CN112113948A - Rapid detection method for pathogenic microorganisms - Google Patents
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- CN112113948A CN112113948A CN202010777142.4A CN202010777142A CN112113948A CN 112113948 A CN112113948 A CN 112113948A CN 202010777142 A CN202010777142 A CN 202010777142A CN 112113948 A CN112113948 A CN 112113948A
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- 244000000010 microbial pathogen Species 0.000 title claims abstract description 50
- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 37
- 108091023037 Aptamer Proteins 0.000 claims abstract description 30
- 238000001069 Raman spectroscopy Methods 0.000 claims abstract description 20
- 239000002105 nanoparticle Substances 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 33
- 239000000047 product Substances 0.000 claims description 23
- 239000011324 bead Substances 0.000 claims description 22
- 244000005700 microbiome Species 0.000 claims description 18
- 241000191967 Staphylococcus aureus Species 0.000 claims description 16
- 238000012360 testing method Methods 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 12
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(I) nitrate Inorganic materials [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 10
- 230000008859 change Effects 0.000 claims description 8
- 238000011534 incubation Methods 0.000 claims description 8
- 239000002245 particle Substances 0.000 claims description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 5
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- 239000012279 sodium borohydride Substances 0.000 claims description 5
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 239000006185 dispersion Substances 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 241000186779 Listeria monocytogenes Species 0.000 claims description 3
- 241000607142 Salmonella Species 0.000 claims description 3
- 108010090804 Streptavidin Proteins 0.000 claims description 3
- 229960002685 biotin Drugs 0.000 claims description 3
- 235000020958 biotin Nutrition 0.000 claims description 3
- 239000011616 biotin Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 241000193755 Bacillus cereus Species 0.000 claims description 2
- 241001135265 Cronobacter sakazakii Species 0.000 claims description 2
- 241000607272 Vibrio parahaemolyticus Species 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 238000007885 magnetic separation Methods 0.000 claims description 2
- 238000004445 quantitative analysis Methods 0.000 claims description 2
- 101710134784 Agnoprotein Proteins 0.000 claims 1
- 238000004321 preservation Methods 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 9
- 238000005516 engineering process Methods 0.000 abstract description 4
- 238000011065 in-situ storage Methods 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000000926 separation method Methods 0.000 abstract description 4
- 238000010276 construction Methods 0.000 abstract description 2
- 230000001404 mediated effect Effects 0.000 abstract 1
- 239000002131 composite material Substances 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 238000004611 spectroscopical analysis Methods 0.000 description 5
- 208000019331 Foodborne disease Diseases 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000001237 Raman spectrum Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000003368 label free method Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000013077 target material Substances 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- Life Sciences & Earth Sciences (AREA)
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- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a rapid detection method of pathogenic microorganisms, which comprises the following steps: unwinding an aptamer into a single chain, preparing magnetic bead-aptamer-target microorganism @ Ag, drawing a standard working curve, and detecting a sample to be detected. The rapid detection of pathogenic microorganisms is realized by means of a portable Raman spectrometer by combining a rapid and efficient magnetic capture separation technology with Ag nano particles in-situ enhanced pathogenic microorganism intrinsic Raman signals synthesized by an aptamer in a mediated mode. The sensor construction process and the detection process do not need large-scale instruments, the operation is simple, the whole process can be completed within 1h, the sensitivity is high, and the selectivity is good.
Description
Technical Field
The invention relates to the technical field of microorganism detection, in particular to a pathogenic microorganism unmarked SERS detection method constructed by Ag nano particles synthesized in situ on the surfaces of aptamers and aptamers based on a magnetic capture separation technology.
Background
Pathogenic microorganisms are the leading cause of food-borne diseases, which not only seriously harm people's life and health, but also cause a great deal of economic loss. Therefore, in order to reduce or control the occurrence of food-borne diseases and to reduce or avoid serious influences and damages caused by the food-borne diseases, it is very necessary to detect the content of pathogenic microorganisms as soon as possible before the food or agricultural products enter the market circulation.
