CN107488718A - Detect C60Nanoparticles generate the method and device influenceed to Microcystin - Google Patents
Detect C60Nanoparticles generate the method and device influenceed to Microcystin Download PDFInfo
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- CN107488718A CN107488718A CN201710815783.2A CN201710815783A CN107488718A CN 107488718 A CN107488718 A CN 107488718A CN 201710815783 A CN201710815783 A CN 201710815783A CN 107488718 A CN107488718 A CN 107488718A
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- microcystin
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- frustule
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- SRUWWOSWHXIIIA-UKPGNTDSSA-N Cyanoginosin Chemical compound N1C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](C)[C@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C(=C)N(C)C(=O)CC[C@H](C(O)=O)N(C)C(=O)[C@@H](C)[C@@H]1\C=C\C(\C)=C\[C@H](C)[C@@H](O)CC1=CC=CC=C1 SRUWWOSWHXIIIA-UKPGNTDSSA-N 0.000 title claims abstract description 99
- 108010067094 microcystin Proteins 0.000 title claims abstract description 99
- 238000000034 method Methods 0.000 title claims abstract description 42
- 239000002105 nanoparticle Substances 0.000 claims abstract description 82
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 80
- 239000003053 toxin Substances 0.000 claims abstract description 78
- 231100000765 toxin Toxicity 0.000 claims abstract description 71
- 108700012359 toxins Proteins 0.000 claims abstract description 71
- 241000195493 Cryptophyta Species 0.000 claims abstract description 62
- 238000001514 detection method Methods 0.000 claims abstract description 56
- 235000015097 nutrients Nutrition 0.000 claims abstract description 53
- 241000700605 Viruses Species 0.000 claims abstract description 34
- 239000006228 supernatant Substances 0.000 claims abstract description 32
- 241000192700 Cyanobacteria Species 0.000 claims abstract description 29
- 238000004519 manufacturing process Methods 0.000 claims abstract description 29
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- 239000000243 solution Substances 0.000 claims description 56
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- 239000002299 complementary DNA Substances 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 20
- 238000012408 PCR amplification Methods 0.000 claims description 19
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 18
- 238000005119 centrifugation Methods 0.000 claims description 15
- 230000004087 circulation Effects 0.000 claims description 15
- 239000002953 phosphate buffered saline Substances 0.000 claims description 13
- 108010049746 Microcystins Proteins 0.000 claims description 12
- 230000003834 intracellular effect Effects 0.000 claims description 12
- 239000000047 product Substances 0.000 claims description 12
- 238000010839 reverse transcription Methods 0.000 claims description 12
- 241000192710 Microcystis aeruginosa Species 0.000 claims description 11
- 238000011529 RT qPCR Methods 0.000 claims description 11
- 238000000605 extraction Methods 0.000 claims description 11
- 238000010790 dilution Methods 0.000 claims description 10
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- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
- 238000000746 purification Methods 0.000 claims description 8
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- 102000004190 Enzymes Human genes 0.000 claims description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 4
- 238000000137 annealing Methods 0.000 claims description 4
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 4
- 230000001900 immune effect Effects 0.000 claims description 4
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
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- 239000011780 sodium chloride Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
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- -1 4 μ L Substances 0.000 description 2
- 241001062009 Indigofera Species 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
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- 239000000499 gel Substances 0.000 description 2
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- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 210000003705 ribosome Anatomy 0.000 description 2
- 235000015170 shellfish Nutrition 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 208000003443 Unconsciousness Diseases 0.000 description 1
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- 239000002775 capsule Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- NWFNSTOSIVLCJA-UHFFFAOYSA-L copper;diacetate;hydrate Chemical compound O.[Cu+2].CC([O-])=O.CC([O-])=O NWFNSTOSIVLCJA-UHFFFAOYSA-L 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000000254 damaging effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000012776 electronic material Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 231100000784 hepatotoxin Toxicity 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000008239 natural water Substances 0.000 description 1
- VIKNJXKGJWUCNN-XGXHKTLJSA-N norethisterone Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](C)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 VIKNJXKGJWUCNN-XGXHKTLJSA-N 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
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- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 244000038651 primary producers Species 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
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- 235000009566 rice Nutrition 0.000 description 1
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Classifications
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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Abstract
The invention discloses one kind to detect C60Nanoparticles generate the method and device influenceed to Microcystin, and this method includes：The C of various concentrations is added in the blue-green algae nutrient solution of producing microcystic toxins60Nanoparticles are cultivated；Take the C of various concentrations60The blue-green algae nutrient solution of nanoparticles culture, frustule is collected, and then detect the production virus gene of the Microcystin in testing sample mcyD gene copy number；Take the C of various concentrations60The blue-green algae nutrient solution of the producing microcystic toxins of nanoparticles culture, collect supernatant, the content of detection wherein Microcystin；Correlation analysis is carried out to the result of above-mentioned two step, establishes various concentrations C60The relation of Algae toxins yield and mcyD gene copy number in the blue-green algae of nanoparticles culture；Using the relation of Algae toxins yield and mcyD gene copy number, the growing amount of Microcystin under different condition of culture is determined by determining mcyD gene copy number.
Description
Technical field
The present invention relates to one kind to detect C60Nanoparticles generate the method and device influenceed on Microcystin, particularly
It is related to a kind of Algae toxins production virus gene detection C60Nanoparticles generate the method and device influenceed to Microcystin.
Background technology
Microcystic aeruginosa is blue-green algae most commonly seen in water body, and it is the primary producer in water body, is sent out in water environment
Wave important effect.Microcystic aeruginosa can produce Microcystin (microcystin, MC), and it is a kind of widow of ring-type
Peptide hepatotoxin, Microcystin can be detected in the natural water body of many countries in the whole world.MC influences phytoplankton, without ridge
Vertebrate and the growth of vertebrate.MC can cause water Endophytic bacteria, protozoan and other phytoplanktons (including diatom and
Green alga) poisoning, suppress their growth and breedings, change food chain structure so that microcystic aeruginosa turns into advantage algae kind.MC is not only right
Other biological has damaging effect in water body, but also brings negative shadow to lake, river ecological, livestock and mankind's water source supply
Ring.
