CN107435064A - The Quantitative detection qPCR methods of chrysophyceae are endangered in chlorella culture - Google Patents
The Quantitative detection qPCR methods of chrysophyceae are endangered in chlorella culture Download PDFInfo
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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
- C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
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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
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Abstract
Present invention firstly provides application of the chrysophyceae CoI genes in the molecular labeling as the Quantitative detection qPCR methods that chrysophyceae is endangered in being cultivated for chlorella, and DNA molecular marker and specific primer as shown in SEQ ID No.1；A kind of Quantitative detection qPCR methods that chrysophyceae is endangered in chlorella culture are then provided, using above-mentioned specific primer, quantitative fluorescent PCR is carried out to the genomic DNA of testing sample.The solution have the advantages that finding a kind of molecular labeling and designing specific primer, identify under the conditions of effectively chrysophyceae can be had in chlorella incubation existing for other pollution protozoans.Overcome the deficiency of Standard PCR qualitative detection, drastically increase the Sensitivity and Specificity of detection, shorten test period, simplify operation, and the result of fluoroscopic examination is analyzed by computer software, the pollution caused by product postprocessing process is avoided, more conventional PCR is more objective, sensitive, accurate.
Description
Technical field
The present invention relates to microbial molecules detection technique field, more particularly, in chlorella large-scale culture to chrysophyceae
Carry out the qPCR methods of early monitoring.
Background technology
Chrysophyceae is a kind of important pollution protozoan present in chlorella large-scale culture, it be a kind of flagellate-
Poterioochromonas sp..The flagellate has the characteristics of autotrophy and mixotrophism, easily infects and quickly swallows bead
Algae, once infection, chlorella large-scale culture can be made completely routed in 2 to 4 days, so as to the large-scale culture band to chlorella
Carry out high risks.Therefore for chlorella culture present in chrysophyceae early monitoring and method for identifying molecules determination to close weight
Will.
It is to chrysophyceae research at present more be confined to cultivate it and its phagocytosis habit to Bloom-causing Algal kind in terms of, it is and right
The research of its progress context of detection is very few, only rests on traditional micro- sem observation, DNA direct Sequencings and polymerase chain reaction
(PCR) stage.Traditional morphological observation sensitivity is low, protozoan exist it is large numbers of under the premise of can just complete,
And because protozoan substantial amounts, also have much morphologically that similar species can not except known morphological knowledge in addition to
Distinguish, and DNA direct Sequencings and polymerase chain reaction (PCR) have that detection efficiency is low, there is the phenomenon of false positive in testing result,
Also the judgement to actual conditions is often influenceed.And when detection using three of the above method to chrysophyceae, with regard to explanation
The presence of chrysophyceae has been observed in microscope, and due to chrysophyceae cell individual very little, and microscope highest detection efficiency is 1
Individual every microlitre of cell, also imply that when the presence of micro- sem observation chrysophyceae is that the cell density of chrysophyceae has been at least up to 1000
It is individual every milliliter, and be enough to make the generation of chlorella large-scale culture completely routed under this cell density.Therefore, to chrysophyceae
The method of early monitoring and Molecular Identification is particularly important.
With the development of immunology and molecular Biological Detection technology, develop in terms of protozoic detection means fast
Speed, real-time fluorescence quantitative PCR (Quantitative Real-time PCR, qPCR), high sensitivity fast with its detection speed are special
The characteristics of different in nature strong, has been widely used in protozoic Molecular Detection and in shellfish Pathogen test.On shellfish
Protozoan detection sensitivity is to reach 30 copies.
The content of the invention
The invention aims to the presence of pollutant is quickly monitored in chlorella culture early stage, there is provided a kind of chrysophyceae
Fluorescent quantitative PCR detection method.The more traditional PCR method of this method has higher sensitivity, and easy to operate, can carry out
Substantial amounts of sample detection, and chrysophyceae is quantified, provided so as to cultivate the detection of early stage chrysophyceae pollution for chlorella and monitor
Effective ways.