Common pathogenic microorganisms in food and agricultural products mainly comprise staphylococcus aureus, salmonella, escherichia coli, listeria monocytogenes and the like, and common detection methods mainly comprise three major methods, namely a plate colony counting method, a PCR method and an ELISA method. The culture method is used as a gold-labeled method for detecting microorganisms, and although the result is accurate, the method usually needs to be subjected to links such as separation, identification, culture, counting and the like, is complex to operate and time-consuming, and generally needs 3-5 days. The PCR method and the ELISA method have the disadvantages of greatly shortened detection time, dependence on expensive special instruments and professional technicians, complicated steps, high requirements on experimental environment and easy occurrence of false positive or false negative in detection results. In conclusion, the methods all have difficulty in meeting the urgent need of the rapid field detection of pathogenic microorganisms at present, and a novel pathogenic microorganism detection method which is simple, rapid, cheap, accurate and highly sensitive is urgently needed to be developed.
The surface enhanced raman scattering spectroscopy (SERS) technology has the advantages of being simple and rapid in operation, narrow in spectral peak, high in sensitivity, small in influence of water, capable of providing molecular fingerprint information, portable in instruments and the like, and has recently become a research hotspot for detecting pathogenic microorganisms. SERS-based detection of pathogenic microorganisms typically involves both labeling and unlabeling methods. The former requires modification of additional signal molecules, and usually requires immunological means for detection of microorganisms, and the whole process is relatively complicated. The label-free method reflects intrinsic Raman spectrum information of microorganisms, is also called as a direct method, and is favored in the field of field detection due to relatively few steps.
Disclosure of Invention
The invention provides a rapid detection method of pathogenic microorganisms, which utilizes a magnetic capture separation technology and aptamer mediation to synthesize Ag nano particles on the surfaces of the microorganisms in situ, enhances intrinsic Raman signals of the pathogenic microorganisms and realizes simple, rapid and accurate detection of the pathogenic microorganisms.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a rapid detection method for pathogenic microorganisms comprises the following steps:
s1, performing denaturation treatment on the aptamer solution of the pathogenic microorganism to be detected to ensure that double chains in the aptamer structure are uncoiled into single chains;
s2, incubating the treated aptamer solution with a series of pathogenic microorganisms to be detected with known concentrations for a period of time, then adding magnetic beads for incubation, carrying out magnetic separation and washing, resuspending the obtained product, and storing, wherein the obtained product is marked as magnetic bead-aptamer-target microorganism;
s3, magnetically separating and washing the product of S2, and then suspending the product to AgNO3Incubating in solution, magnetically separating, washing, removing supernatant, and resuspending to NaBH4Reacting in the solution, storing the obtained product in a dark place, and recording the obtained product as magnetic bead-aptamer-target microorganism @ Ag;
s4, taking the product obtained in the step S3 by using a capillary tube, carrying out Raman test in an external magnetic field, and drawing a quantitative analysis standard working curve by taking the logarithm of the concentration of the pathogenic microorganism as a horizontal coordinate and the intensity change value of one characteristic Raman peak of the pathogenic microorganism as a vertical coordinate; the intensity change value refers to the intensity of the Raman signal of the system relative to a blank value, namely the intensity of the Raman signal of the system when no pathogenic microorganism exists;
s5, preparing the pathogenic microorganism sample to be detected with unknown concentration into magnetic beads, aptamer, target microorganism and @ Ag, detecting the intensity change value of the characteristic Raman peak by adopting the method S4, and calculating the concentration of the pathogenic microorganism to be detected in the sample according to the working curve.
Preferably, the 5' -end of the aptamer of step S1 is modified with biotin; s2, modifying streptavidin on the surface of the magnetic bead, wherein the particle size of the magnetic bead is 30-100 nm.
