CN101974632B - Method for quantitatively detecting toxigenic microcystis and special standard substance thereof - Google Patents
Method for quantitatively detecting toxigenic microcystis and special standard substance thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting toxigenic microcystis and a special standard substance thereof. The invention provides a standard substance for detecting toxigenic microcystis in a sample, which is a recombinant plasmid or a recombinant bacterium or a recombinant cell containing a mcyA gene; the sequence of the mcyA gene is sequence 1 in a sequence table. The starting plasmid in the recombinant plasmid is pMD-18T. The standard substance for detecting toxigenic microcystis has the characteristics of combining broad-spectrum identification with specificity, high sensitivity, simple and convenient preparation method, long-term storage, good purity and wide linear detection range, and can be used for quickly and quantitatively detecting toxigenic microcystis in a water environment sample.
Description
Technical field
The present invention relates to a kind of detection by quantitative and produce method and the specialized standard article thereof of malicious Microcystis aeruginosa.
Background technology
Microcystin (Microsystems, be called for short MCs) is blue-green algae (cyanobacteria), single ring seven peptide of producing of some Microcystis aeruginosas (Microcystis) especially, ubiquity in the water body of outburst blue-green alga bloom.Microcystin is a kind of hepatotoxin, and long-term drinking contains the water of Microcystin, can cause liver injury even liver cancer.
Microcystin is one type and produced virus gene and regulate and control, the toxin that produces at born of the same parents' intracellular metabolite.Research shows, is not that all Microcystis aeruginosas can both produce Microcystin, and Microcystis aeruginosa produces the toxicity of strain (Toxic strains) and avirulent strain (Nontoxic strains) by the heredity decision, and both are not significantly difference on form.Microcystin is the non-ribosomal synthetic, is accomplished by peptide synthetase complex body gene cluster (mcy).Discovered 11 with the synthetic relevant gene (mcy A-J) of Microcystin; The sequential analysis in mcy zone has disclosed the structure (mcy A-C and mcy D-J) of both-way operation; Mcy A-C genes encoding polypeptide synthetic enzyme (NRPS); Act on two peptidyl intermediates and extend to seven peptidyls, and the cyclisation of peptide subsequently, many ketone of mcy D-J genes encoding heterozygosis-polypeptide synthetic enzyme (PKS-NRPS).
There has been the researchist to investigate the distribution process of Microcystin between frustule and substratum in the laboratory culture test; The result shows; Be in the Microcystis aeruginosa culture of logarithmic phase discharge 10%-20% to multipotency toxin in substratum, most of toxin still are present in the cell; When cell gets into stationary growth phase, maybe be along with the increase of the dead quantity of frustule, the ratio that is discharged into the toxin in the substratum also can increase to some extent.Research also finds, even finish at logarithmic phase, and the living weight of algal cultures possibly be that seldom a part of frustule can be dead very large the time.Therefore, the most of the time of algal grown in the cycle, the content of The dissolved toxin maintains lower level.Microcystin is discharged into the season that generally appears at a large amount of declines of wawter bloom in the water column in a large number in natural water, has perhaps used in the water body all can cause the release that born of the same parents' intracellular toxin is a large amount of after the algicide.Therefore; The content that directly detects Microcystin in the water body can't satisfy the needs of blue algae bloom prealarming and water quality safety control; The separation of Microcystin synthase gene and functional study have not only disclosed the hereditary basis of Microcystin synthetic, and provide accurate detection to produce the method for malicious Microcystis aeruginosa.
Quantitative PCR (quantitative PCR) technology has experienced the transition from the relative quantification to the absolute quantitation, and is wherein maximum with the research of real-time quantitative PCR technical application.Real-time quantitative PCR technology and present used several method have more advantage, for example rapid speed to recently seeing; And need not use microscopic examination, have very high reproducibility, and this technological omnidistance stopped pipe operation; The last handling process that does not have PCR, pollution probability reduces.In addition, because of the quantitatively robotization of process, thereby make the prospect scope of applying more wide.
The malicious Microcystis aeruginosa of single celled product only contains the mcyA gene of a copy.There are some researches show, the product poison Microcystis aeruginosa of under laboratory condition, cultivating, the frustule number is consistent with mcyA gene number.
Summary of the invention
An object of the present invention is to provide the standard substance that produce malicious Microcystis aeruginosa in a kind of test sample.
Standard substance provided by the invention are recombinant plasmid or reorganization bacterium or the reconstitution cell that contains the mcyA gene; The sequence of said mcyA gene is the sequence 1 in the sequence table.
The plasmid that sets out in the said recombinant plasmid is pMD-18T.
Another object of the present invention provides the method for producing malicious Microcystis aeruginosa in a kind of test sample.