First technical purpose of the present invention is to provide chrysophyceae CoI genes as endangering chrysophyceae in being cultivated for chlorella
Quantitative detection qPCR methods molecular labeling in application.It is such as straight because protozoan gene order similarity is larger
Connect and use DNA direct Sequencings and polymerase chain reaction (PCR), often occur that detection efficiency is low, and false positive occurs in testing result
Phenomenon.And the present invention after studying repeatedly, find using CoI genes as molecular labeling progress qPCR, can effectively by
Chrysophyceae identifies under the conditions of in chlorella incubation having existing for other pollution protozoans.
Second technical purpose of the present invention is to provide a kind of DNA molecular marker, and its sequence is as shown in SEQ ID No.1.
Then a kind of primer is provided, the molecular labeling that it can be described in specific amplification.
Primer sequence is：
Sense primer CoI-F：5 '-TATATATCATCTTCGGAGCATTCTC-3 ', as shown in SEQ ID NO.2；
Anti-sense primer CoI-R：5 '-TTATTTAGCCTTGGGAACG-3 ', as shown in SEQ ID NO.3.
Above-mentioned molecular labeling or primer can be applicable in chlorella culture the Quantitative detection qPCR methods for endangering chrysophyceae
In.
Further, the present invention provides a kind of Quantitative detection qPCR methods that chrysophyceae is endangered in chlorella culture, adopts
With above-mentioned specific primer, fluorescence quantitative PCR detection is carried out to the genomic DNA of testing sample.The more traditional PCR of this method
Method has higher sensitivity, and easy to operate, can carry out substantial amounts of sample detection, and chrysophyceae is quantified, so as to for
The detection and monitoring of chlorella culture early stage chrysophyceae pollution provide effective ways.
Described Quantitative detection qPCR methods, in addition to step：Using described specific primer, purpose will be included
Into plasmid PGEM-T, the recombinant plasmid PGEMT-CoI of structure determines the gene cloning of amplified fragments as detection chrysophyceae concentration
Measure standard items.
Described Quantitative detection qPCR methods, wherein specific primer：The concentration of sense primer and anti-sense primer is equal
For 10 μm of ol/L.
The fluorescent dye that quantitative fluorescent PCR uses is preferably SYBR Green dyestuffs, and fluoroscopic examination wavelength is 465-
510nm。
Preferably, the reaction system of the quantitative fluorescent PCR is：Included in 20 μ l reaction systems：2 ×
I Master of LightCycler 480SYBR Green 10 μ l, 10 μm of ol/L the μ l of sense primer 0.5,10 μm of ol/L downstream
The μ l of primer 0.5, the μ l of DNA profiling 1, add water to the μ l of cumulative volume 20.
The response procedures of the quantitative fluorescent PCR are：95 DEG C of 5min, 1 circulation；95 DEG C of 10s, 62 DEG C of 20s, 72 DEG C of 30s,
40 circulations.
The quantitative fluorescent PCR, it is as follows with the determination methods of result：
1) the Ct values of positive control should be less than 33.0, and the Ct values of negative control should be greater than 35.0, and positive template is determined for plasmid
Standard items PGEMT-CoI is measured, negative template is aseptic double-distilled water；
2) if Ct values are less than 33.0, and typical amplification curve is presented, then it is determined as positive findings, shows to detect in sample
Contain chrysophyceae；
3) if Ct values are more than 35.0 or without amplified signal, it is determined as negative findings, shows to detect in sample without chrysophyceae；
If 4) Ct values are between 33.0~35.0, it is considered as suspect results, sample needs duplicate test once, if result still exists
In the range of this, then it is determined as negative findings；
5) sample that result is positive, calculate the chrysophyceae in sample according to its Ct value and the quantitation curves established and contain
Amount；
Calculation formula is as follows：
Cell number (cells/ml)=conc/N, N of the sample containing chrysophyceae represents the copy number of CoI genes in each cell.
