CN107502678A - A kind of method and device for detecting blue algae producing microcystic toxins - Google Patents
A kind of method and device for detecting blue algae producing microcystic toxins Download PDFInfo
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Abstract
The invention discloses a kind of method and device for detecting blue algae producing microcystic toxins, this method includes：Surface water water sample is taken, after micro porous filtration, filter membrane is subjected to Genome DNA extraction purifying, obtains DNA sample；Enter performing PCR amplification by template of the DNA sample of acquisition；Pcr amplification product is purified；DNA after purification is subjected to plasmid conversion process, obtains the white colony for having converted plasmid；Choose full single bacterium colony and be inoculated in shaken cultivation in nutrient solution and stay overnight, for bacterium solution PCR identifications and sequencing analysis；Plasmid will be extracted by the white colony of identification and analysis, determine gradient dilution after plasmid concentration, using the plasmid after dilution as standard sample, establish standard sample concentration standard curve corresponding with critical cycle number；The DNA sample obtained in step 1 is taken to carry out quantitative PCR measure as template and obtain sample to be tested, the DNA concentration in sample to be tested is determined according to the standard curve.
Description
Technical field
The present invention relates to a kind of method and device for detecting blue algae producing microcystic toxins, and algae poison is utilized more particularly to one kind
The method and device of element production virus gene detection blue algae producing microcystic toxins.
Background technology
Water body in lake eutrophication can trigger the outburst of blue-green alga bloom, and blue-green alga bloom has turned into China or even the world is faced
One of great environmental pollution problem.There is part blue-green algae to produce Algae toxins in blue-green alga bloom, such as microcystic aeruginosa, verdigris are micro-
Capsule algae is blue-green algae most commonly seen in water body, and it is the primary producer in water body, is played an important role in water environment.
Microcystic aeruginosa can produce Microcystin (microcystin, MC), and it is a kind of oligopeptides hepatotoxin of ring-type, in full generation
Microcystin can be detected in the natural water body of many countries of boundary.Microcystin (microcystin, MC) influences to swim
The growth of plant, invertebrate and vertebrate.MC can cause water Endophytic bacteria, protozoan and other phytoplanktons
(including diatom and green alga) is poisoned, and suppresses their growth and breedings, changes food chain structure so that microcystic aeruginosa turns into advantage algae
Kind.MC not only has damaging effect to other biological in water body, but also lake, river ecological, livestock and mankind water source are supplied
To being negatively affected, research shows, lives in the crowd of blue-green alga bloom hotspot, and onset of liver cancer rate will be apparently higher than other
Area, therefore, the research for the detection of blue algae producing microcystic toxins have turned into the study hotspot of field of Environment Protection.
Production poison is distinguished and not produce malicious blue-green algae be extremely difficult by the traditional microbiological such as morphological observation method.It is existing
Result of study shows that MC is compiled by Microcystin gene cluster (microcystin synthetase gene clusters, mcy)
Code generation, gene cluster is located on chromosome, comprising the opposite operator of the both direction being made up of 10 genes, except gene
McyA and mcyB, gene mcyD have also assisted in MC coding synthesis, and wherein gene mcyD participates in coding synthesis MC Adda (Adda
It is group necessary to the expression of Microcystin toxicity), and Adda and MC toxicity is closely related, in each blue-green algae for producing MC only
There is mcyD gene copy, therefore blue algae producing microcystic toxins can be detected by detecting the copy number of mcyD genes.
Prior art is typically expanded using PCR (Polymerase Chain Reaction, PCR) technology
McyD fragments produce the detection of malicious blue-green algae to realize, but it is blue to use in general round pcr amplification mcyD fragments qualitative can only distinguish
Algae whether producing microcystic toxins, it is impossible to quantitatively detect water body in blue algae producing microcystic toxins concentration.
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 producing microcystic toxins
The method and device of blue-green algae, to provide, one kind is easy to operate, is easy to grasp and can fast and accurately determine to produce micro-capsule in surface water
The quantitative measurement technology of the blue algae producing microcystic toxins of the concentration of Algae toxins blue-green algae.