Preferably, the aptamer denaturation treatment method in step S1 specifically comprises: heating at 90-95 deg.C for 2-5 min.
Preferably, the incubation conditions of the aptamer solution and the pathogenic microorganism in step S2 are as follows: incubating for 20-30 min at 4 ℃; the mass concentration of the magnetic beads is 30mg/mL, the volume of the magnetic beads is 5-20 mu L, the dispersion liquid of the magnetic beads is PB buffer solution, and the incubation time after the magnetic beads are added is 20-30 min;
preferably, the AgNO3The concentration of the solution is 10mM, the volume is 50-400 mu L, and the solution is oscillated for 15-20 min at room temperature; NaBH4The concentration of the solution is 0.1mM, the volume is 100-400 mu L, and the solution is shaken for 15-20 min at room temperature.
Preferably, in the product magnetic bead-aptamer-pathogenic microorganism @ Ag, the particle size of Ag nanoparticles is 5-20 nm.
Preferably, the solvent of the aptamer solution in step S1 is PB buffer or deionized water, the dispersion of the pathogenic microorganism in step S2 is PB buffer, both solutions used for washing and resuspension in step S2 are PB buffer, and the solution used for washing in step S3 is deionized water. More preferably, the concentration of the PB buffer solution is 0.01-0.1M, and the pH value is 7.0-7.4.
Preferably, the raman test conditions in step S4 are: the laser wavelength is 633nm, the exposure time is 5-10 s, and the average measurement is 3-5 times.
Preferably, the pathogenic microorganism is one of staphylococcus aureus, salmonella, escherichia coli, listeria monocytogenes, vibrio parahaemolyticus, bacillus cereus and enterobacter sakazakii.
The invention has the beneficial effects that:
(1) the aptamer is denatured, de-spun into a single chain and then acted with the target microorganism, so that the aptamer can be better wound around the target microorganism, and the Ag nano particles synthesized in the later period are more close to the surface of the target microorganism, so that the intrinsic signal of the microorganism is better enhanced, and the detection sensitivity of the method is improved; (2) the aptamer is adopted to firstly identify the microorganism and then is captured by the magnetic beads under the action of biotin and streptavidin, so that not only is the full action between the aptamer and the microorganism preferentially ensured and the microorganism not influenced by the steric hindrance of the magnetic beads, but also the problem of reduced identification capability caused by chemical bonding modification of the aptamer on the magnetic beads is effectively avoided, and the capture efficiency of the magnetic beads is indirectly improved; (3) the use of the magnetic beads in the system not only simplifies the operation process and avoids the use of a centrifuge, but also has excellent enrichment performance and is beneficial to further enhancing the intrinsic signals of microorganisms during Raman testing; (4) the Ag nano particles are synthesized in situ on the surfaces of pathogenic microorganisms by adopting aptamer mediation to enhance the intrinsic Raman signals of the microorganisms to construct an SERS detection platform, so that not only is the modification of additional signal molecules removed and the operation flow simplified, but also the detection sensitivity of the method can be greatly improved by combining the excellent enrichment capacity of magnetic beads, and the lowest detectable concentration of the pathogenic microorganisms can be as low as 100 CFU/mL; (5) the detection method provided by the invention does not need to depend on large-scale instruments and professional operators in the using process, is simple and rapid to operate, controls the whole process within 1h, and is expected to be widely applied to rapid quantitative detection of pathogenic microorganisms in small and medium-sized enterprises and primary laboratories.
Detailed Description
The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention, but are merely illustrative.
Example 1
Taking staphylococcus aureus as an example, a construction process for quickly detecting staphylococcus aureus is provided.