Most important have the technical effect that of the invention finds a kind of molecular labeling and designs specific primer, can be effective
Identify under the conditions of chrysophyceae is had existing for other pollution protozoans in chlorella incubation.Used fluorescence is determined
Amount round pcr cleverly make use of the DNA efficient amplifications of round pcr, the high specific of specific primer and spectral technique
The advantages of sensitiveness and real-time quantitative, some shortcomings of Standard PCR qualitative detection are overcome, drastically increase the sensitivity of detection
Property and specificity, shorten test period, simplify experimental implementation, and the result of fluoroscopic examination is analyzed by computer software, is kept away
The pollution caused by product postprocessing process is exempted from, more conventional PCR is more objective, sensitive, accurate.
The method of the present invention is also designed to chrysophyceae early stage Rapid identification kit.It has to the low requirement of sample, efficiently
Rate, accurate detection, time saving and energy saving province's expense, the features such as significant effect.Described specific primer is included in the kit.
Specifically, the kit can include detection reagent and extracting genome DNA reagent.The detection reagent includes
Fluorescent dye, specific primer, positive criteria product.The extracting genome DNA reagent preferably includes Roche High Pure
Template Preparation Kit kits.
Detection of the detection method provided by the invention to chrysophyceae has the characteristics of high specificity, high sensitivity, and this method can
The least concentration detected respectively reaches presence and the 0.15cells/ μ l gold of 21copies/ μ l chrysophyceae mitochondria CoI genes
The presence of frustule, and the presence of 1 chrysophyceae cell can be quickly detected in 100000 chlorella cells, to bead
The early monitoring of algae culture pollutant is significant.
Sensitivity, stability, specificity to the inventive method and the validity progress overall merit to environmental sample
It is as follows：
(1) sensitivity：Plasmid plasmid standards for quantitation PGEMT-CoI is diluted 10 respectively with sterilizing distilled water9、108、107、
106、105、104、103、102、101Opies/ μ l, are detected using the system after optimized, the spirit of the established method of checking
Sensitivity.This detection method is 102~109There is good linear relationship, phase relation in the range of the copies/reaction orders of magnitude
Number R2=0.9974, its detection range can reach 8 orders of magnitude, and its sensitivity can be following to 100 copies.
(2) stability：Evaluated in terms of interior repetition two is repeated and criticized between criticizing.Repeatability in batch：Choose with a
Sample, if 5 parallel reaction pipes are reacted simultaneously, examine the coefficient of variation of its Ct value.Repeatability between batch：Choose 2 differences
The recombinant plasmid standard items of concentration, duplicate test 3 times in one week, examine the coefficient of variation of its Ct value.
(3) it is specific：This research have chosen 22 parts of samples and be detected：5 parts of wheel animalcule samples, 8 parts of infusorian samples, 2 parts
Amoeba sample, 1 part of flagellate sample, 2 parts of environmental samples from Wuhan City, Hubei Province East Lake, 2 parts come from Wuhan City, Hubei Province
In aquatile research institute of the Chinese Academy of Sciences of city the environmental sample in pond and from Dafeng City of Jiangsu Province spirulina breeding base cultivate
Each 1 part of the environmental sample of pond and soil.Detected using PCR method, testing result is shown in addition to chrysophyceae, and remaining sample is equal
For negative findings.