In view of the above and other objects, the present invention proposes a kind of method for detecting blue algae producing microcystic toxins, including it is as follows
Step：
Step 1, surface water water sample is taken, after micro porous filtration, filter membrane is subjected to Genome DNA extraction purifying, obtains DNA samples
This；
Step 2, enter performing PCR amplification by template of the DNA sample that step 1 obtains；
Step 3, pcr amplification product is purified；
Step 4, DNA after purification is subjected to plasmid conversion process, obtains the white colony for having converted plasmid；
Step 5, choose full single bacterium colony and be inoculated in nutrient solution, and shaken cultivation is stayed overnight, and is reflected for bacterium solution PCR
Fixed and sequencing analysis；
Step 6, plasmid will be extracted by the white colony of identification and analysis, after determining plasmid concentration, gradient dilution will be dilute
Plasmid after releasing establishes the concentration one-variable linear regression curve corresponding with critical cycle number of standard sample as standard sample,
Obtain standard curve；
Step 7, the DNA sample obtained in step 1 is taken to carry out quantitative PCR measure as template and obtain sample to be tested, root
The DNA concentration in sample to be tested is determined according to the standard curve.
Further, in step 2, the PCR reaction systems are used to be for 50 μ L, primer information：Sense primer 5 '-
GCCCACAAAACCCCAACTC-3 ' ,-TTTCTGCCTAATCTTTTCCCCTCT-3 ' of anti-sense primer 5 '；Response procedures are：95℃
Pre-degeneration 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
Extension 10 minutes；Confirm pcr amplification product finally by 1.5% agarose gel electrophoresis.
Further, in step 3, glue reclaim is carried out using DNA glue reclaims kit, pcr amplification product is carried out pure
Change, and confirm whether recovery succeeds by 1.5% agarose gel electrophoresis.
Further, in step 4, will be inserted through the DNA of step 3 after purification in plasmid vector, and further convert
To DH5 α competence Escherichia coli solution, it is then applied to and adds on the LB solid mediums of Amp resistances, sieved by blue hickie
Choosing, selects the white colony for having converted plasmid.
Further, in step 5, choose full single bacterium colony and be inoculated in the liquid LB nutrient solutions containing Amp, in
37 DEG C, 200 revs/min of shaken cultivations stay overnight, for bacterium solution PCR identification and sequencing analysis.
Further, step 7 further comprises：
Reagent needed for real-time quantitative PCR is added, reaction system is 20 μ L, 2 μ LDNA samples；
Set and carry out negative control and positive control, using deionized water as negative control, with blue algae producing microcystic toxins
DNA as positive control；
Real-time quantitative PCR reaction is carried out, enters performing PCR amplification；
After reaction terminates, by the cycle threshold Ct and standard curve control of DNA sample, the mcyD genes of DNA sample are calculated
Copy number, so as to obtain the copy number of mcyD genes in raw water, and analyze and obtain the dense of blue algae producing microcystic toxins in water sample
Degree.
Further, in step 7, 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.
To reach above-mentioned purpose, the present invention also provides a kind of device for detecting blue algae producing microcystic toxins, including：
DNA sample extraction unit, for by taking surface water water sample, after micro porous filtration, filter membrane progress STb gene to be carried
Purifying is taken, obtains DNA sample；
PCR amplification units, enter performing PCR amplification by template of the DNA sample that the DNA sample extraction unit obtains；
Purification unit, for being purified to pcr amplification product；
Plasmid conversion unit, for DNA after purification to be carried out into plasmid conversion process, obtain the white bacterium for having converted plasmid
Fall；
Identification and analysis unit, the single bacterium colony for choosing full is inoculated in nutrient solution, and shaken cultivation is stayed overnight, for
Bacterium solution PCR is identified and sequencing analysis；
Standard curve establishes unit, for plasmid will to be extracted by the white colony of identification and analysis, after determining plasmid concentration,
Gradient dilution, using the plasmid after dilution as standard sample, establish the concentration unitary corresponding with critical cycle number of standard sample
Linear regression curves, that is, obtain standard curve；
Quantitative PCR detection unit, for taking the DNA sample that the DNA sample extraction unit obtains to be determined as template
Measure PCR measure and obtain sample to be tested, the DNA concentration in sample to be tested is determined according to the standard curve.
Further, the PCR amplification units use the PCR reaction systems to be for 50 μ L, primer information：Sense primer 5 '-
GCCCACAAAACCCCAACTC-3 ' ,-TTTCTGCCTAATCTTTTCCCCTCT-3 ' of anti-sense primer 5 '；Response procedures are：95℃
Pre-degeneration 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
Extension 10 minutes；Confirm pcr amplification product finally by 1.5% agarose gel electrophoresis.