Produce the method for malicious Microcystis aeruginosa in the test sample provided by the invention, comprise the steps:
1) is the standard substance template with recombinant plasmid or reorganization bacterium or reconstitution cell, carries out real-time fluorescence quantitative PCR with the system of pcr amplification and increase, set up the concentration one-variable linear regression curve corresponding of standard substance, promptly obtain typical curve with the critical cycle number;
2) with testing sample replacement step 1) in standard substance, carry out real-time fluorescence quantitative PCR amplification, according to the critical cycle number and the typical curve of amplification, obtain the quantity of mcyA gene in the testing sample, promptly obtain producing in the testing sample quantity of malicious Microcystis aeruginosa;
The system of said pcr amplification by real-time fluorescence quantitative PCR amplification buffer, primer to forming with template; The concentration of each primer in system of said primer centering is 0.1 μ mol/L, and a primer of said primer centering is the sequence 2 in the sequence table, and another primer is the sequence 3 in the sequence table.
The concentration of said standard substance template in the system of said pcr amplification is 1.12 * 10
2Copy/μ L, 1.12 * 10
3Copy/μ L, 1.12 * 10
4Copy/μ L, 1.12 * 10
5Copy/μ L, 1.12 * 10
6Copy/μ L, 1.12 * 10
7Copy/μ L or 1.12 * 10
8Copy/μ L.
Annealing temperature in the said PCR reaction is 59 ℃.
Said recombinant plasmid or reorganization bacterium or reconstitution cell are recombinant plasmid or reorganization bacterium or the reconstitution cell that contains the mcyA gene; The sequence of said mcyA gene is the sequence 1 in the sequence table.
Sequence 1 in the sequence table is made up of 376 deoxyribonucleotides.
The plasmid that sets out in the said recombinant plasmid is pMD-18T.
Said sample is specially water sample.
The 3rd purpose of the present invention provides a kind of test kit that produces malicious Microcystis aeruginosa that detects.
The test kit of malicious Microcystis aeruginosa is produced in detection provided by the invention, comprises that primer is right, and a primer of said primer centering is the sequence 2 in the sequence table, and another primer is the sequence 3 in the sequence table.
The 4th purpose of the present invention provides a kind of PCR reagent.
PCR reagent provided by the invention comprises that real-time fluorescence quantitative PCR amplification buffer, primer are right; The primer that each each primer concentration in reagent of said primer centering is the said primer centering of 0.1 μ mol/L. is the sequence 2 in the sequence table, and another primer is the sequence 3 in the sequence table.
McyA gene, primer also are the scopes that the present invention protects to the application that, recombinant plasmid or reconstitution cell produce in test sample in the malicious Microcystis aeruginosa; The sequence of said mcyA gene is the sequence 1 in the sequence table; A primer of said primer centering is the sequence 2 in the sequence table, and another primer is the sequence 3 in the sequence table; Said recombinant plasmid or reorganization bacterium or reconstitution cell are recombinant plasmid or reorganization bacterium or the reconstitution cell that contains the mcyA gene, and the plasmid that sets out in the said recombinant plasmid is pMD-18T.
Experiment of the present invention proves; The standard substance of detection by quantitative Microcystis aeruginosa of the present invention have the advantages that wide spectrum is discerned and specificity combines, and susceptibility is high, and the preparation method is easy; Can prolonged preservation; Purity is good, and linear detection range is wide, can be used for the fast quantification detection that the water surrounding sample produces malicious Microcystis aeruginosa.The present invention is producing design specific primers on the malicious Microcystis aeruginosa gene mcyA, adopts in the real-time PCR detection by quantitative water surrounding and produces malicious Microcystis aeruginosa, and its crucial technology is the outer standard substance of preparation, optimizes real-time PCR reaction conditions.Because quantitative PCR has fast, the sensitive advantage, therefore, this technology can be used for the malicious Microcystis aeruginosa of a spot of product in the rapid detection water surrounding, for the quick early warning of blue-green alga bloom provides technical support.
Description of drawings
Fig. 1 utilizes primer (sequence 2 and sequence 3) to detect green alga chlorella (avirulent strain) and microcystic aeruginosa (product strain)
Fig. 2 quantitative pcr amplification curve
Fig. 3 quantitative PCR typical curve
Fig. 4 quantitative PCR melting curve figure
Fig. 5 standard substance quantitative PCR gel electrophoresis figure
Fig. 6 produces malicious microcystic aeruginosa gene (mcyA) concentration ratio for utilizing microscope count method and quantifying PCR method to detect
The melting curve of Fig. 7 actual water sample quantitative PCR detection
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The standard substance pMD-18T-mcyA plasmid standard preparation of embodiment 1, detection by quantitative Microcystis aeruginosa
1. the cultivation of microcystic aeruginosa
Produce strain microcystic aeruginosa (Microcystis aeruginosa) and be numbered FACHB-905, for available from Wuhan hydrobiont institute of the Chinese Academy of Sciences.According to the cultural method that Inst. of Hydrobiology, Chinese Academy of Sciences provides, cultivate microcystic aeruginosa.Prescription (table 1) configuration BG11 substratum according to providing promptly in the 1000mL deionized water, adds following material, the back 4 ℃ of preservations of sterilizing respectively.