1. Preparation of magnetic bead-aptamer-staphylococcus aureus @ Ag composite structure
(1) Denaturation of the aptamer:
the sequence of the selected staphylococcus aureus aptamer is as follows: 5' -Biotin- (CH)2)6-TCCCT ACGGC GCTAA CCTCC CAACC GCTCC ACCCT GCCTC CGCCT CGCCA CCGTG CTACA AC-3’。
The above aptamer was dispersed in PB solution (0.01M, pH7.4), then heat treated at 95 ℃ for 2min, allowed to cool naturally, and finally stored at-20 ℃ in a refrigerator.
(2) Synthesis of magnetic bead-aptamer-staphylococcus aureus composite structure
1mL Staphylococcus aureus (10)7CFU/mL, PB (0.01M, pH7.4) added with 500nM aptamer solution, mixed well, incubated at 4 ℃ for 20 min; then, 10. mu.L of streptavidin-functionalized magnetic beads (30mg/mL, particle size about 50nm) were added, and incubation was performed at room temperature for 30min with shaking; and finally, washing the product for 2-3 times by using the PB solution, suspending the product into 100 mu L of PB solution, and storing the product for later use at 4 ℃ in a refrigerator.
(3) Synthesis of magnetic bead-aptamer-staphylococcus aureus @ Ag composite structure
Adding 10 mu L of the magnetic bead-aptamer-staphylococcus aureus solution synthesized in the step (2) into 90 mu L of PB solution, washing the solution for 2 times by deionized water, and then resuspending the solution to 100 mu L of 0.01M AgNO3After shaking vigorously at room temperature for 20min, washed 2 times with deionized water, supernatant removed, and resuspended to 100. mu.L of 0.1mM NaBH4After further shaking for 15min, the product was stored in a refrigerator at 4 ℃ in the dark.
And (3) testing the products obtained in the steps (2) and (3), and finding that the staphylococcus aureus surface in the composite structure synthesized in the step (3) is loaded with a plurality of particles with the particle size of less than 20nm besides the loaded magnetic beads, and the EDS test shows that the composition of the composite structure is silver, which indicates that the target material is successfully synthesized.
2. Rapid detection process for staphylococcus aureus
And (2) taking 10 mu L of the magnetic bead-aptamer-staphylococcus aureus @ Ag obtained in the step (1) to a capillary, and carrying out Raman test under the action of an external magnet. And (3) testing conditions are as follows: laser wavelength 633nm, exposure time 10s, average measurement 3 times. Record it at 735cm-1The intensity change value of the Raman peak (the peak is one of the characteristic peaks of the microbial cell membrane). Then, a series of staphylococcus aureus with known concentration is used for synthesizing a corresponding magnetic bead-aptamer-staphylococcus aureus @ Ag compositeStructure; then, testing according to the Raman testing conditions; finally according to the concentration of staphylococcus aureus and 735cm-1And drawing a corresponding quantitative standard working curve according to the change condition of the Raman peak-to-peak intensity, wherein the concentration of the detected staphylococcus aureus can be known to be as low as 100CFU/mL according to the standard working curve.
Example 2
Testing of staphylococcus aureus mimetic samples
First, normal temperature milk (3000rpm, 5min) purchased from a supermarket is centrifuged to remove impurities. It was then treated with 10-fold dilution with PB solution. 1000 and 10000CFU/mL of standard Staphylococcus aureus solution were added thereto, and the mixture was mixed well to perform the detection of Staphylococcus aureus as shown in example 1. By measuring it at 735cm-1The change values of the raman peak intensities were calculated from the working curves obtained in example 1 to find that the concentrations of staphylococcus aureus in the mock samples were 968.2 and 10121CFU/mL, respectively, and further, the recovery rates of the two samples were 96.82% and 101.21%, respectively. Therefore, the method can be verified to have good feasibility and accuracy in detection in actual samples.
The above examples are merely illustrative of the present invention, but the embodiments of the present invention are not limited by the above examples. Various other changes and modifications to the above-described embodiments and concepts will become apparent to those skilled in the art from the above description, and all such changes and modifications are intended to be included within the scope of the present invention as defined in the appended claims.
