shown in Figure 2, the sour thermoduric bacteria of having a liking for of different initial concentrations finally produces and obviously is different from the amber of blank in the DNAzyme reaction system, and variable concentrations initially to add the sour thermoduric bacteria of having a liking for of bacteria concentration to produce color different.
(6) absorbance detection
Detect the absorbance of different bacteria concentration sample coloring reaction systems, testing result as shown in Figure 3.
What take variable concentrations in this embodiment step (1) has a liking for sour thermoduric bacteria as horizontal ordinate, and its corresponding coloring reaction system ultraviolet absorption value is ordinate, the production standard curve, as shown in Figure 4.
Detect the absorbance of the coloring reaction system in embodiment 1, the content that utilizes this absorbance to look on typical curve shown in Fig. 4 to get bacterium in samples of juice to be detected is 112cfu/mL.
Comparative Examples:
This Comparative Examples is to adopt traditional microbe growth separation method to be detected the samples of juice in embodiment 1.
(1) Liquid Culture of initial fruit juice actual sample
Under aseptic technique, pipette the 1mL samples of juice with aseptic transfer pipet, add in liquid 402 nutrient culture media, under 45 ℃ of conditions, after upper shaking table shaken cultivation 18h, the centrifugal 5min of 5000rpm, abandoning supernatant, now use thalline in 10mL sterilized water dilution tube, makes bacteria suspension standby.
(2) bacteria suspension solid culture
Sterile working pipettes initial bacteria suspension 1mL, puts into the test tube that the 9mL sterilized water is housed, and makes bacterium liquid fully vibrate and mix, and microbial cell is disperseed, standing 20～30s, 10
-1dilution; Use again the 1mL aseptic straw, draw 10
-1dilution 1mL, move into and to be equipped with in the test tube of 9mL sterilized water, and pressure-vaccum 3 times, allow bacterium liquid mix, 10
-2dilution; Change again an aseptic straw, draw 10
-2dilution 1mL, move into and to be equipped with in the test tube of 9mL sterilized water, and also pressure-vaccum is 3 times, 10
-3dilution; By that analogy, serial dilution, make 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, 10
-9etc. a series of dilution bacterium liquid.Draw respectively 10 with three 1ml aseptic straws again
-4, 10
-5, and 10
-6each 1ml of dilution bacteria suspension, check the number and to put into the aseptic plate of the number of finishing, each plate is put 0.2ml.Finally, flat-plate inverted is placed in to 45 ℃ of constant incubators and cultivates 24h, observe the colony morphology characteristic in flat board.
(3) result is judged
In plate, there is single bacterium colony occur and occur that bacterium colony is larger, the smooth of the edge, projection, thickness, easily provoke, and is the colony characteristics of wire drawing shape, judges in fruit juice to contain and have a liking for sour thermoduric bacteria, consistent with the testing result of embodiment 1.
Claims (10)
1. have a liking for the detection method of sour thermoduric bacteria in fruit juice, it is characterized in that, comprise the following steps:
Induce cultivation: utilize derivant to be induced cultivation to samples of juice to be detected; Described derivant comprises vanillic acid and nutrient culture media, and described nutrient culture media is 402 nutrient culture media, YSG nutrient culture media, BSSA nutrient culture media, BAT nutrient culture media or KShi nutrient culture media.
Color developing detection: utilize developer and the samples of juice to be detected after inducing cultivation to carry out chromogenic reaction, illustrate in samples of juice to be detected to contain while in reaction system, amber compound occurring and have a liking for sour thermoduric bacteria; Described developer comprises DNA enzyme and hydrogen peroxide.
2. have a liking for the detection method of sour thermoduric bacteria in fruit juice as claimed in claim 1, it is characterized in that, the consumption of described vanillic acid and nutrient culture media is: l milliliter samples of juice to be detected need be used vanillic acid and 100 milliliters of nutrient culture media of 0.1～0.17 gram.
3. have a liking for the detection method of sour thermoduric bacteria in fruit juice as claimed in claim 1, it is characterized in that, described DNA enzyme comprises the Acetate-acetate buffer solution that single-chain nucleic acid sequence 5'-GTGGGTAGGGCGGGTTGG-3', protohemin, potassium chloride or ammonium chloride, sodium chloride, pH are 5.
4. have a liking for the detection method of sour thermoduric bacteria in fruit juice as claimed in claim 1, it is characterized in that,
Described hydrogen peroxide consumption is: the 4mM hydrogen peroxide: 50 μ L,
The component of described DNA enzyme is:
0.3 μ M single-chain nucleic acid sequence 5'-GTGGGTAGGGCGGGTTGG-3':50 μ L;
1 μ M protohemin: 50 μ L;
20mM Klorvess Liquid: 100 μ L;
150mM sodium chloride solution: 100 μ L;
The Acetate-acetate buffer solution that pH is 5: 600 μ L.
5. have a liking for the detection method of sour thermoduric bacteria in fruit juice as claimed in claim 1, it is characterized in that, the described condition of cultivation of inducing is: 20～60 ℃, cultivate 13～15 hours.
6. have a liking for the detection method of sour thermoduric bacteria in fruit juice as claimed in claim 1, it is characterized in that, the condition of described color developing detection is: 20～30 ℃, 15～45 minutes.
7. have a liking for the detection method of sour thermoduric bacteria in fruit juice as claimed in claim 1, it is characterized in that, the pol in described samples of juice to be detected is 8～12 ° of Brix.
8. have a liking for the detection method of sour thermoduric bacteria in fruit juice, it is characterized in that, method comprises the following steps:
(1) make and have a liking for the typical curve between sour thermoduric bacteria concentration and uv absorption intensity:
(2) measure the content of having a liking for sour thermoduric bacteria in samples of juice to be detected: comprise and utilize the described derivant of claim 1 or 2 to be induced cultivation to samples of juice to be detected, utilize claim 1,3 or 4 described developers to carry out chromogenic reaction with the samples of juice to be detected after inducing cultivation, detect the ultraviolet absorption value of coloring reaction system, with, read from typical curve the content of having a liking for sour thermoduric bacteria in samples of juice to be detected.
9. one kind for detection of detecting the derivant have a liking for sour thermoduric bacteria in fruit juice, it is characterized in that, this derivant is a kind of in claim 1 and 2 described derivants.
10. one kind for detection of detecting the developer have a liking for sour thermoduric bacteria in fruit juice, it is characterized in that, this developer is a kind of in claim 1,3 and 4 described developers.
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| CN104614531A (en) * | 2015-02-09 | 2015-05-13 | 甘肃出入境检验检疫局检验检疫综合技术中心 | Alicyclobacillus colloidal gold label test strip and preparation method thereof |
| CN109868304A (en) * | 2017-12-04 | 2019-06-11 | 沈阳药科大学 | Glucose oxidase-DNAzyme composite hydrogel and its application in glucose detection |
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