Table 1BG11 culture medium prescription
The cultivation of algae kind must guard against that a spot of algae kind is seeded in a large amount of substratum.The whole absolute aseptic technique of range request excessively of cultivation algae kind is cultivated and the needed experimental tool of transferring must be sterilized, and under aseptic condition, preserves; Switching algae kind must be carried out in super clean bench or aseptic inoculation tank; The algae kind need be cultivated at airtight sterilized space, can not cultivate in the space of opening wide; The light dark period of algal species cultivation is a light: dark=14h: 10h.Must regularly whether pollute in microscopically microscopy algae kind.Primary vaccination is seeded in 10mL algae liquid in the 20mL substratum, according to cultivating down, can help the algae kind to adapt to new substratum and culture environment at the low light level, treat the adaptation of algae kind after, along with the increase of algae kind cell density, can progressively improve illumination, enlarged culturing.
2. to the design of Microcystis aeruginosa Auele Specific Primer
In GenBank, download the sequence of all mcyA of Microcystis aeruginosa, with carrying out its homology of multisequencing comparative analysis in these sequence inputs DNAMAN software, carry out design of primers with synthetic, the GC content of primer is between 40-60%, and PCR product length is 200-500bp.According to Microcystis aeruginosa (numbering: AB019578.2) design primer, primer sequence is following:
McyA-F:5-TTTCATCTCCATCGCCGCA-3 (sequence 2);
McyA-R:5-GGACTCCCACTTGTTTACCGA-3 (sequence 3).
With aseptic double-distilled water primer is mixed with the storage liquid of 200 μ M, packing ,-20 ℃ of preservations.
3. the extraction of Microcystis aeruginosa total genomic dna
Utilize Universal Genomic DNA Extraction Kit Ver.3.0 (TaKaRa Code:DV811A) test kit to extract total DNA of Microcystis aeruginosa, operation steps is with reference to specification sheets, and concrete steps are:
(1) get the microcystic aeruginosa 905 that 100 μ L cultivate, behind the gradient dilution, add the Solution A of 650 μ L, room temperature left standstill 1 minute.
(2) the Solution B of adding 400 μ L, vibration mixes.
(3) the Solution C solution of 4 ℃ of precoolings of adding 1mL, behind the abundant mixing, 12, centrifugal 2 minutes of 000rpm.
Annotate: the preparation method of Solution C solution: before using first; Please the Solution C stoste with 1.1mL moves in the Solution C empty bottle of 125mL; Add the Virahol of 68.75mL, the isopropylcarbinol of 41.25mL then, mix, use after being mixed with Solution C solution.Solution C solution is please in 4 ℃ of preservations.
(4) discard upper organic phase, add the Solution C solution of 4 ℃ of precoolings of 1mL again, behind the abundant mixing, 12, centrifugal 2 minutes of 000rpm.
(5) discard upper organic phase, then aqueous phase solution (colourless lower floor) is transferred to the Filter Cup that places on the Collection Tube, 12, centrifugal 1 minute of 000rpm.
Annotate: organic phase (upper strata) has color, do eliminate, and is attached to DNA and prepares on the film otherwise can hinder DNA.INTERPHASE CARBIDE PRECIPITATION needn't be considered, when filtering, will be removed.
(6) abandon Filter Cup, in filtrating, add the DB Buffer of 400 μ L, mix.
(7) the Spin Column in the test kit is placed on the Collection Tube.Aforesaid operations 6 mixing solutionss are transferred among the Spin Column, 12, centrifugal 1 minute of 000rpm abandons filtrating.
(8) the Rinse A with 500 μ L is added among the Spin Column, and 12,000rpm centrifugal 30 seconds, abandon filtrating.
(9) the Rinse B with 700 μ L is added among the Spin Column, 12.000rpm centrifugal 30 seconds, abandons filtrating.
Notes) Rinse B adds 100% ethanol of 56mL clearly before using first, mixes.Please around the SpinColumn tube wall, add Rinse B, help complete flushing to be built-up in the salt on the tube wall like this.
(10) repetitive operation step 9.
(11) Spin Column is placed on the Collection Tube, 12, centrifugal 1 minute of 000rpm.
(12) Spin Column is placed on the centrifuge tube of new 1.5mL, adds sterile purified water or the Elution Buffer of 50 μ L in the centre of Spin Column film, room temperature left standstill 1 minute.
Annotate) be heated to 65 ℃ to sterile purified water or Elution Buffer and help improving elution efficiency when using.