Brief description of the drawings
Fig. 1 is the standard curve of chrysophyceae CoI gene absolute quantitations, and y=-4.0439 × log (conc)+45.344 (is noted：
Conc represents the standard plasmid copy number in reaction system)；
Fig. 2 is the fluorescence signal figure of quantitative fluorescent PCR susceptibility detection, and standard concentration from left to right is respectively 109
~102copies/μl；
Fig. 3 is the PCR primer agarose gel electrophoresis figure of primer specificity checking,
Wherein M：100bp DNA Ladder；1：Jiangxi brachionus plicatilis；2：Hainan brachionus plicatilis；3：Yunnan pleat
Wrinkle Brachionus；4：Blood cell algae spinning roller worm；5：Beijing water wheels worm；6：Tetrahymena；7：Hainan abdomen post worm；8：Beijing
Sterkiella；9：Yunnan prosopyle worm；10：Beijing paramecium；11：Vorticella；12：CBN Vannella；13：Wuhan
Heterolobosean；14：Hainan flagellate Suigetsumonas clinomigrationis；15：Jiangxi Spathidium
stammeri；16：Jiangxi Oxytricha sp.；17：East Lake water sample 1；18：East Lake water sample 3；19：Aquatic institute's water sample 1；20：Water
Raw institute water sample 2；21：Environmental sample 43#；22：Environmental sample 2-2；23：Water compares；24：Chrysophyceae；
Fig. 4 is the fluorescence signal figure of detection of each period, cell proportion from left to right be respectively adjustment period, logarithmic phase and
Stationary phase；
Fig. 5 is that the cell of chlorella is set as 106The fluorescence signal figure of detection is tested in individual addition, from left to right chlorella
Cell proportion with chrysophyceae is respectively 0：1、102:1、103:1、104:1、105:1、106:1；
Fig. 6 is that the cell of chlorella is set as 107The fluorescence signal figure of detection is tested in individual addition, from left to right chlorella
Cell proportion with chrysophyceae is respectively 0：1、102:1、103:1、104:1、105:1、106:1、107:1；
Fig. 7 is that the cell of chlorella is set as 108The fluorescence signal figure of detection is tested in individual addition, from left to right chlorella
Cell proportion with chrysophyceae is respectively 0：1、102:1、103:1、104:1、105:1、106:1、107:1、108:1。
Embodiment
Following examples further illustrate present disclosure, but should not be construed as limiting the invention.Without departing substantially from
In the case of of the invention spirit and essence, the modifications or substitutions made to the inventive method, step or condition belong to the present invention
Scope.
Unless otherwise specified, the conventional meanses that technological means used in embodiment is well known to those skilled in the art.
Embodiment 1.
Design primer, step：
Molecular barcode method is commonly used for identifying microorganism pollution, mainly studies at present among all eucaryotes all
The part of an existing gene --- cytochrome oxidase I (CoI cytochrome c oxidase I) gene.
This partial dna sequence can accurately distinguish out species difference, have the specificity of each species, just as " identity card " of species is compiled
Number.During algae culture, it may appear that the not pollution of same species of microorganism, for the information of clear and definite pollutant, we are by CoI bases
Because of the prioritizing selection as monitoring.
The chrysophyceae CoI genes for occurring polluting in chlorella incubation are entered into performing PCR amplification, obtain aim sequence and selection
Molecular labeling be CoI genes, using the CoI gene orders (table 1) of known 3 chrysophyceae, and GenBank obtain 43
The common protozoic CoI gene orders (table 2) of bar, compared and analyzed by bioinformatics, choose aim sequence specificity area
The target-gene sequence that domain expands as qPCR, using the softwares of Primer Primer 5.0, a pair of qPCR primers are designed, purpose expands
Increasing fragment is 250bp, and the extension increasing sequence is the specific regions of purpose fragment, and in the specific amplification of primer, we select
22 parts of common microbiological samples carry out primer specificity checking, including 5 parts of wheel animalcule samples, 8 parts of infusorian samples, 2 parts of changes
Shape worm sample, 1 part of flagellate sample, 2 parts of environmental samples from Wuhan City, Hubei Province East Lake, 2 parts come from Wuhan City, Hubei Province
The environmental sample in pond and from Dafeng City of Jiangsu Province spirulina breeding base culture pond in aquatile research institute of the Chinese Academy of Sciences
Each 1 part with the environmental sample of soil.Detected using PCR method, testing result is shown in addition to chrysophyceae, and remaining sample is
Negative findings.Result above absolutely proves that the primer possesses the specificity of amplification purpose fragment.