With the further investigation to nucleic acid detection technique, the base of Microcystin synzyme is clearly encoded substantially now
Cause.Can synthesize specific primer and probe by using production virus gene mcyD, establish molecular biological assay detecting and
Differentiate the production strain of the low concentration in environmental water sample.Real-time quantitative PCR detection method can with it is sensitive, quick, accurately and efficiently detect
Go out species, it to instrument without very high requirement, can particularly on the preceding processing time of sample and the amount that needs to gather sample
To save substantial amounts of manpower and time.
With the birth of nanometer technology, nano material is increasingly applied in the production and life of people, more next
It is more it was recognized that unconscious discharge of the nano material in nature.C60As one of carbon nanomaterial, because its is strange
Special Wuli-Shili-Renli system approach is widely used in multiple production fields such as optical material, electronic material and biomedicine.It is in ring
Cumulative concentration in border is as the gradual increase in nano material market, the continuous research and development of new product list and gradually increase.This leads
Cause C60The possibility being discharged into water body increases significantly, C60 nanoparticles is formed by water body, to water body environment and biology
Harm also corresponding increase.There is research to point out, the possible food chain transfer mode of nanoparticles is in water body, by algae,
Flea class (bed mud biology), fish, may finally enter human body.
Therefore, it is necessary to propose a kind of technological means, to study C60Nanoparticles are to Growth of Microcystis aeruginosa and algae
Influenceed caused by toxin secretion.
The content of the invention
To overcome above-mentioned the shortcomings of the prior art, the purpose of the present invention is to provide a kind of detection C60Nanoparticles
The method and device influenceed on Microcystin generation, to pass through the C toward addition various concentrations in nutrient solution60Nanoparticles,
The content of Microcystin and Microcystin production virus gene mcyD copy number are detected, is established a kind of by detecting Algae toxins conjunction
C is studied into enzyme gene mcyD copy numbers60The method that nanoparticles have an impact to Algae toxins generation, it enormously simplify algae poison
The continuous mode of element, saves substantial amounts of manpower and time.
In view of the above and other objects, the present invention proposes a kind of detection C60Nanoparticles generate on Microcystin to be influenceed
Method, comprise the following steps：
Step 1, the C of various concentrations is added in the blue-green algae nutrient solution of producing microcystic toxins60Nanoparticles are trained
Support；
Step 2, take the C of various concentrations60The blue-green algae nutrient solution of the producing microcystic toxins of nanoparticles culture, collect algae
Cell, and then detect the production virus gene of the Microcystin in testing sample mcyD gene copy number；
Step 3, take the C of various concentrations60The blue-green algae nutrient solution of the producing microcystic toxins of nanoparticles culture, in collection
Clear liquid, the content of detection wherein Microcystin；
Step 4, correlation analysis is carried out to the result of step 2 and step 3, establishes various concentrations C60Nanoparticles
The relation of Algae toxins yield and Microcystin production virus gene mcyD gene copy number in the blue-green algae of culture；
Step 5, the relation of virus gene mcyD gene copy number is produced using Algae toxins yield and Microcystin, is led to
Measure Microcystin production virus gene mcyD gene copy number is crossed to determine the generation of Microcystin under different condition of culture
Amount.
Further, step 2 includes：
Step S1, take the C of various concentrations60The microcystic aeruginosa nutrient solution of nanoparticles culture, removes supernatant, collects
Frustule；
Step S2, frustule is transferred in centrifuge tube, and supernatant is removed in centrifugation again after washing, collects frustule；
Step S3, the extraction of intracellular total serum IgE is carried out to the frustule in step S2；
Step S4, RNA reverse transcription is carried out, synthesize cDNA；
Step S5, enter performing PCR amplification by template of the cDNA in step S4；
Step S6, is purified to pcr amplification product；
Step S7, DNA after purification is subjected to plasmid conversion process, obtains the white colony for having converted plasmid；
Step S8, plasmid is extracted using the white colony in step S7, after determining plasmid concentration, carry out gradient dilution, will
Plasmid after dilution establishes the concentration one-variable linear regression curve corresponding with critical cycle number of standard items as standard sample,
Obtain standard curve；
Step S9, using the cDNA samples obtained in step S4 as the standard sample in testing sample alternative steps S8, enter
The measure of row quantitative PCR, the DNA concentration in testing sample is determined according to standard curve, and detect the micro-capsule in testing sample
Algae toxins production virus gene mcyD gene copy number.
Further, in step S1, the C of various concentrations is taken60The m of nanoparticles culture1Milliliter microcystic aeruginosa training
Nutrient solution, using low-temperature centrifugation, remove supernatant.
Further, in step S2, frustule is transferred to m using phosphate buffered saline solution2In milliliter centrifuge tube, adopt
Remove supernatant with low-temperature centrifugation, collect frustule, and frustule is subjected to low temperature or Liquid nitrogen storage, for subsequent operation.
Further, in step S3, the frustule in step S2 is carried out using RNA extracts kits intracellular total
RNA extraction.
Further, in step S5, performing PCR amplification is entered by template of cDNA, reaction system is 50 μ L, and primer information is such as
Under：Upstream primer sequence is 5 '-GCCCACAAAACCCCAACTC-3 ' (SEQ IDNO.1), downstream primer sequence 5 '-
TTTCTGCCTAATCTTTTCCCCTCT-3 ' (SEQ IDNO.2), response procedures are：95 DEG C of pre-degenerations 5 minutes, subsequent 94 DEG C 30
Second, annealing temperature 60 DEG C extends 40 seconds for 40 seconds and 72 DEG C, carries out 30 circulations, and final 72 DEG C extend 10 minutes, pass through 1.5%
(wt/vol) agarose gel electrophoresis confirms product.
Further, in step S9, reagent needed for real-time quantitative PCR is added, reaction system is 20 μ L, 2 μ LDNA samples
Product, real-time quantitative PCR reaction is carried out, performing PCR amplification is entered using two-step method, reaction condition is：Pre-degeneration, 1 circulation, 95 DEG C 30
Second；PCR react, 40 circulation, 95 DEG C 5 seconds, 60 DEG C 30 seconds.