Centrifugal 1 minute eluted dna of (13) 12,000rpm obtains total DNA of microcystic aeruginosa 905.
4. plasmid standard preparation
A. the segmental preparation of purpose
Get the template of the DNA of the above-mentioned microcystic aeruginosa that obtains 905 of 20 μ L as the PCR reaction; The PCR reaction system is 150 μ L: 4 μ L, 20 μ M upstream primers (sequence 2), 4 μ L, 20 μ M downstream primers (sequence 3), 4 μ L10 μ M dNTP; 7 μ L TaqDNA polysaccharases (5u/ μ L); 12 μ L, 25 μ M MgCl2,15 μ L, 10 * PCR damping fluid, 84 μ L aseptic double-distilled waters.
The PCR response procedures is 94 ℃ of 4min of elder generation; 94 ℃ of 1min then, 59 ℃ of 40s, 72 ℃ of 1.5min carry out 35 circulations; 72 ℃ are extended 10min again.Get 5 μ L PCR products and carry out 2% agarose gel electrophoresis, the result shows that the amplified production size is 376bp.
The B.PCR product purification
Get the above-mentioned PCR product of 140 μ L through 2% agarose gel electrophoresis, cut and utilize behind the glue TaKaRa to cut glue to reclaim test kit and reclaim (TaKaRa Code:DV805A), concrete grammar is following:
(1) use the TAE damping fluid to make sepharose, then the PCR product is carried out agarose gel electrophoresis, and under uv lamp, cut out the sepharose that contains target DNA, paper towel exhausts the liquid of gel surface.Should notice that excision does not as far as possible contain the gel of target DNA part this moment, reduce gel volume as far as possible, improve the DNA recovery.
(2) chopping blob of viscose according to calculating with 1mg=1 μ L, adds isopyknic blob of viscose and melts liquid DR-I Buffer in blob of viscose.
(3) 75 ℃ of heating and melting blob of viscoses (the low melting-point agarose gel only needs 45 ℃ of heating) behind the uniform mixing.Should be interrupted vibration and mix this moment, makes blob of viscose fully melt (about 6-10min).
(4) the DR-II Buffer of 1/2 volume of adding DR-I Buffer amount in above-mentioned blob of viscose thawing liquid, uniform mixing.When the dna fragmentation that separates less than 400bp, should in this solution, add final concentration again and be 20% Virahol.
(5) the Spin Column in the test kit is placed on the Collection Tube.
(6) solution with aforesaid operations 4 is transferred among the Spin Column, and 12, the centrifugal 1min of 000rpm abandons filtrating.
(7) the Rinse A of 500 μ L is added among the Spin Column, 12,000rpm centrifugal 30 seconds, abandon filtrating.
(8) the Rinse B of 700 μ L is added among the Spin Column, 12,000rpm centrifugal 30 seconds, abandon filtrating.Repetitive operation step 8.
(9) Spin Column is placed on the Collection Tube, 12, the centrifugal 1min of 000rpm.
(10) Spin Column is placed on the centrifuge tube of new 1.5mL, adds sterile purified water or the Elution Buffer of 25 μ L in the centre of Spin Column film, room temperature leaves standstill 1min, obtains the PCR product of purifying.
The C.PCR product is connected with plasmid
With the PCR product of above-mentioned purifying with
18-T Vector (TaKaRa Code:D101A) is at T
4Connect under the condition of dna ligase and damping fluid, 4 ℃ of connections are spent the night.Operation steps is with reference to
18-T Vector product service manual, and concrete steps are following:
(1) preparation following DNA ligation solution (20 μ L) in Eppendorf tube:
18-T Vector 1 μ L; PCR purified product 4 μ L, Solution I 5 μ L.
(2) 16 ℃ of reaction 30min.
(3) full dose (20 μ L) connection solution is added to 100 μ L intestinal bacteria competence bacteria DH5a, places 30min in the ice.
Behind (4) 42 ℃ of heating 45s, in ice, place 1min again.
(5) add 1mL LB liquid nutrient medium, 37 ℃ of shaking culture 60min.
(6) the centrifugal 2min of 8000rpm, supernatant discarded, mixing is got to be coated in right amount and is contained Amp
+LB solid culture ware.
(7) 37 ℃ of incubated overnight (12-16h) form single bacterium colony.
D.PCR identifies positive colony
In 0.2mL PCR pipe, add 10 μ L dH in advance
2O with sterilization toothpick picking single bacterium colony, reserves seed for planting containing on the LB solid medium of Amp, and is dipped into dH
2Among the O, 95 ℃ of thermo-cracking 10min obtain DNA.With this DNA is masterplate, is that primer increases with mcyA-F (sequence 2) and mcyA-R (sequence 3), and the result is for obtaining the segmental positive bacterium colony of 376bp.