Further, the quantitative PCR detection unit is achieved by the steps of：
Reagent needed for real-time quantitative PCR is added, reaction system is 20 μ L, 2 μ LDNA samples；
Set and carry out negative control and positive control, using deionized water as negative control, with blue algae producing microcystic toxins
DNA as positive control；
Real-time quantitative PCR reaction is carried out, enters performing PCR amplification；
After reaction terminates, by the cycle threshold Ct and standard curve control of DNA sample, the mcyD genes of DNA sample are calculated
Copy number, so as to obtain the copy number of mcyD genes in raw water, and analyze and obtain the dense of blue algae producing microcystic toxins in water sample
Degree.
Compared with prior art, a kind of method and device for detecting blue algae producing microcystic toxins of the present invention is by designing micro-capsule
Algae toxins synthase gene mcyD primer, the DNA of sample to be detected is extracted, quantitative PCR detection is carried out, according to standard curve meter
The copy number of mcyD genes and the concentration of blue algae producing microcystic toxins of sample to be tested are calculated, is realized a kind of easy to operate, easy
Examined in the blue algae producing microcystic toxins grasped and can fast and accurately determine the concentration of blue algae producing microcystic toxins in surface water
Survey technology.
Brief description of the drawings
Fig. 1 is a kind of step flow chart for the method for detecting blue algae producing microcystic toxins of the present invention；
Fig. 2 is a kind of system architecture diagram for the device for detecting blue algae producing microcystic toxins of the present 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 is a kind of step flow chart for the method for detecting blue algae producing microcystic toxins of the present invention.As shown in figure 1, this hair
A kind of bright method for detecting blue algae producing microcystic toxins, comprises the following steps：
Step 101, surface water water sample is taken, after micro porous filtration, filter membrane is subjected to Genome DNA extraction purifying, obtains DNA samples
This.
Step 102, performing PCR amplification is entered by template of the DNA sample that step 101 obtains.Specifically, PCR reaction systems are
50 μ L, primer information are as follows：- the GCCCACAAAACCCCAACTC-3 ' of sense primer 5 ' anti-sense primers 5 '-
TTTCTGCCTAATCTTTTCCCCTCT-3’.Response procedures are：95 DEG C of pre-degenerations 5 minutes, it is subsequent 94 DEG C 30 seconds, annealing temperature
60 DEG C extend 40 seconds for 40 seconds and 72 DEG C, carry out 30 circulations, final 72 DEG C extend 10 minutes.Pass through 1.5% (wt/vol) fine jade
Sepharose electrophoresis confirms product.
Step 103, pcr amplification product is purified：Utilize DNA glue reclaims kit (such as Axygen DNA pillars
Glue reclaim kit) glue reclaim is carried out, pcr amplification product is purified, passes through 1.5% (wt/vol) Ago-Gel electricity
Swimming confirms whether recovery succeeds；
Step 104, DNA after purification is subjected to plasmid conversion process, obtains the white colony for having converted plasmid.Specifically
Ground, the DNA in step 103 after purification is inserted in plasmid vector pMD18-T, and be further transformed into DH5 α competence large intestine bars
Bacterium solution, it is then applied to and adds on the LB solid mediums of Amp resistances, screened by blue hickie, select and converted plasmid
White colony；
Step 105, choose full single bacterium colony (the single white colony converted in selecting step 104) and be inoculated in and contain
Have in Amp liquid LB nutrient solutions, stayed overnight in 37 DEG C, 200 revs/min of shaken cultivations, identify and be sequenced for bacterium solution PCR and divide
Analysis, in the specific embodiment of the invention, step 105 is by PCR method and company's sequencing is identified and sequencing analysis, specifically
Ground, the single white colony of picking is expanded into culture, enter performing PCR amplification as template, pcr amplification product is then subjected to electricity
Swimming analysis, observes purpose fragment size, is confirmed whether purpose fragment being successfully transferred in plasmid, while PCR primer glue is returned
Purifying is received, sequencing is sent to, whether is successfully transferred to further verifying purpose fragment in plasmid；
Step 106, plasmid will be extracted by the white colony in the step 104 of identification and analysis, after determining plasmid concentration, entered
Row gradient dilution, using the plasmid after dilution as standard sample, establish the concentration unitary corresponding with critical cycle number of standard items
Linear regression curves, that is, obtain standard curve；
Step 107, taking the DNA sample obtained in step 101, test sample is treated in the measure acquisition for carrying out quantitative PCR as template
This, the DNA concentration in sample to be tested is determined according to standard curve.Specifically, add real-time quantitative PCR needed for reagent (such as by
According toPremix Ex TaqTMKit explanation is operated), reaction system is 20 μ L, 2 μ LDNA samples, is set simultaneously
Put and carry out negative control and positive control, using deionized water as negative control, sun is used as using the DNA of blue algae producing microcystic toxins
Property control, carry out real-time quantitative PCR reaction (such as Stepone plus Detection System using ABI companies), adopt
Enter performing PCR with two-step method to expand, reaction condition is：Pre-degeneration, 1 circulation, 95 DEG C 30 seconds；PCR reacts, 40 circulations, 95 DEG C 5
Second, 60 DEG C 30 seconds；After reaction terminates, by the cycle threshold Ct and standard curve control of DNA sample, the mcyD of DNA sample is calculated
The copy number of gene, so as to obtain the copy number of mcyD genes in raw water, and then analyze to obtain producing microcystic toxins indigo plant in water sample
The concentration of algae.