Further, step 3 includes：
Take m3Milliliter frustule nutrient solution, carries out low-temperature centrifugation, supernatant is collected, for determining microcystin in nutrient solution
Cellulose content；
Frustule is transferred to m with phosphoric acid buffer salt buffer2In milliliter centrifuge tube, low-temperature centrifugation is carried out, washed once；
The frustule for the precipitation that suspended using phosphoric acid buffer salt buffer, liquid nitrogen multigelation several times, carry out low-temperature centrifugation,
Supernatant is collected, for determining Microcystins in Microcystis aeruginosa Strains；
Each sample of acquisition is set into three Duplicate Samples, determined according to enzyme linked immunological kit operating instruction in nutrient solution
Microcystins in Microcystins and Microcystis aeruginosa Strains.
Further, in determination sample before Microcystins, preliminary experiment is carried out, to determine the dilution of testing sample
Multiple so that Microcystin measured value is multiplied by extension rate, you can calculate sample in kit detection range when finally calculating
Actual Microcystins in product.
To reach above-mentioned purpose, the present invention also provides a kind of detection C60Nanoparticles generate what is influenceed to Microcystin
Device, including：
Nutrient solution culture unit, the C of various concentrations is added in the blue-green algae nutrient solution of producing microcystic toxins60Nanoparticles
Cultivated；
McyD copy number detection units, for taking the C of various concentrations60The indigo plant of the producing microcystic toxins of nanoparticles culture
Algae culturing liquid, frustule is collected, and then detect the production virus gene of the Microcystin in testing sample mcyD gene copy number；
Algae toxins content detection unit, for taking the C of various concentrations60The indigo plant of the producing microcystic toxins of nanoparticles culture
Algae culturing liquid, collect supernatant, the content of detection wherein Microcystin；
Dependency analysis unit, the mcyD copy numbers detection unit and the result of Algae toxins content detection unit are carried out
Correlation analysis, establish various concentrations C60In the blue-green algae of the producing microcystic toxins of nanoparticles culture Algae toxins yield with it is micro-
The relation of capsule Algae toxins production virus gene mcyD gene copy number；
Algae toxins growing amount determining unit, the gene for being produced virus gene mcyD with Microcystin using Algae toxins yield are copied
The relation of shellfish number, micro-capsule under different condition of culture is determined by determining Microcystin production virus gene mcyD gene copy number
The growing amount of Algae toxins.
Compared with prior art, a kind of detection C of the present invention60Nanoparticles on Microcystin generate influence method and
The C that device passes through the addition various concentrations in nutrient solution60Nanoparticles, detect the content and Microcystin of Microcystin
Virus gene mcyD gene copy number is produced, establishes the C of various concentrations60Nanoparticles, Microcystins and Algae toxins
Correlation between synthase gene mcyD copy numbers, containing for Microcystin is studied eventually through detection gene copy number
Amount, realize one kind and study C by detecting Algae toxins synthase gene mcyD copy numbers60Nanoparticles are to Microcystin
The method having an impact is generated, simplifies the continuous mode of Microcystin.
Brief description of the drawings
Fig. 1 detects C for the present invention is a kind of60Nanoparticles generate the step flow of the method influenceed on Microcystin
Figure；
Fig. 2 is the thin portion flow chart of step 102 in the specific embodiment of the invention；
Fig. 3 detects C for the present invention is a kind of60Nanoparticles generate the system architecture of the device influenceed on Microcystin
Figure；
Fig. 4 is the detail structure chart of mcyD copy number detection units in the specific embodiment of the invention.
Embodiment
Below by way of specific instantiation and embodiments of the present invention are described with reference to the drawings, those skilled in the art can
Understand the further advantage and effect of the present invention easily by content disclosed in the present specification.The present invention can also pass through other differences
Instantiation implemented or applied, the various details in this specification also can be based on different viewpoints with application, without departing substantially from
Various modifications and change are carried out under the spirit of the present invention.
Fig. 1 detects C for the present invention is a kind of60Nanoparticles generate the step flow of the method influenceed on Microcystin
Figure.A kind of as shown in figure 1, detection C of the present invention60Nanoparticles generate the method influenceed, including following step on Microcystin
Suddenly：
Step 101, the C of various concentrations is added in the blue-green algae nutrient solution of producing microcystic toxins60Nanoparticles are trained
Support.In the specific embodiment of the invention, the C of various concentrations is added in microcystic aeruginosa nutrient solution60Nanoparticles are cultivated.
Step 102, the C of various concentrations is taken60The blue-green algae nutrient solution of the producing microcystic toxins of nanoparticles culture, collect algae
Cell, and then detect the production virus gene of the Microcystin in sample to be tested mcyD copy number.
Specifically, as shown in Fig. 2 step 102 further comprises：
Step S1, take the C of various concentrations60The blue-green algae nutrient solution of the producing microcystic toxins of nanoparticles culture, remove on
Clear liquid.In the specific embodiment of the invention, the C of various concentrations is taken6025 milliliters of microcystic aeruginosa cultures of nanoparticles culture
Liquid, centrifuged 10 minutes with 10000 revs/min, remove supernatant, by centrifugation, frustule is changed into precipitating, supernatant is removed,
Obtain frustule.
Step S2, frustule is transferred in centrifuge tube, is washed that (algae that will be obtained is thin with phosphate buffer solution
Born of the same parents' precipitation is hanged again), centrifuge again, remove supernatant, collect frustule.Specifically, phosphate buffered saline solution is utilized
Frustule is transferred in 1.5 milliliters of centrifuge tubes by (phosphate buffer saline, PBS), and in 10000 turns, 4 DEG C centrifuge
10 minutes, remove supernatant, collect frustule, and frustule is put in -80 DEG C of refrigerators or Liquid nitrogen storage, for subsequent operation.
Step S3, carrying for intracellular total serum IgE (Ribonucleic Acid, ribosomal ribonucleic acid) is carried out to the frustule in step S2
Take.In the specific embodiment of the invention, intracellular total serum IgE is carried out to the frustule in step S2 using RNA extracts kits
Extraction.