Primer sequence is：
Sense primer CoI-F：5’-TATATATCATCTTCGGAGCATTCTC-3’
Anti-sense primer CoI-R：5’-TTATTTAGCCTTGGGAACG-3’
Table 1：The chrysophyceae CoI gene sequence information tables obtained on GenBank：
Table 2：The information of the representative protozoan CoI gene orders obtained on GenBank
Embodiment 2.
The structure of plasmid plasmid standards for quantitation and preparation
Experimental procedure：
1. the structure of plasmid standards for quantitation
Clone to obtain the CoI genetic fragments containing target-gene sequence using PCR method, recombinate in carrier PGEM-T, and
Carry out determined dna sequence.The recombinant plasmid of structure is named as PGEMT-CoI as plasmid standards for quantitation.
2. the preparation of plasmid standards for quantitation
Plasmid PGEMT-CoI carries out extraction purification with Omega plasmid extraction kits, is determined according to Nanodrop8000
The concentration and quality and Avgadro constant of plasmid are converted into copy number.
Calculation formula is as follows：
The mean molecule quantity of one base-pair is 660g/mol；
The total length of plasmid adds the length of Insert Fragment for the total length of carrier；
N：Represent Avgadro constant (6.02 × 1023copies/mol)。
Embodiment 3.
The extraction of genomic DNA
Experimental procedure：
1. the enrichment of chrysophyceae cell
The condition of Concentration of Gold frustule is optimized, different centrifugal rotational speeds and centrifugation time are set respectively to pure culture
The nutrient solution of chrysophyceae centrifuged, after by supernatant microscopy under the microscope, seen whether cell residence, condition is shown in Table 3,
As a result obtain carrying out centrifugal enrichment cell to the nutrient solution of the chrysophyceae of pure culture under conditions of rotating speed is 12000g centrifugations 5min
Retained afterwards by the way that sediments microscope inspection is acellular.
The condition and result of the Concentration of Gold frustule of table 3
2. the extraction of genomic DNA
Compare different DNA extraction kit extraction chrysophyceae DNA extraction efficiency, DNeasy Blood＆ have been respectively adopted
Tissue Kit (QIAGEN), DNeasy Plant Mini Kit (QIAGEN) and High Pure Template
Three kinds of kits of Preparation Kit (Roche), as a result obtain High Pure Template Preparation Kit
(Roche) extraction efficiency highest.
The nutrient solution of chrysophyceae and chlorella to pure culture carries out cell count, obtains both cell densities.Respectively take 2ml
High Pure Template Preparation Kit (Roche) and DNeasy Plant are respectively adopted in cell culture fluid
Mini Kit (QIAGEN) DNA extraction kit both DNA of extraction, concentration and quality using Nanodrop8000 to DNA
It is measured.Choose concentration and the optimal DNA of quality is saved backup.
Embodiment 4.
The foundation and optimization of fluorescent quantitative PCR detection method
Experimental procedure：
1. the foundation of absolute quantitation standard curve
1) plasmid PGEMT-CoI carries out extraction purification with Omega plasmid extraction kits, is determined according to Nanodrop8000
Plasmid concentration and quality and Avgadro constant be converted into copy number.
2) quantitative PCR reaction condition optimization
Pass through the quantification PCR primer and the supermix of different company and response procedures to various concentrations in reaction system
The experimental result of middle different annealing temperature and annealing time etc. is compared, and selects that reaction sensitivity is high, background fluorescence signal
It is low, have typical S types amplification fluorescent signal curve, reaction system and reaction condition of the reaction efficiency close to 1.The present invention uses
Quantitative real time PCR Instrument be Roche companies LightCycle480.The program of II fluoroscopic examinations is arranged on each circulation second
Fluorescence signal, Detection wavelength 465-510nm are gathered during the end of the step.
Quantitative PCR circulation uses three-step approach, and annealing and extension are separately carried out.