The detection process of the present invention will be further illustrated by a specific embodiment below：
1) July, August and September surface water water sample is taken to carry out the quantitative analysis of blue algae producing microcystic toxins.Water sample passes through micropore
After filtering, filter membrane is preserved to -20 DEG C, analyzed for subsequent experimental.Experiment carries out Genome DNA extraction purifying after terminating, and obtains DNA
Sample.
2) performing PCR amplification is entered by template of the DNA sample in step 1), reaction system is 50 μ L.Primer information is as follows：On
Swim-the GCCCACAAAACCCCAACTC-3 ' of primer 5 ' ,-TTTCTGCCTAATCTTTTCCCCTCT-3 ' of anti-sense primer 5 '.Reaction interval
Sequence is：95 DEG C of pre-degenerations 5 minutes, it is subsequent 94 DEG C 30 seconds, annealing temperature 60 DEG C extends 40 seconds for 40 seconds and 72 DEG C, carries out 30 and follows
Ring, final 72 DEG C extend 10 minutes.Product is confirmed by 1.5% (wt/vol) agarose gel electrophoresis；
3) target gene (i.e. pcr amplification product) is purified：Entered using Axygen DNA gel extraction kits
Row glue reclaim, is purified to target gene, by 1.5% (wt/vol) agarose gel electrophoresis confirm recovery whether into
Work(；
4) plasmid conversion process：DNA in step 3) 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；
5) choose full single bacterium colony to be inoculated in the liquid LB nutrient solutions containing Amp, in 37 DEG C, 200 revs/min of vibrations
Overnight incubation, for bacterium solution PCR identifications and sequencing analysis；
6) plasmid will be extracted by the white colony in the step 4) of identification and analysis, after determining plasmid concentration, gradient dilution,
Using the plasmid after dilution as standard sample, the concentration one-variable linear regression corresponding with critical cycle number for establishing standard items is bent
Line, that is, obtain standard curve y=-0.3123x+14.002, R2=0.9961；
7) taking the DNA sample obtained in step 1), the measure for carrying out quantitative PCR obtains sample to be tested as template, according to
Standard curve determines the DNA concentration in sample to be tested.According toPremix Ex TaqTMKit explanation is operated,
Reagent needed for real-time quantitative PCR is added, reaction system is 20 μ L, 2 μ LDNA samples.Set simultaneously and carry out negative control and the positive
Control, using deionized water as negative control, positive control is used as using the DNA of blue algae producing microcystic toxins.Using ABI companies
Stepone plus Detection System carry out real-time quantitative PCR reaction, enter performing PCR amplification using two-step method, react bar
Part is：Pre-degeneration, 1 circulation, 95 DEG C 30 seconds；PCR react, 40 circulation, 95 DEG C 5 seconds, 60 DEG C 30 seconds；, will after reaction terminates
The cycle threshold Ct and standard curve control of DNA sample, the copy number (being shown in Table 1) of the mcyD genes of DNA sample is calculated, so as to
The copy number of mcyD genes into raw water, and then analyze and obtain the concentration of blue algae producing microcystic toxins in water sample.