Step S4, RNA reverse transcription is carried out, synthesize cDNA.In the specific embodiment of the invention, application specific is fixed in real time
PCR (Polymerase Chain Reaction, PCR) reverse transcription reagent box is measured, carries out RNA reversion
Record, synthesize cDNA；
Step S5, enter performing PCR amplification by template of the cDNA in step S4.Specifically, using the cDNA in step S3 as mould
Plate enter performing PCR amplification, reaction system be 50 μ L (the μ L of 0.25 μ L, 10 × Ex Taq Buffer of TakaRa Ex Taq HS 5,
4 μ L, cDNA templates of dNTP Mixture 4 μ, each 1 μ L of upstream and downstream primer, remaining uses sterile purified water polishing).Primer information is such as
Under：Upstream primer sequence is 5 '-GCCCACAAAACCCCAACTC-3 ' (SEQ IDNO.1), downstream primer sequence 5 '-
TTTCTGCCTAATCTTTTCCCCTCT-3’(SEQ IDNO.2).Response procedures are：95 DEG C of pre-degenerations 5 minutes, subsequent 94 DEG C 30
Second, annealing temperature 60 DEG C extends 40 seconds for 40 seconds and 72 DEG C, carries out 30 circulations, and final 72 DEG C extend 10 minutes, pass through 1.5%
(wt/vol) agarose gel electrophoresis confirms product.
Step S6, is purified to pcr amplification product：Utilize DNA glue reclaims kit (such as Axygen DNA pillars
Glue reclaim kit) glue reclaim is carried out, target gene is purified, passes through 1.5% (wt/vol) agarose gel electrophoresis
Confirm whether recovery succeeds.
Step S7, DNA after purification is subjected to plasmid conversion process, obtains the white colony for having converted plasmid.Specifically,
DNA in step S6 after purification is inserted in plasmid vector pMD18-T, and it is molten to be further transformed into DH5 α competence Escherichia coli
Liquid, it is then applied to and adds on the LB solid mediums of Amp resistances, screened by blue hickie, select the white for having converted plasmid
Bacterium colony.
Step S8, plasmid is extracted using the white colony in step S7, after determining plasmid concentration, carry out gradient dilution, will
Plasmid after dilution establishes the concentration one-variable linear regression curve corresponding with critical cycle number of standard items as standard sample,
Obtain standard curve；
Step S9, using the cDNA samples obtained in step S4 as the standard sample in testing sample alternative steps S8, enter
The measure of row quantitative PCR, the DNA concentration in testing sample is determined according to standard curve, and detect the micro-capsule in testing sample
Algae toxins production virus gene mcyD copy number.Specifically, add real-time quantitative PCR needed for reagent (according toPremix
Ex TaqTMKit explanation is operated), reaction system is 20 μ L, 2 μ LDNA samples.The reaction of progress real-time quantitative PCR (such as
Using the Stepone plus Detection System of ABI companies), performing PCR amplification is entered using two-step method, reaction condition is：
Pre-degeneration, 1 circulation, 95 DEG C 30 seconds；PCR react, 40 circulation, 95 DEG C 5 seconds, 60 DEG C 30 seconds.
Step 103, the C of various concentrations is taken60The blue-green algae nutrient solution of the producing microcystic toxins of nanoparticles culture, in collection
Clear liquid, the content of detection wherein Microcystin.
In the specific embodiment of the invention, using enzyme linked immunosorbent assay (enzyme-linked immunosorbent
Assay, ELISA) measure Microcystin (microcystin, MC) content.Specifically, step 103 further comprises walking as follows
Suddenly：
Take 50 milliliters of frustule nutrient solution (i.e. C of various concentrations60The blue-green algae of the producing microcystic toxins of nanoparticles culture
Nutrient solution), centrifuge 10 minutes under the conditions of 10000 turns, 4 DEG C, supernatant is collected, for determining Microcystin in nutrient solution
(Extracellular microcystin, Extra MC) content；
Frustule is transferred in 1.5 milliliters of centrifuge tubes with PBS, 10000 turns, 4 DEG C centrifuge 10 minutes, washing one
It is secondary；
With 1 milliliter, the frustule of PBS suspension precipitation, three times, in 10000 turns, 4 DEG C centrifuge liquid nitrogen multigelation
10 minutes, supernatant is collected, for determining Microcystin (Intracellular in Microcystis aeruginosa Strains
Microcystin, Intra MC) content；
Each sample (sample that i.e. 50ml frustules nutrient solution is formed by above-mentioned steps) sets three parallel samples, presses
Extra MC and IntraMC contents are determined according to enzyme linked immunological kit (ELISA kit) operating instruction.It is preferred that determining
, it is necessary to preliminary experiment be carried out, to determine sample extension rate so that MC measured values are in kit detection range before MC contents in sample
It is interior, it is multiplied by extension rate when finally calculating, you can calculate actual MC contents in sample.
Step 104, correlation analysis is carried out to the result of step 102 and step 103, establishes various concentrations C60Nano particle
The relation of Algae toxins yield and Microcystin production virus gene mcyD gene copy numbers in the microcystic aeruginosa of thing culture.
Step 105, the relation of virus gene mcyD gene copy numbers is produced using above-mentioned Algae toxins yield and Microcystin,
Virus gene mcyD gene copy numbers are produced to determine the growing amount of Algae toxins under different condition of culture by determining Microcystin.
It can be seen that the present invention utilizes the C of various concentrations60Nanoparticles act on microcystic aeruginosa, then by C60Nano particle
Thing is associated with Microcystin production virus gene mcyD copy numbers to Microcystins, establishes C60Nanoparticles are dense
Correlation between degree, Algae toxins content and Microcystin production virus gene mcyD.Establish after correlation, survey can be passed through
Determine Microcystin production virus gene mcyD copy number quick detections C60Nanoparticles produce the influence of Algae toxins to Microcystis aeruginosa.Can
To greatly simplify the continuous mode of Algae toxins, substantial amounts of manpower and time are saved.