E. order-checking
Above-mentioned D is accredited as the male colony inoculation in 5mL LB (Amp
+) in the nutrient solution, cultivate 10-12h at 37 ℃, get 2mL bacterium liquid and send the order-checking of order-checking company.Sequencing result contains the Nucleotide shown in the sequence 1 in the ordered list in the plasmid of this bacterium colony; And this plasmid is for inserting the plasmid that
18-T Vector obtains with the sequence in the sequence table 1; Called after pMD-18T-mcyA, the bacterium that will contain pMD-18T-mcyA is named DH5a/pMD-18T-mcyA.
Further with the Microcystis aeruginosa mcyA sequence alignment homology among sequence 1 and the NCBI; The result is as shown in table 2 below; Can find out; The mcyA sequence of multiple Microcystis aeruginosa has the homology more than 98% among sequence 1 and the Gene Bank, do not have homology with other gene, so pMD-18T-mcyA is the standard substance of detection by quantitative Microcystis aeruginosa.
Microcystis aeruginosa mcyA sequence alignment information among table 2. sequence 1 and the GenBank
F. a large amount of acquisitions of plasmid standard
1, the extraction of plasmid
DH5a/pMD-18T-mcyA is inoculated in 150mL contains Amp
+The LB liquid nutrient medium in, incubated overnight (12-16h) is utilized plasmid to extract test kit in a large number and is extracted plasmid (prestige lattice Lars), operation steps is with reference to operation instruction, concrete steps are following:
(1) get incubated overnight bacterium liquid 150mL, in the suitable aseptic centrifugal bottle of packing into, 4 ℃ of centrifugal 30min of 8000rpm, deposition thalline, reject supernatant fully;
(2) add 5mL Buffer I, fully suspendible vibration bacterial sediment scatter it fully and does not exist to there being the wadding piece, and bacterial suspension is moved in the 50mL sterilization centrifuge tube;
(3) add 5mL Buffer II, put upside down centrifuge tube 6-8 time gently, room temperature is placed 5min, makes the complete cracking of bacterium, and solution is transparence;
(4) add 5mL Buffer III, put upside down centrifuge tube 6-8 time immediately, abundant mixing is to white floss generation.Place 10-15min on ice;
(5) above-mentioned lysate is in 4 ℃ of centrifugal 15min of 14000rpm, and careful sucking-off supernatant moves in the new 50mL sterilization centrifuge tube;
(6) add the 10mL Virahol, put upside down centrifuge tube, fully mixing is positioned over 10-15min on ice;
(7) in 4 ℃ of centrifugal 10min of 14000rpm, careful supernatant discarded is inverted and is drained residual liquid gently, adds 0.5mL TE, fully dissolution precipitation agglomerate (available wide mouthful of suction pipe blown and beaten assist in dissolving gently).Move in the centrifuge tube of new 1.5mL.
(8) the plasmid crude extract is with desk centrifuge room temperature high speed centrifugation 2min, in the centrifuge tube of the 1.5mL that the supernatant immigration is new (every pipe 500 μ L).
(9) add 100 μ L contaminant removal liquid A (Buffer IV), mixing gently, the centrifugal 2min of 14000rpm is transferred to supernatant in the new centrifuge tube.
(10) add 100 μ L contaminant removal liquid A (Buffer IV) again, mixing gently, the centrifugal 5min of 14000rpm is transferred to supernatant in the new centrifuge tube.
(11) add 70 μ L contaminant removal liquid B (Buffer V), mixing gently, the centrifugal 5min of 14000rpm is transferred to supernatant in the new centrifuge tube.
(12) add 500 μ L Virahols, mixing, room temperature is placed 10min.The centrifugal 10min of 14000rpm room temperature, abandoning supernatant is washed with 70% ethanol 1mL gently, discards liquid, and room temperature is inverted and is dried 5min.
(13) 500 μ L TE dissolution precipitations (can in 37 ℃ of water-baths, vibrate or blow and beat assist in dissolving gently) with wide mouthful suction pipe.
(14) add 200 μ L contaminant removal liquid C (Buffer VI), place 10-30min behind the mixing on ice, the centrifugal 10min of 14000rpm room temperature, abandoning supernatant adds 1mL 70% washing with alcohol twice gently, and 5-10min is dried in the room temperature inversion evaporates ethanol fully.
(15) add an amount of TE (being generally 500 μ L) dissolution precipitation (can in 37 ℃ of water-baths, vibrate or blow and beat assist in dissolving gently), obtain plasmid standard pMD-18T-mcyA with wide mouthful suction pipe.
(16) with spectrophotometer and agarose electrophoresis plasmid standard pMD-18T-mcyA is carried out quantitatively, quantitatively the back packing is kept at-20 ℃.