Producing microcystic toxins gene mcyD copy number in the water sample of table 1
Fig. 2 is a kind of system architecture diagram for the device for detecting blue algae producing microcystic toxins of the present invention.As shown in Fig. 2 this hair
A kind of bright device for detecting blue algae producing microcystic toxins, including：DNA sample extraction unit 201, PCR amplification units 202, purifying
Unit 203, plasmid conversion unit 204, identification and analysis unit 205, standard curve establish unit 206 and quantitative PCR detection list
Member 207.
DNA sample extraction unit 201, for by taking surface water water sample, after micro porous filtration, filter membrane being carried out total
DNA extraction purifications, obtain DNA sample.
PCR amplification units 202, expand for entering performing PCR as template using the DNA sample that DNA sample extraction unit 201 obtains
Increase.Specifically, PCR reaction systems are 50 μ L, and primer information is as follows：Under-the GCCCACAAAACCCCAACTC-3 ' of sense primer 5 '
Swim-the TTTCTGCCTAATCTTTTCCCCTCT-3 ' of primer 5 '.Response procedures are：95 DEG C of pre-degenerations 5 minutes, it is subsequent 94 DEG C 30 seconds,
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.
Purification unit 203, for being purified to pcr amplification product.In the specific embodiment of the invention, purification unit
203 carry out glue reclaim using DNA glue reclaims kit (such as Axygen DNA gel extraction kits), and PCR is expanded and produced
Thing is purified, and confirms whether recovery succeeds by 1.5% (wt/vol) agarose gel electrophoresis.
Plasmid conversion unit 204, for DNA after purification to be carried out into plasmid conversion, obtain the white bacterium for having converted plasmid
Fall.Specifically, the DNA of purification unit 203 after purification is inserted in plasmid vector pMD18-T, and is further transformed into DH5 α senses
By state Escherichia coli solution, it is then applied to and adds on the LB solid mediums of Amp resistances, screened by blue hickie, selects and turn
The white colony of plasmid is changed.
Identification and analysis unit 205, (select what is converted in plasmid conversion unit 204 for choosing full single bacterium colony
Single white colony) be inoculated in the liquid LB nutrient solutions containing Amp, stayed overnight in 37 DEG C, 200 revs/min of shaken cultivations, with
In bacterium solution PCR identifications and sequencing analysis, in the specific embodiment of the invention, identification and analysis unit 205 passes through PCR method and company
Sequencing is identified and sequencing analysis, and specifically, the single white colony of picking is expanded culture by identification and analysis unit 205, with
This is that template enters performing PCR amplification, and pcr amplification product then carried out into electrophoretic analysis, observes purpose fragment size, be confirmed whether by
Purpose fragment is successfully transferred in plasmid, while PCR primer glue reclaim is purified, and sends to sequencing, with further verifying purpose piece
Whether section is successfully transferred in plasmid；
Standard curve establishes unit 206, for will extract plasmid by the white colony of identification and analysis, determines plasmid concentration
Afterwards, gradient dilution is carried out, using the plasmid after dilution as standard sample, the concentration for establishing standard items is corresponding with critical cycle number
One-variable linear regression curve, that is, obtain standard curve.
Quantitative PCR detection unit 207, for taking the DNA sample obtained in DNA sample extraction unit 201 to enter as template
The measure of row quantitative PCR obtains sample to be tested, and the DNA concentration in sample to be tested is determined according to standard curve.Specifically, it is quantitative
Reagent needed for the addition real-time quantitative PCR of PCR detection units 207 (such as according toPremix Ex TaqTMKit is said
It is bright to be operated), reaction system is 20 μ L, 2 μ LDNA samples, while sets and carry out negative control and positive control, with deionization
Water is as negative control, and using the DNA of blue algae producing microcystic toxins as positive control, carrying out real-time quantitative PCR reaction (such as should
With the Stepone plus Detection System of ABI companies), performing PCR amplification is entered using two-step method, reaction condition is：In advance
Denaturation, 1 circulation, 95 DEG C 30 seconds；PCR react, 40 circulation, 95 DEG C 5 seconds, 60 DEG C 30 seconds；After reaction terminates, by DNA sample
Cycle threshold Ct and standard curve control, the copy numbers of the mcyD genes of DNA sample is calculated, so as to obtain mcyD bases in raw water
The copy number of cause, and then analyze and obtain the concentration of blue algae producing microcystic toxins in water sample.