Fig. 3 detects C for the present invention is a kind of60Nanoparticles generate the system architecture of the device influenceed on Microcystin
Figure.A kind of as shown in figure 3, detection C of the present invention60Nanoparticles generate the device influenceed on Microcystin, including：Nutrient solution
Cultivate unit 301, mcyD copy numbers detection unit 302, Algae toxins content detection unit 303, dependency analysis unit 304 and
Algae toxins growing amount determining unit 305.
Nutrient solution culture unit 301, for adding the C of various concentrations in the blue-green algae nutrient solution toward producing microcystic toxins60Receive
Rice grain thing, obtain the C of various concentrations60The blue-green algae nutrient solution of the producing microcystic toxins of nanoparticles culture, have in the present invention
In body embodiment, nutrient solution culture unit 301, in the C toward addition various concentrations in microcystic aeruginosa nutrient solution60Nano particle
Thing, obtain the C of various concentrations60The microcystic aeruginosa nutrient solution of nanoparticles culture.
McyD copy numbers detection unit 302, for taking the C of various concentrations60The producing microcystic toxins of nanoparticles culture
Blue-green algae nutrient solution, collect frustule, and then detect the production of the Microcystin in sample to be tested virus gene mcyD copy number.
Specifically, as shown in figure 4, mcyD copy numbers detection unit 302 further comprises：
Nutrient solution acquiring unit 3021, take the C of various concentrations60The blue-green algae training of the producing microcystic toxins of nanoparticles culture
Nutrient solution, remove supernatant.In the specific embodiment of the invention, the C of various concentrations is taken6025 milliliters of verdigris of nanoparticles culture
Micro-capsule algae culturing liquid, centrifuged 10 minutes with 10000 revs/min, remove supernatant, by centrifugation, frustule is changed into precipitating, will be upper
Clear liquid removes, and obtains frustule.
Frustule collector unit 3022, for frustule to be transferred in centrifuge tube, washed with phosphate buffer solution
(the frustule precipitation that will be obtained is hanged again) is washed, supernatant is removed in centrifugation again, collects frustule.Specifically, phosphorus is utilized
Frustule is transferred in 1.5 milliliters of centrifuge tubes by acid buffering salting liquid (phosphate buffer saline, PBS), in
10000 turns, 4 DEG C centrifuge 10 minutes, remove supernatant, collect frustule, and frustule is put in into -80 DEG C of refrigerators or liquid nitrogen guarantor
Deposit, for subsequent operation.
Total RNAs extraction unit 3023, intracellular total serum IgE is carried out to the frustule in frustule collector unit 3022
The extraction of (Ribonucleic Acid, ribosomal ribonucleic acid).It is thin to algae using RNA extracts kits in the specific embodiment of the invention
Frustule in born of the same parents' collector unit 3022 carries out the extraction of intracellular total serum IgE.
Reverse transcription unit 3024, for carrying out RNA reverse transcription, synthesize cDNA.In the specific embodiment of the invention, application
The reverse transcription reagent box of real-time quantitative PCR (Polymerase Chain Reaction, PCR) is exclusively used in, is entered
Row RNA reverse transcription, synthesize cDNA；
PCR amplification units 3025, enter performing PCR amplification by template of the cDNA in reverse transcription unit 3024.Specifically, with anti-
CDNA in transcriptional units 3024 enters performing PCR amplification for template, reaction system be 50 μ L (the μ L of TakaRa Ex Taq HS 0.25,
10 × Ex Taq Buffer, 5 μ L, dNTP Mixture, 4 μ L, cDNA templates 4 μ, each 1 μ L of upstream and downstream primer, remaining uses sterilizing
Distilled water polishing).Primer information is as follows：Upstream primer sequence be 5 '-GCCCACAAAACCCCAACTC-3 ' (SEQ IDNO.1),
Downstream primer sequence is 5 '-TTTCTGCCTAATCTTTTCCCCTCT-3 ' (SEQ IDNO.2).Response procedures are：95 DEG C of pre- changes
Property 5 minutes, it is subsequent 94 DEG C 30 seconds, annealing temperature 60 DEG C 40 seconds and 72 DEG C extends 40 seconds, carries out 30 circulations, final 72 DEG C of extensions
10 minutes, product is confirmed by 1.5% (wt/vol) agarose gel electrophoresis.
Purification unit 3026, for being purified to pcr amplification product：Utilize DNA glue reclaims kit (such as Axygen
DNA gel extraction kits) carry out glue reclaim, target gene is purified, passes through 1.5% (wt/vol) agarose
Gel electrophoresis confirms whether recovery succeeds.
Plasmid conversion unit 3027, for DNA after purification to be carried out into plasmid conversion process, acquisition has converted the white of plasmid
Color bacterium colony.Specifically, the DNA in step 106 after purification is inserted in plasmid vector pMD18-T, and is further transformed into DH5 α
Competence Escherichia coli solution, it is then applied to and adds on the LB solid mediums of Amp resistances, screened, selected by blue hickie
The white colony of plasmid is converted.
Standard curve establishes unit 3028, extracts plasmid using the white colony in plasmid conversion unit 3027, determines matter
Grain concentration after, carry out gradient dilution, using the plasmid after dilution be used as standard sample, establish standard items concentration and critical cycle number
Corresponding one-variable linear regression curve, that is, obtain standard curve；
Quantitative PCR determination unit 3029, the cDNA samples for reverse transcription unit 3024 to be obtained replace as testing sample
The standard sample established for standard curve in unit 3028, carries out the measure of quantitative PCR, and testing sample is determined according to standard curve
In DNA concentration, and detect the production of the Microcystin in testing sample virus gene mcyD copy number.Specifically, add real
When reagent needed for quantitative PCR (according toPremix Ex TaqTMKit explanation is operated), reaction system is 20 μ
L, 2 μ LDNA samples.The reaction of progress real-time quantitative PCR (such as the Stepone plus Detection using ABI companies
System), performing PCR amplification is entered using two-step method, reaction condition is：Pre-degeneration, 1 circulation, 95 DEG C 30 seconds；PCR reacts, 40
Circulation, 95 DEG C 5 seconds, 60 DEG C 30 seconds.