2, the detection of plasmid
Behind the 1 * TE gradient dilution of the above-mentioned plasmid standard pMD-18T-mcyA that obtains, utilize ultramicron nucleic acid-protein determinator (NanoDrop ND-2000C, the U.S.) to measure the concentration of plasmid standard with pH 8.0.Carry out the calculating of plasmid copy number according to formula, calculation formula is following:
The copy number of DNA=(molar mass of quality/DNA of DNA) * 6.02 * 10
23
DNA mass concentration=A260 * nucleic acid extension rate * 50/1000 wherein
1bp=649Da in the double-stranded DNA;
After the calculating, confirm the copy number of plasmid standard pMD-18T-mcyA: 5.6 * 10
9Copies/ μ L.
The foundation of embodiment 2, Microcystis aeruginosa quantitative PCR detecting method
1. the optimization of quantitative PCR reaction conditions
Utilize the DNA of the method extraction microcystic aeruginosa (being numbered FACHB-905) (product strain) among the embodiment 1.According to
Premix Ex Taq
TM(Code:DRR041S) specification sheets, the reaction system of qPCR are (20 μ L):
Premix Ex Taq
TM10 μ L, each 0.1-0.5 μ L of upstream and downstream primer (mcyA-F, mcyA-R) (final concentration 0.1-0.5 μ mol/L), by the above-mentioned microcystic aeruginosa that obtains (being numbered FACHB-905) (product strain) DNA 2 μ L, distilled water 7.6 μ L supply volume to 20 μ L.
The qPCR response procedures is following, 1 circulation: 95 ℃, and 5min; 40 circulations: 95 ℃, 5s, 50-60 ℃, 30s, 72 ℃, 30s collects fluorescence in annealing process; The process of melting curve is: 95 ℃, and 1min, 65 ℃ of 1min raise 0.5 ℃ since 65 ℃ of every 30s temperature, and it is 95 ℃ that knot comes temperature.Reaction finishes back 4 ℃ of preservations.
Experimental result shows that the optimum annealing temperature of primer mcyA-F, mcyA-R is 59 ℃, and when best primer concentration was 0.1 μ mol/L, expanding effect was best.Utilize Blast in gene pool, to compare primer (mcyA-F and mcyA-R), show that the algae with other kind does not have similarity (table 2).
Simultaneously, for verifying used primer (mcyA-F and mcyA-R) high specificity, extract green alga chlorella (avirulent strain) (Chlorella sp.; Numbering FACHB-1298 is available from Chinese Academy of Sciences typical case culture collection council algae kind storehouse) DNA is as template, is primer with mcyA-F and mcyA-R; Optimum annealing temperature is 59 ℃; When best primer concentration is 0.1 μ mol/L, carry out PCR, DNA is contrast with microcystic aeruginosa (being numbered FACHB-905) (product strain).
The result is as shown in Figure 1, M:DNA Marker (DL 2000); 1: the green alga chlorella; 2: microcystic aeruginosa (being numbered FACHB-905).From figure, find out to have only microcystic aeruginosa that the fragment of 376bp is arranged, show this primer and green alga chlorella avirulent strain no cross reaction.
2. reach typical curve between quantitative PCR detection limit, quantification area
With the plasmid standard pMD-18T-mcyA that obtains by embodiment 1 behind 10 times of gradient dilutions as quantitative PCR (qPCR) masterplate DNA, the PCR reaction system is 20 μ L, adding 2 μ L concentration is 5.6 * 10
0-5.6 * 10
8The plasmid standard of copy/ul is as reaction template, so ultimate density is 1.12 * 10
1, 1.12 * 10
2, 1.12 * 10
3, 1.12 * 10
4, 1.12 * 10
5, 1.12 * 10
6, 1.12 * 10
7, 1.12 * 10
8With 1.12 * 10
9Copy/ul carries out the qPCR reaction with the top condition of optimizing (obtaining in above-mentioned 1).Negative control is a distilled water, and positive control is the DNA of microcystic aeruginosa (being numbered FACHB-905).Logarithmic value with the standard substance dilution titer is an X-coordinate, and (Threshold Cycle Ct) sets up the typical curve of real-time quantitative PCR for ordinate zou with the critical cycle number.
The pcr amplification curve is as shown in Figure 2, and as can be seen from the figure, amplification curve is level and smooth, and expanding effect is better.
Utilize this qPCR reaction conditions to detect plasmid standard pMD-18T-mcyA, lowest detection is limited to 1.12 * 10
1The copies/ reaction, the detection by quantitative interval is 1.12 * 10
2-1.12 * 10
8Copies/ul is specially 1.12 * 10
2, 1.12 * 10
3, 1.12 * 10
4, 1.12 * 10
5, 1.12 * 10
6, 1.12 * 10
7With 1.12 * 10
8Copy/ul, it is-3.2275 that the typical curve equation is respectively slope of standard curve, R
2=0.9865, amplification efficiency E=10
1/3.2275-1=1.041, i.e. 104.1% (Fig. 3).