The screening of reaction system：
| Reagent | Screening conditions |
| supermix | Roche, Bio-Rad, Kang Wei |
| Quantification PCR primer | 0.3μmol/L、0.25μmol/L、0.2μmol/L、0.15μmol/L |
The screening of reaction condition：
| Condition | Screening conditions |
| Annealing temperature | 58℃、60℃、62℃、64℃ |
| Annealing time | 10s、20s、30s |
Optimized quantitative PCR program is：
Optimized quantitative PCR reaction system (20 μ l reaction systems) is：
3) the amplification standard curve of absolute quantitation is established
With sterilizing 10 times of gradient dilution plasmid standards for quantitation PGEMT-CoI of distilled water.Plasmid concentration is respectively 10 after dilution9、
108、107、106、105、104、103、102、101Copies/ μ l, reacted using the system after optimized and condition.Detection knot
The software (LightCycler480) carried after beam using fluorescent PCR draws standard curve, refers to Fig. 1.
This detection method is 102~109There is good linear relationship in the range of the copies/reaction orders of magnitude, it is related
It is R=0.9974, its detection range can reach 8 orders of magnitude, and its sensitivity can be following to 100 copies, refers to Fig. 2.
The calculation formula of the copy number of chrysophyceae CoI genes in detection reaction：
Conc=10(-0.248×Ct+11.213)
The calculation formula of Gold Samples concentration of algae：
Cell number (cells/ml)=conc/N (note of the sample containing chrysophyceae：N represents the copy of CoI genes in each cell
Number)
2. stability analysis
From from batch between repeatability and batch in evaluated quantitative fluorescent PCR reaction system as possible in terms of repeatability two
Stability.
1) Repeatability checking in criticizing:
Choose with a sample, nucleic acid is extracted to it according to the method described in embodiment 2 and 3.Using the body after optimized
System and 5 parallel reaction pipes of condition setting are reacted simultaneously, and the coefficient of variation of its Ct value is examined using statistical method.In batch
The coefficient of variation (CV) of the Ct values of repeatability detection is 1.69%.
2) Repeatability checking between criticizing：
Choose the recombinant plasmid standard items of 2 various concentrations, using the reaction system and condition described in embodiment 4, one week
It is interior to repeat detection 3 times, the coefficient of variation of its Ct value is examined using statistical method.The variation lines of the Ct values of repeatability detection between batch
Number (CV) is respectively 2.07% and 1.58%.Show that the detection method that the present invention establishes has good stability and repeatability.
3. specificity analysis
To examine the applicability for the fluorescence quantifying PCR method established, this research have chosen 22 parts of samples and be detected：5 parts
Wheel animalcule sample, 8 parts of infusorian samples, 2 parts of amoeba samples, 1 part of flagellate sample, 2 parts from Wuhan City, Hubei Province East Lake
Environmental sample, the environmental sample in 2 parts of ponds in aquatile research institute of the Wuhan City, Hubei Province Chinese Academy of Sciences and from Jiangsu
Each 1 part of the environmental sample of Dafeng City of province spirulina breeding base culture pond and soil.Detected using PCR method, detection knot
Fruit shows that in addition to chrysophyceae remaining sample is negative findings, as a result sees Fig. 3.
4. the judgement of testing result and quantifying for chrysophyceae
1) the Ct values of positive control should be less than 33.0, and the Ct values of negative control should be greater than 35.0.Positive template is determined for plasmid
Standard items PGEMT-CoI is measured, negative template is aseptic double-distilled water.
2) if Ct values are less than 33.0, and typical amplification curve is presented, then it is determined as positive findings, shows to detect in sample
Contain chrysophyceae.
3) if Ct values are more than 35.0 or without amplified signal, it is determined as negative findings, shows to detect in sample without chrysophyceae.
If 4) Ct values are between 33.0~35.0, it is considered as suspect results, sample needs duplicate test once.If result still exists
In the range of this, then it is determined as negative findings.
5) sample that result is positive, calculate the chrysophyceae in sample according to its Ct value and the quantitation curves established and contain
Amount.
Calculation formula is as follows：
Cell number (cells/ml)=conc/N (note of the sample containing chrysophyceae：N represents the copy of CoI genes in each cell
Number)
Embodiment 5.