Algae toxins content detection unit 303, for choosing the C of various concentrations60The producing microcystic poison of nanoparticles culture
The blue-green algae nutrient solution of element, collect supernatant, the content of detection wherein Microcystin.
In the specific embodiment of the invention, the application enzyme linked immunosorbent assay of Algae toxins content detection unit 303 (enzyme-
Linked immunosorbent assay, ELISA) measure Microcystin (microcystin, MC) content.Specifically, algae
Content of toxins detection unit 303 is realized especially by following steps：
50 milliliters of frustule nutrient solutions are taken, are centrifuged 10 minutes under the conditions of 10000 turns, 4 DEG C, supernatant are collected, for determining
Microcystin (extracellular microcystin, Extra MC) content in nutrient solution；
Frustule is transferred in 1.5 milliliters of centrifuge tubes with PBS, 10000 turns, 4 DEG C centrifuge 10 minutes, washing one
It is secondary；
With 1 milliliter, the frustule of PBS suspension precipitation, three times, in 10000 turns, 4 DEG C centrifuge liquid nitrogen multigelation
10 minutes, supernatant is collected, for determining Microcystin (intracellular in Microcystis aeruginosa Strains
Microcystin, Intra MC) content；
Each sample sets three Duplicate Samples, is determined according to enzyme linked immunological kit (ELISA kit) operating instruction
Extra MC and Intra MC contents.It is preferred that, it is necessary to preliminary experiment be carried out, to determine sample before MC contents in determination sample
Extension rate so that MC measured values are multiplied by extension rate, you can calculate in sample in kit detection range when finally calculating
Actual MC contents.
Dependency analysis unit 304, for mcyD copy numbers detection unit 302 and Algae toxins content detection unit 303
Result carry out correlation analysis, establish various concentrations C60In the microcystic aeruginosa of nanoparticles culture Algae toxins yield with
The relation of mcyD gene copy numbers.
Algae toxins growing amount determining unit 305, for the pass using above-mentioned Algae toxins yield and mcyD gene copy numbers
System, the growing amount of Algae toxins under different condition of culture is determined by determining mcyD gene copy numbers.
The present invention is further illustrated below by way of a specific embodiment：
1) C of various concentrations60The microcystic aeruginosa of nanoparticles culture, it produces the content of Microcystin and algae poison
Correlation before plain synthase gene mcyD copy numbers is：Y=kx+b, wherein y are Algae toxins content, and unit is that μ g/L, x are
Algae toxins synthase gene mcyD copy numbers, unit copies/mL, k and b are constant.
2) C of various concentrations is used60Nanoparticles act on microcystic aeruginosa, take 25 milliliters of microcystic aeruginosa nutrient solutions, use
10000 revs/min centrifuge 10 minutes, remove supernatant；
3) frustule is transferred to 1.5 milliliters with phosphate buffered saline solution (phosphate buffer saline, PBS)
In centrifuge tube, in 10000 turns, 4 DEG C centrifuge 10 minutes, remove supernatant, collect frustule, frustule is put in into -80 DEG C of refrigerators,
For subsequent operation；
4) extraction of intracellular total serum IgE is carried out to the frustule in step 3) using RNA extracts kits；
5) application specific carries out RNA reverse transcription, synthesizes cDNA in the reverse transcription reagent box of real-time quantitative PCR；
6) performing PCR amplification is entered by template of the cDNA in step 5), reaction system is 50 μ L (TakaRa Ex Taq HS
0.25 μ L, 10 × Ex Taq Buffer, 5 μ L, dNTP Mixture, 4 μ L, cDNA templates 4 μ, each 1 μ L of upstream and downstream primer, its
Remaining sterile purified water polishing).Primer information is as follows：Upstream primer sequence is 5 '-GCCCACAAAACCCCAACTC-3 ' (SEQ
IDNO.1) ,-TTTCTGCCTAATCTTTTCCCCTCT-3 ' of downstream primer sequence 5 ' (SEQ IDNO.2).Response procedures are：
95 DEG C of pre-degenerations 30s, 60 DEG C of 30s, 72 DEG C of 1min, 30 circulations, 72 DEG C of extension 10min.Pass through 1.5% (wt/vol) agar
Sugared gel electrophoresis confirms product；
7) target gene is purified：Glue reclaim is carried out using Axygen DNA gel extraction kits, to target
Gene is purified, and confirms whether recovery succeeds by 1.5% (wt/vol) agarose gel electrophoresis；
8) plasmid conversion process：DNA in step 7) after purification is inserted in plasmid vector pMD18-T, and further turned
Change, to DH5 α competence Escherichia coli solution, to be then applied to and add on the LB solid mediums of Amp resistances, sieved by blue hickie
Choosing, selects the white colony for having converted plasmid；
9) the white colony extraction plasmid in step 8), after determining plasmid concentration, gradient dilution, by the matter after dilution are utilized
Grain is used as standard sample, establishes the concentration one-variable linear regression curve corresponding with critical cycle number of standard items, that is, obtains standard
Curve；
10) testing sample alternative steps 9 are used) in standard items, carry out the measure of quantitative PCR, determined according to standard curve
DNA concentration in sample；According toPremix Ex TaqTMKit explanation is operated, and adds real-time quantitative PCR
Required reagent, reaction system are 20 μ L, 2 μ LDNA samples.Using the Stepone plus Detection System of ABI companies
Real-time quantitative PCR reaction is carried out, performing PCR amplification is entered using two-step method, reaction condition is：Pre-degeneration, 1 circulation, 95 DEG C of 30s；
PCR reacts, 40 circulations, 95 DEG C of 5s, 60 DEG C of 30s.Amplification curve analysis finds, the exponential amplification phase of mcyD genes and plateau
All fairly obvious, fluorescent quantitation kinetic curve is smooth, is preferable amplification curve, and the PCR method for illustrating the present invention is success
, the quantitative analysis available for mcyD genes.Solubility curve analysis finds that single peak is presented in all purposes gene, and
Solubility curve Tm values are consistent.Calibration curve coefficient correlation is 0.9995, and copying for mcyD genes in sample is determined according to standard curve
Shellfish number；
11) detection of Algae toxins
Microcystis aeruginosa is determined using enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA)
Toxin (microcystin, MC) content.50 milliliters of frustule nutrient solutions are taken, are centrifuged 10 minutes under the conditions of 10000 turns, 4 DEG C, are received
Collect supernatant, for determining Microcystin in nutrient solution (extracellular microcystin, Extra MC) content.