Sequence listing
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Claims (10)
1. A rapid detection method for pathogenic microorganisms is characterized by comprising the following steps:
s1, performing denaturation treatment on the aptamer solution of the pathogenic microorganism to be detected to ensure that double chains in the aptamer structure are uncoiled into single chains;
s2, incubating the treated aptamer solution with a series of pathogenic microorganisms to be detected with known concentrations for a period of time, then adding magnetic beads for incubation, finally carrying out magnetic separation and washing, and carrying out resuspension and preservation on the product; the obtained product is marked as magnetic bead-aptamer-target microorganism;
s3, magnetically separating and washing the product of S2, and then suspending the product to AgNO3Incubating in solution, magnetically separating, washing, removing supernatant, and resuspending to NaBH4Reacting in the solution to obtain a product magnetic bead-aptamer-target microorganism @ Ag, and storing the product in a dark place;
s4, taking the product obtained in the step S3 by using a capillary tube, carrying out Raman test in an external magnetic field, and drawing a quantitative analysis standard working curve by taking the logarithm of the concentration of the pathogenic microorganism as a horizontal coordinate and the intensity change value of one characteristic Raman peak of the pathogenic microorganism as a vertical coordinate;
s5, preparing the pathogenic microorganism sample to be detected with unknown concentration into magnetic beads, aptamer, target microorganism and @ Ag, detecting the intensity change value of the characteristic Raman peak by adopting the method S4, and calculating the concentration of the pathogenic microorganism to be detected in the sample according to the working curve.
2. The method for rapidly detecting pathogenic microorganisms according to claim 1, wherein biotin is modified at the 5' end of the aptamer of step S1; s2, modifying streptavidin on the surface of the magnetic bead, wherein the particle size of the magnetic bead is 30-100 nm.
3. The method for rapidly detecting pathogenic microorganisms according to claim 1, wherein the method for denaturing the aptamer of step S1 specifically comprises: heating at 90-95 deg.C for 2-5 min.
4. The method for rapidly detecting pathogenic microorganisms according to claim 1, wherein the incubation conditions of the aptamer solution and the pathogenic microorganisms in the step S2 are as follows: incubating for 20-30 min at 4 ℃; the dispersion liquid of the magnetic beads is a PB buffer solution, the incubation time after the magnetic beads are added is 20-30 min, and the incubation temperature is 20-25 ℃.
5. The method for rapidly detecting pathogenic microorganisms according to claim 1, wherein the AgNO is AgNO3The concentration of the solution is 10mM, the volume is 50-400 mu L, and the solution is oscillated for 15-20 min at room temperature; the NaBH4The concentration of the solution is 0.1mM, the volume is 100-400 mu L, and the solution is shaken for 15-20 min at room temperature.
6. The method for rapidly detecting the pathogenic microorganisms according to claim 1, wherein in the product magnetic bead-aptamer-pathogenic microorganism @ Ag, the particle size of Ag nanoparticles is 5-20 nm.
7. The method for rapidly detecting pathogenic microorganisms according to claim 1, wherein the dispersion liquid of pathogenic microorganisms in step S2 is PB buffer, the solutions for washing and resuspending in step S2 are both PB buffer, and the solution for washing in step S3 is deionized water.
8. The method for rapidly detecting pathogenic microorganisms according to claim 4 or 7, wherein the concentration of the PB buffer solution is 0.01-0.1M, and the pH is 7.0-7.4.
9. The method for rapidly detecting pathogenic microorganisms according to claim 1, wherein the conditions of the raman test in step S4 are as follows: the laser wavelength is 633nm, the exposure time is 5-10 s, and the average measurement is 3-5 times.