In summary, a kind of method and device for detecting blue algae producing microcystic toxins of the present invention is by designing Microcystin
Synthase gene mcyD primer, the DNA of sample to be detected is extracted, carry out quantitative PCR detection, calculated and treated according to standard curve
The copy number of mcyD genes and the concentration of blue algae producing microcystic toxins of test sample sheet, realize it is a kind of it is easy to operate, be easy to grasp
And it can fast and accurately determine the blue algae producing microcystic toxins detection technique of the concentration of blue algae producing microcystic toxins in surface water.
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.
Claims (10)
1. a kind of method for detecting blue algae producing microcystic toxins, comprises the following steps：
Step 1, surface water water sample is taken, after micro porous filtration, filter membrane is subjected to Genome DNA extraction purifying, obtains DNA sample；
Step 2, enter performing PCR amplification by template of the DNA sample that step 1 obtains；
Step 3, pcr amplification product is purified；
Step 4, DNA after purification is subjected to plasmid conversion process, obtains the white colony for having converted plasmid；
Step 5, choose full single bacterium colony and be inoculated in nutrient solution, and shaken cultivation is stayed overnight, for bacterium solution PCR identifications and
Sequencing analysis；
Step 6, plasmid will be extracted by the white colony of identification and analysis, after determining plasmid concentration, gradient dilution, after dilution
Plasmid as standard sample, establish the concentration one-variable linear regression curve corresponding with critical cycle number of standard sample, produce
To standard curve；
Step 7, the DNA sample obtained in step 1 is taken to carry out quantitative PCR measure as template and obtain sample to be tested, according to institute
State standard curve and determine DNA concentration in sample to be tested.
A kind of 2. method for detecting blue algae producing microcystic toxins as claimed in claim 1, it is characterised in that：In step 2,
The PCR reaction systems are used to be for 50 μ L, primer information：- the GCCCACAAAACCCCAACTC-3 ' of sense primer 5 ', anti-sense primer
5’-TTTCTGCCTAATCTTTTCCCCTCT-3’；Response procedures are：95 DEG C of pre-degenerations 5 minutes, it is subsequent 94 DEG C 30 seconds, annealing temperature
60 DEG C of degree extends 40 seconds for 40 seconds and 72 DEG C, carries out 30 circulations, final 72 DEG C extend 10 minutes；Finally by 1.5% agar
Sugared gel electrophoresis confirms pcr amplification product.
A kind of 3. method for detecting blue algae producing microcystic toxins as claimed in claim 1, it is characterised in that：In step 3,
Glue reclaim is carried out using DNA glue reclaims kit, pcr amplification product is purified, and passes through 1.5% Ago-Gel electricity
Swimming confirms whether recovery succeeds.
A kind of 4. method for detecting blue algae producing microcystic toxins as claimed in claim 1, it is characterised in that：In step 4,
It will be inserted through the DNA of step 3 after purification in plasmid vector, and be further transformed into DH5 α competence Escherichia coli solution, then
It is applied to and adds on the LB solid mediums of Amp resistances, is screened by blue hickie, select the white colony for having converted plasmid.
A kind of 5. method for detecting blue algae producing microcystic toxins as claimed in claim 1, it is characterised in that：In step 5,
Choose full single bacterium colony to be inoculated in the liquid LB nutrient solutions containing Amp, stayed overnight in 37 DEG C, 200 revs/min of shaken cultivations,
For bacterium solution PCR identifications and sequencing analysis.
6. a kind of method for detecting blue algae producing microcystic toxins as claimed in claim 1, it is characterised in that step 7 is further
Including：
Reagent needed for real-time quantitative PCR is added, reaction system is 20 μ L, 2 μ LDNA samples；
Set and carry out negative control and positive control, using deionized water as negative control, with the DNA of blue algae producing microcystic toxins
As positive control；
Real-time quantitative PCR reaction is carried out, enters performing PCR amplification；
After reaction terminates, by the cycle threshold Ct and standard curve control of DNA sample, the mcyD genes for calculating DNA sample are copied
Shellfish number, so as to obtain the copy number of mcyD genes in raw water, and analyze and obtain the concentration of blue algae producing microcystic toxins in water sample.