The efficient of these standard substance of qPCR amplification of above this research of presentation of results the primer is higher, and linear relationship is good, meets the requirement of preparation real-time quantitative PCR typical curve.
3. the specificity analyses of quantitative PCR detecting method
Whether the melting point curve of quantitative PCR mainly is to be specificity purpose product in order to detect the PCR product.The based composition of dna double chain is different, and melting temperature is different.When the melting temperature that arrives product causes the dna double chain to be opened, discharge with the SYBR Green optical dye of dna double chain combination and to cause fluorescent value and reduce.If it is fine that reaction system and condition optimizing get, the specificity of primer is very high, and the PCR product purity is high, and then the fusing point peak of melting point curve is narrow and sharp, if product is impure, nonspecific reaction product and primer dimer is arranged, and then there are several peaks at the fusing point peak.The primer and quantitative fluorescent PCR condition and program are consistent with top condition in 1, detect plasmid standard pMD-18T-mcyA, with the negative contrast of distilled water, and the positive contrast of DNA of microcystic aeruginosa (being numbered FACHB-905).
The result is as shown in Figure 4, can find out that curve is steady, peak point and narrow, and melting temperature (Tm) is 83 ± 1 ℃, proves that this pcr amplification product is very special.
The PCR product is carried out electrophoresis, and the result is as shown in Figure 5, M:DNA Marker (DL 2000); 1-10; Plasmid standard pMD-18T-mcyA concentration is 1.12 * 10
0-1.1 * 10
9Copy/μ L; P: positive control; N: negative control.From figure, find out that pcr amplification product length is 376bp, this qPCR atopic is strong.
4. the repeatability of quantitative PCR detecting method
Adopt the method for embodiment 1 to make 7 batches plasmid standard pMD-18T-mcyA respectively.
With the plasmid standard pMD-18T-mcyA multigelation of above-mentioned different batches 6 times, carry out qPCR and measure (condition is identical with 1), according to the variation coefficient (CV) of cycle number repeatability is estimated.
The result is as shown in table 3, and the cycle number variation coefficient of plasmid standard pMD-18T-mcyA is respectively 1.86%-4.52%, and these results show that the typical curve repeatability of plasmid standard pMD-18T-mcyA is good.
Repeatability is analyzed between table 3pMD-18T-mcyA different batches
5, the accuracy of quantitative PCR detecting method
Under laboratory condition, cultivate microcystic aeruginosa (being numbered FACHB-905), cultural method reference implementation example 1 will be cultured to concentration and be about 10
8The microcystic aeruginosa gradient dilution of individual/mL is cultivated; After cultivating a week, get nutrient solution respectively, gradient dilution; Utilize the concentration of blood counting chamber at microscopically (amplifying 200 times) counting microcystic aeruginosa; Get corresponding 100ul microcystic aeruginosa nutrient solution then respectively, utilize quantitative PCR to measure the concentration of mcyA behind the extraction DNA, the detailed results of counting and quantitative PCR detection is as shown in table 4.
For more above-mentioned two kinds of methods detect the consistence of microcystic aeruginosa concentration, the concentration mapping of the microcystic aeruginosa that two kinds of methods are detected, the result is as shown in Figure 6, and X-coordinate is for utilizing microcystic aeruginosa concentration (Log after the microscopic counting
10Individual/mL), ordinate zou is to utilize the quantitative PCR detection microcystic aeruginosa to produce the concentration (Log of virus gene mcyA
10Copy/mL), the result of two kinds of detection methods is consistent (Y=1.0117X) almost, the good (R of dependency
2=0.8863), explains that the quantifying PCR method of this patent invention is accurately credible.
Table 4 microscopic counting and quantitative PCR detection are produced the concentration of malicious microcystic aeruginosa
1. actual water sample collection
Actual water sample is taken from Taihu Lake and river on every side thereof, and be in August, 2010 10-11 number sample time.Sampling method is according to the standard sampling system, the water surface once 0.5m get at the place.Sample is put into ice chest and is transported the laboratory back, 4 ℃ of preservations.
2. the mensuration of chlorophyll a and algae density
Be the season of Taihu Lake basin blue-green alga bloom outburst August, utilizes instrument YSI6600V2 to detect the algae density and the chlorophyll a of water sample, and the result is as shown in table 5, and chlorophyll-a concentration is 1.7 * 10
-3-1.35 * 10
-2Mg/L, algae density is 8.2 * 10
5-2.4 * 10
7Individual/L.(chlorophyll a and algae density are to weigh the conventional index of body eutrophication.)