10. The method for rapidly detecting the pathogenic microorganisms according to claim 1, wherein the pathogenic microorganisms are one of staphylococcus aureus, salmonella, escherichia coli, listeria monocytogenes, vibrio parahaemolyticus, bacillus cereus and enterobacter sakazakii.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719353A (en) * | 2012-06-13 | 2012-10-10 | 湖南大学 | Device and method for capturing specificity of circulating cancer cells in peripheral blood |
| CN104597240A (en) * | 2015-02-02 | 2015-05-06 | 广西医科大学 | Biosensing method for detecting leukemia by graphene/mimetic peroxidase double-signal amplification |
| CN105779398A (en) * | 2015-01-12 | 2016-07-20 | 北京亿森宝生物科技有限公司 | Immunomagnetic bead purification kit and method for 12 kinds of swine common viruses and germs |
| CN107238699A (en) * | 2017-05-10 | 2017-10-10 | 江南大学 | A kind of colorimetric methods that magnetic bead and gold nano grain analogue enztme activity detection kanamycins are modified based on aptamers |
| CN107462704A (en) * | 2017-09-21 | 2017-12-12 | 清华大学深圳研究生院 | A kind of biology sensor and preparation method thereof, concentration of target molecules detection method |
| CN108072643A (en) * | 2017-12-28 | 2018-05-25 | 厦门大学 | A kind of target detection method and system based on digital microfluidic technology and Surface enhanced Raman scattering technology |
| CN109813701A (en) * | 2019-04-10 | 2019-05-28 | 徐州医科大学附属医院 | A method of quickly without label surface enhancing Raman scattering detection staphylococcus aureus and escherichia coli |
| CN111024938A (en) * | 2019-11-27 | 2020-04-17 | 武汉市农业科学院 | Rapid detection method for pathogenic microorganisms |
-
2020
- 2020-08-05 CN CN202010777142.4A patent/CN112113948A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719353A (en) * | 2012-06-13 | 2012-10-10 | 湖南大学 | Device and method for capturing specificity of circulating cancer cells in peripheral blood |
| CN105779398A (en) * | 2015-01-12 | 2016-07-20 | 北京亿森宝生物科技有限公司 | Immunomagnetic bead purification kit and method for 12 kinds of swine common viruses and germs |
| CN104597240A (en) * | 2015-02-02 | 2015-05-06 | 广西医科大学 | Biosensing method for detecting leukemia by graphene/mimetic peroxidase double-signal amplification |
| CN107238699A (en) * | 2017-05-10 | 2017-10-10 | 江南大学 | A kind of colorimetric methods that magnetic bead and gold nano grain analogue enztme activity detection kanamycins are modified based on aptamers |
| CN107462704A (en) * | 2017-09-21 | 2017-12-12 | 清华大学深圳研究生院 | A kind of biology sensor and preparation method thereof, concentration of target molecules detection method |
| CN108072643A (en) * | 2017-12-28 | 2018-05-25 | 厦门大学 | A kind of target detection method and system based on digital microfluidic technology and Surface enhanced Raman scattering technology |
| CN109813701A (en) * | 2019-04-10 | 2019-05-28 | 徐州医科大学附属医院 | A method of quickly without label surface enhancing Raman scattering detection staphylococcus aureus and escherichia coli |
| CN111024938A (en) * | 2019-11-27 | 2020-04-17 | 武汉市农业科学院 | Rapid detection method for pathogenic microorganisms |
Non-Patent Citations (3)
| Title |
|---|
| CHEN, J等: "Jellylike flexible nanocellulose SERS substrate for rapid in-situ non-invasive pesticide detection in fruits/vegetables", 《CARBOHYDRATE POLYMERS》, vol. 205, 1 February 2019 (2019-02-01), pages 596 - 600 * |
| 卢向阳: "《分子生物学》", 31 January 2004, 中国农业出版社, pages: 67 - 71 * |
| 白向茹: "复杂体系中高可靠性SERS检测新策略及传感分析应用", 《中国博士学位论文全文数据库工程科技Ⅰ辑》, no. 12, 15 December 2018 (2018-12-15), pages 014 - 254 * |
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