A kind of 7. method for detecting blue algae producing microcystic toxins as claimed in claim 6, it is characterised in that：In step 7,
Performing PCR amplification is entered using two-step method, reaction condition is：Pre-degeneration, 1 circulation, 95 DEG C 30 seconds；PCR reacts, 40 circulations, and 95
DEG C 5 seconds, 60 DEG C 30 seconds.
8. a kind of device for detecting blue algae producing microcystic toxins, including：
DNA sample extraction unit, for by taking surface water water sample, after micro porous filtration, it is pure that filter membrane to be carried out into Genome DNA extraction
Change, obtain DNA sample；
PCR amplification units, enter performing PCR amplification by template of the DNA sample that the DNA sample extraction unit obtains；
Purification unit, for being purified to pcr amplification product；
Plasmid conversion unit, for DNA after purification to be carried out into plasmid conversion process, obtain the white colony for having converted plasmid；
Identification and analysis unit, the single bacterium colony for choosing full is inoculated in nutrient solution, and shaken cultivation is stayed overnight, for bacterium solution
PCR is identified and sequencing analysis；
Standard curve establishes unit, for plasmid will to be extracted by the white colony of identification and analysis, after determining plasmid concentration, and gradient
Dilution, using the plasmid after dilution as standard sample, the concentration unitary corresponding with critical cycle number for establishing standard sample is linear
Regression curve, that is, obtain standard curve；
Quantitative PCR detection unit, for taking the DNA sample that the DNA sample extraction unit obtains to be quantified as template
PCR measure obtains sample to be tested, and the DNA concentration in sample to be tested is determined according to the standard curve.
A kind of 9. device for detecting blue algae producing microcystic toxins as claimed in claim 8, it is characterised in that：The PCR amplifications
Unit uses the PCR reaction systems to be for 50 μ L, primer information：- the GCCCACAAAACCCCAACTC-3 ' of sense primer 5 ', downstream are drawn
- the TTTCTGCCTAATCTTTTCCCCTCT-3 ' of thing 5 '；Response procedures are：95 DEG C of pre-degenerations 5 minutes, it is subsequent 94 DEG C 30 seconds, annealing
Temperature 60 C extends 40 seconds for 40 seconds and 72 DEG C, carries out 30 circulations, and final 72 DEG C extend 10 minutes；Finally by 1.5% fine jade
Sepharose electrophoresis confirms pcr amplification product.
A kind of 10. device for detecting blue algae producing microcystic toxins as claimed in claim 8, it is characterised in that the quantitative PCR
Detection unit is achieved by the steps of：
Reagent needed for real-time quantitative PCR is added, reaction system is 20 μ L, 2 μ LDNA samples；
Set and carry out negative control and positive control, using deionized water as negative control, with the DNA of blue algae producing microcystic toxins
As positive control；
Real-time quantitative PCR reaction is carried out, enters performing PCR amplification；
After reaction terminates, by the cycle threshold Ct and standard curve control of DNA sample, the mcyD genes for calculating DNA sample are copied
Shellfish number, so as to obtain the copy number of mcyD genes in raw water, and analyze and obtain the concentration of blue algae producing microcystic toxins in water sample.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108796103A (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 standard items and preparation, detection method of detection production 2- methyl isoborneol bacterial strains |
| CN109706212A (en) * | 2019-01-24 | 2019-05-03 | 苏州高新区自来水有限公司 | Cyanobacteria rapid detection method in water |
| CN111902546A (en) * | 2018-03-26 | 2020-11-06 | 巴克曼实验室国际公司 | Method for quantifying bioburden in a substance |
| WO2023035334A1 (en) * | 2021-09-13 | 2023-03-16 | 上海城市水资源开发利用国家工程中心有限公司 | Method for quantitatively measuring phycocyanin and special standard product therefor |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111902546A (en) * | 2018-03-26 | 2020-11-06 | 巴克曼实验室国际公司 | Method for quantifying bioburden in a substance |
| CN108796103A (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 standard items and preparation, detection method of detection production 2- methyl isoborneol bacterial strains |
| CN109706212A (en) * | 2019-01-24 | 2019-05-03 | 苏州高新区自来水有限公司 | Cyanobacteria rapid detection method in water |
| WO2023035334A1 (en) * | 2021-09-13 | 2023-03-16 | 上海城市水资源开发利用国家工程中心有限公司 | Method for quantitatively measuring phycocyanin and special standard product therefor |
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Application publication date: 20171222 |