Chlorophyll a concentration and algae density in table 5 water sample
3. the Microcystis aeruginosa in the quantitative PCR detection actual water sample
(1) water sample concentrates
According to algae density, the algae in the centrifugal concentrated water sample.Get the 100ml water sample, 4 ℃, the centrifugal 10min of 4000rpm.Remove supernatant, collecting precipitation probably concentrates most 1ml in the 1.5ml centrifuge tube.And then 4 ℃, the centrifugal 5min of 4000rpm removes supernatant, keeps the about 100-200 μ of remaining sample L, is used for DNA extraction.
(2) DNA extraction
Utilize Universal Genomic DNA Extraction Kit Ver.3.0 (TaKaRa Code:DV811A) test kit to extract the DNA of Microcystis aeruginosa, operation steps is with reference to specification sheets, concrete steps reference implementation example 1, and the volume that finally obtains DNA is 50 μ L.
(3) quantitative PCR detection
Get the DNA 1 μ L of extraction, carry out quantitative PCR detection, 1 of quantitative PCR detection system and procedure reference embodiment 2.It is as shown in table 6 to record the concentration (mcyA gene number) of producing malicious Microcystis aeruginosa in the water sample, and 3.39 * 10
5-1.46 * 10
6Copy/L.
The quantitative PCR melting curve shows (seeing shown in Figure 7), and curve is steady, and the fusing point peak of melting point curve is narrow and sharp, and melting temperature (Tm) is 83 ± 1 ℃, proves that this pcr amplification product is very special.The above results shows that the result of detection of real-time quantitative PCR provided by the invention is more reliable, shows that also plasmid standard prepared among the present invention has better linearity relation and sensing range.
Table 6 utilizes qPCR to detect the content of Microcystis aeruginosa in the water sample
Claims (9)
1. producing the standard substance of malicious Microcystis aeruginosa in the test sample, is recombinant plasmid or reorganization bacterium or the reconstitution cell that contains the mcyA gene; The sequence of said mcyA gene is the sequence 1 in the sequence table.
2. standard substance according to claim 1 is characterized in that: the plasmid that sets out in the said recombinant plasmid is pMD-18T.
3. produce the method for malicious Microcystis aeruginosa in the test sample, comprise the steps:
1) is the standard substance template with recombinant plasmid or reorganization bacterium or reconstitution cell, carries out real-time fluorescence quantitative PCR with the system of pcr amplification and increase, set up the concentration one-variable linear regression curve corresponding of standard substance, promptly obtain typical curve with the critical cycle number; Said recombinant plasmid or reorganization bacterium or reconstitution cell are recombinant plasmid or reorganization bacterium or the reconstitution cell that contains the mcyA gene; The sequence of said mcyA gene is the sequence 1 in the sequence table;
2) with testing sample replacement step 1) in standard substance, carry out real-time fluorescence quantitative PCR amplification, according to the critical cycle number and the typical curve of amplification, obtain the quantity of mcyA gene in the testing sample, promptly obtain producing in the testing sample quantity of malicious Microcystis aeruginosa;
The system of said pcr amplification by real-time fluorescence quantitative PCR amplification buffer, primer to forming with template; The concentration of each primer in system of said primer centering is 0.1 μ mol/L, and a primer of said primer centering is the sequence 2 in the sequence table, and another primer is the sequence 3 in the sequence table.
4. method according to claim 3 is characterized in that: the concentration of said standard substance template in the system of said pcr amplification is 1.12 * 10
2Copy/μ L, 1.12 * 10
3Copy/μ L, 1.12 * 10
4Copy/μ L, 1.12 * 10
5Copy/μ L, 1.12 * 10
6Copy/μ L, 1.12 * 10
7Copy/μ L or 1.12 * 10
8Copy/μ L.
5. according to claim 3 or 4 described methods, it is characterized in that:
Annealing temperature in the said PCR reaction is 59 ℃.
6. according to claim 3 or 4 described methods, it is characterized in that:
The plasmid that sets out in the said recombinant plasmid is pMD-18T.
7. one kind is detected the test kit that produces malicious Microcystis aeruginosa, comprises that primer is right, and a primer of said primer centering is the sequence 2 in the sequence table, and another primer is the sequence 3 in the sequence table.
8. PCR reagent comprises that real-time fluorescence quantitative PCR amplification buffer, primer are right; Each each primer concentration in reagent of said primer centering is 0.1 μ mol/L, and a primer of said primer centering is the sequence 2 in the sequence table, and another primer is the sequence 3 in the sequence table.
9.mcyA gene, primer produce the application in the malicious Microcystis aeruginosa to, recombinant plasmid or reconstitution cell in test sample;
The sequence of said mcyA gene is the sequence 1 in the sequence table;
A primer of said primer centering is the sequence 2 in the sequence table, and another primer is the sequence 3 in the sequence table;
Said recombinant plasmid or reorganization bacterium or reconstitution cell are recombinant plasmid or reorganization bacterium or the reconstitution cell that contains the mcyA gene; The plasmid that sets out in the said recombinant plasmid is pMD-18T.
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