Frustule is transferred in 1.5 milliliters of centrifuge tubes with PBS, 10000 turns, 4 DEG C centrifuge 10 minutes, washed once.With 1 milli
Rise, the frustule of PBS suspension precipitation, liquid nitrogen multigelation is three times.In 10000 turns, 4 DEG C centrifuge 10 minutes, in collection
Clear liquid, contain for determining Microcystin in Microcystis aeruginosa Strains (intracellular microcystin, Intra MC)
Amount.Each sample sets three Duplicate Samples.Extra MC and Intra MC contents are determined according to ELISA kit operating instruction.
, it is necessary to preliminary experiment be carried out, to determine sample extension rate before MC contents in determination sample so that MC measured values are examined in kit
In the range of survey, extension rate is multiplied by when finally calculating, you can calculate actual MC contents in sample；
12) correlation analysis is carried out to the result of step 10) and step 11).As a result show, the C of various concentrations60Nanometer
After grain thing effect microcystic aeruginosa, meet linear relationship y=kx+b between its Algae toxins content and mcyD gene copy numbers, wherein
K is that 0.3596, b is -2.7686.
A kind of it can be seen that detection C of the present invention60Nanoparticles on Microcystin generate influence method and device by
The C of various concentrations is added in nutrient solution60Nanoparticles, detect the content and Microcystin production virus gene of Microcystin
McyD gene copy number, establish the C of various concentrations60Nanoparticles, Microcystins and Algae toxins synzyme base
Because of the correlation between mcyD copy numbers, the content of Microcystin is studied eventually through detection gene copy number, is realized
One kind studies C by detecting Algae toxins synthase gene mcyD copy numbers60Nanoparticles are generated to Microcystin and produced
The method of influence, simplifies the continuous mode of Algae toxins, and high specificity of the present invention, sensitivity are higher, and required time is shorter, behaviour
Make comparisons simple, required instrument and equipment and reagent are relatively easy.
The above-described embodiments merely illustrate the principles and effects of the present invention, not for the limitation present invention.Any
Art personnel can be modified above-described embodiment and changed under the spirit and scope without prejudice to the present invention.Therefore,
The scope of the present invention, should be as listed by claims.
Sequence table
<110>Shanghai urban water resources development and utilization of National Engineering Center Co., Ltd
<120>Detect C60 nanoparticles and the method and device influenceed is generated on Microcystin
<130> 2017
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
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gcccacaaaa ccccaactc 19
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence
<400> 2
tttctgccta atcttttccc ctct 24
Claims (10)
1. one kind detection C60Nanoparticles generate the method influenceed to Microcystin, comprise the following steps：
Step 1, the C of various concentrations is added in the blue-green algae nutrient solution of producing microcystic toxins60Nanoparticles are cultivated；
Step 2, take the C of various concentrations60The blue-green algae nutrient solution of the producing microcystic toxins of nanoparticles culture, frustule is collected,
And then detect the production virus gene of the Microcystin in testing sample mcyD gene copy number；
Step 3, take the C of various concentrations60The blue-green algae nutrient solution of the producing microcystic toxins of nanoparticles culture, supernatant is collected,
The content of detection wherein Microcystin；
Step 4, correlation analysis is carried out to the result of step 2 and step 3, establishes various concentrations C60Nanoparticles culture
Blue-green algae in Algae toxins yield and Microcystin production virus gene mcyD gene copy number relation；
Step 5, the relation of virus gene mcyD gene copy number is produced using Algae toxins yield and Microcystin, passes through survey
Microcystin production virus gene mcyD gene copy number is determined to determine the growing amount of Microcystin under different condition of culture.
A kind of 2. detection C as claimed in claim 160Nanoparticles generate the method influenceed, its feature to Microcystin
It is, step 2 further comprises：
Step S1, take the C of various concentrations60The microcystic aeruginosa nutrient solution of nanoparticles culture, remove supernatant, it is thin to collect algae
Born of the same parents；
Step S2, frustule is transferred in centrifuge tube, and supernatant is removed in centrifugation again after washing, collects frustule；
Step S3, the extraction of intracellular total serum IgE is carried out to the frustule in step S2；
Step S4, RNA reverse transcription is carried out, synthesize cDNA；
Step S5, enter performing PCR amplification by template of the cDNA in step S4；
Step S6, is purified to pcr amplification product；
Step S7, DNA after purification is subjected to plasmid conversion process, obtains the white colony for having converted plasmid；
Step S8, plasmid is extracted using the white colony in step S7, after determining plasmid concentration, carry out gradient dilution, will dilute
Plasmid afterwards is established the concentration one-variable linear regression curve corresponding with critical cycle number of standard items, produced as standard sample
To standard curve；
Step S9, using the cDNA samples obtained in step S4 as the standard sample in testing sample alternative steps S8, determined
PCR measure is measured, the DNA concentration in testing sample is determined according to standard curve, and detect the microcystin in testing sample
Element production virus gene mcyD gene copy number.
A kind of 3. detection C as claimed in claim 260Nanoparticles generate the method influenceed, its feature to Microcystin
It is：In step S1, the C of various concentrations is taken60The m of nanoparticles culture1Milliliter microcystic aeruginosa nutrient solution, using low temperature
Centrifugation, removes supernatant.
A kind of 4. detection C as claimed in claim 260Nanoparticles generate the method influenceed, its feature to Microcystin
It is：In step S2, frustule is transferred to m using phosphate buffered saline solution2In milliliter centrifuge tube, gone using low-temperature centrifugation
Fall supernatant, collect frustule, and frustule is subjected to low temperature or Liquid nitrogen storage, for subsequent operation.
A kind of 5. detection C as claimed in claim 460Nanoparticles generate the method influenceed, its feature to Microcystin
It is：In step S3, the extraction of intracellular total serum IgE is carried out to the frustule in step S2 using RNA extracts kits.
A kind of 6. detection C as claimed in claim 560Nanoparticles generate the method influenceed, its feature to Microcystin
It is：In step S5, enter performing PCR amplification by template of cDNA, reaction system is 50 μ L, and primer information is as follows：Sense primer
Sequence is SEQ IDNO.1, downstream primer sequence is SEQ IDNO.2, and response procedures are：95 DEG C of pre-degenerations 5 minutes, subsequent 94 DEG C
30 seconds, annealing temperature 60 DEG C extended 40 seconds for 40 seconds and 72 DEG C, carried out 30 circulations, and final 72 DEG C extend 10 minutes, pass through 1.5%
(wt/vol) agarose gel electrophoresis confirms product.
A kind of 7. detection C as claimed in claim 660Nanoparticles generate the method influenceed, its feature to Microcystin
It is：In step S9, reagent needed for real-time quantitative PCR is added, reaction system is 20 μ L, and 2 μ LDNA samples, it is fixed in real time to carry out
PCR reactions are measured, performing PCR amplification are entered using two-step method, reaction condition is：Pre-degeneration, 1 circulation, 95 DEG C 30 seconds；PCR reacts, and 40
Individual circulation, 95 DEG C 5 seconds, 60 DEG C 30 seconds.
A kind of 8. detection C as claimed in claim 260Nanoparticles generate the method influenceed, its feature to Microcystin
It is, step 3 further comprises：
Take m3Milliliter frustule nutrient solution, carries out low-temperature centrifugation, collects supernatant, contains for determining Microcystin in nutrient solution
Amount；
Frustule is transferred to m with phosphoric acid buffer salt buffer2In milliliter centrifuge tube, low-temperature centrifugation is carried out, washed once；
The frustule for the precipitation that suspended using phosphoric acid buffer salt buffer, liquid nitrogen multigelation several times, are carried out low-temperature centrifugation, collected
Supernatant, for determining Microcystins in Microcystis aeruginosa Strains；
Each sample of acquisition is set into three Duplicate Samples, micro-capsule in nutrient solution is determined according to enzyme linked immunological kit operating instruction
Microcystins in Algae toxins content and Microcystis aeruginosa Strains.
A kind of 9. detection C as claimed in claim 860Nanoparticles generate the method influenceed, its feature to Microcystin
It is：In determination sample before Microcystins, preliminary experiment is carried out, to determine the extension rate of testing sample so that micro-
Capsule Algae toxins measured value is multiplied by extension rate, you can calculate actual in sample in kit detection range when finally calculating
Microcystins.
10. one kind detection C60Nanoparticles generate the device influenceed on Microcystin, including：
Nutrient solution culture unit, the C of various concentrations is added in the blue-green algae nutrient solution of producing microcystic toxins60Nanoparticles are carried out
Culture；
McyD copy number detection units, for taking the C of various concentrations60The blue-green algae training of the producing microcystic toxins of nanoparticles culture
Nutrient solution, frustule is collected, and then detect the production virus gene of the Microcystin in testing sample mcyD gene copy number；
Algae toxins content detection unit, for taking the C of various concentrations60The blue-green algae training of the producing microcystic toxins of nanoparticles culture
Nutrient solution, collect supernatant, the content of detection wherein Microcystin；
Dependency analysis unit is related to the result progress of Algae toxins content detection unit to the mcyD copy numbers detection unit
Property analysis, establish various concentrations C60Algae toxins yield and Microcystis aeruginosa in the blue-green algae of the producing microcystic toxins of nanoparticles culture
The relation of toxin production virus gene mcyD gene copy number；
Algae toxins growing amount determining unit, virus gene mcyD gene copy number is produced using Algae toxins yield and Microcystin
Relation, produce virus gene mcyD gene copy number by determining Microcystin to determine microcystin under different condition of culture
The growing amount of element.
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| CN112342279A (en) * | 2020-11-06 | 2021-02-09 | 中国科学院城市环境研究所 | Kit and method for simultaneously detecting specific genes of cyanobacteria bloom Ralstonia and prototheca toxin |
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| CN108796102A (en) * | 2018-07-04 | 2018-11-13 | 上海城市水资源开发利用国家工程中心有限公司 | A kind of method and device of quantitative detection production 2- methyl isoborneol bacterial strains |
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| CN102242191B (en) * | 2011-04-25 | 2013-12-04 | 宁波大学 | PCR (polymerase chain reaction) primer for detecting microcystins in water bodies |
| CN102952876A (en) * | 2012-02-13 | 2013-03-06 | 上海海洋大学 | Specific multiple PCR (polymerase chain reaction) detection method and kit for toxigenic microcystis and kit therefor |
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- 2017-09-12 CN CN201710815784.7A patent/CN107488719A/en active Pending
- 2017-09-12 CN CN201710815782.8A patent/CN107604051A/en active Pending
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| WO2004104211A3 (en) * | 2003-05-21 | 2005-03-03 | Helsingin Yliopisto | Method for detecting toxic and non-toxic cyanobacteria |
| CN104131066A (en) * | 2013-05-03 | 2014-11-05 | 中国科学院生态环境研究中心 | Method for researching influence of nitrogen and phosphorus on generation of microcystis by employing toxin-producing gene of the microcystis |
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| CN112342279A (en) * | 2020-11-06 | 2021-02-09 | 中国科学院城市环境研究所 | Kit and method for simultaneously detecting specific genes of cyanobacteria bloom Ralstonia and prototheca toxin |
| CN112342279B (en) * | 2020-11-06 | 2022-10-14 | 中国科学院城市环境研究所 | Kit and method for simultaneously detecting specific genes of cyanobacteria bloom Ralstonia and prototheca toxin |
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| Publication number | Publication date |
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| CN107488719A (en) | 2017-12-19 |
| CN107604051A (en) | 2018-01-19 |
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