The determination of mitochondria CoI gene copy numbers
Experimental procedure：
Take the chrysophyceae cell suspending liquid of adjustment period, logarithmic phase and stationary phase to carry out cell count respectively, obtain respective thin
Born of the same parents' density.Nucleic acid is extracted to it according to the method described in embodiment 2 and 3.Reacted using the system after optimized and condition.
Detection calculates the CoI gene copy numbers of each self-contained chrysophyceae according to the method combined standard curve described in embodiment 4 after terminating,
The mitochondria CoI gene copy numbers of each period chrysophyceae are calculated in conjunction with cell density.Testing result is shown in Table 3, each period inspection
The fluorescence signal of survey is shown in Fig. 4.
Calculation formula is as follows：
Mitochondria CoI gene copy numbers (copies/cell)=conc/P*V of chrysophyceae0/V1(note：P represents cell density,
V0Represent the Cell suspension volumes taken during extraction DNA, V1Represent the lysate volume of dissolving DNA)
The fluorescent quantitative PCR result of 3. each period of table detection
| Period | Cell density (individual/ml) | Ct values | The CoI gene copy numbers of reckoning |
| Adjustment period | 7.153×105 | 15 | 307.595 |
| Logarithmic phase | 1.501×106 | 16.48 | 56.18 |
| Stationary phase | 1.844×106 | 18.58 | 42.33 |
Embodiment 6.
Chrysophyceae and chlorella addition experiment
Experimental procedure：
1. the cell of chlorella is set as 106It is individual
0,10 are taken respectively0、101、102、103、104Individual chrysophyceae cell, 1 is followed successively by according to chlorella and chrysophyceae cell proportion:
0、106:1、105:1、104:1、103:1、102:1 and 0:1, nucleic acid is extracted to it according to the method described in embodiment 2 and 3.Using
System and condition after optimized are reacted.Detection combines the quantitative mark established according to the method described in embodiment 4 after terminating
Directrix curve checks the validity of testing result.The gold in sample is calculated according to its Ct value and the quantitation curves established simultaneously
Algae content.As a result display is less than or equal to 10 when chlorella and chrysophyceae cell proportion4:The presence of chrysophyceae can be effectively detected when 1,
Testing result is shown in Table 4, and the fluorescence signal of detection is shown in Fig. 5.
The cell of the chlorella of table 4. is set as 106Fluorescent quantitative PCR result when individual
2. the cell of chlorella is set as 107It is individual
0,10 are taken respectively0、101、102、103、104、105Individual chrysophyceae cell, according to chlorella and chrysophyceae cell proportion successively
For 1:0、107:1、106:1、105:1、104:1、103:1、102:1 and 0:1, it is extracted according to the method described in embodiment 2 and 3
Nucleic acid.Reacted using the system after optimized and condition.Detection is combined according to the method described in embodiment 4 after terminating and established
Quantitation curves check testing result validity.Sample is calculated according to its Ct value and the quantitation curves established simultaneously
Chrysophyceae content in product.As a result display is less than or equal to 10 when chlorella and chrysophyceae cell proportion4:Gold can be effectively detected when 1
The presence of algae, testing result are shown in Table 5, and the fluorescence signal of detection is shown in Fig. 6.
The cell of the chlorella of table 5. is set as 107Fluorescent quantitative PCR result when individual
3. the cell of chlorella is set as 108It is individual
0,10 are taken respectively0、101、102、103、104、105、106Individual chrysophyceae cell, according to chlorella and chrysophyceae cell proportion
It is followed successively by 1:0、108:1、107:1、106:1、105:1、104:1、103:1、102:1 and 0:1, according to the side described in embodiment 2 and 3
Method extracts nucleic acid to it.Reacted using the system after optimized and condition.Detect the side described according to embodiment 4 after terminating
Method combines the validity that the quantitation curves established check testing result.Simultaneously according to its Ct value and the quantitative criterion established
Curve calculates the chrysophyceae content in sample.As a result display is less than or equal to 10 when chlorella and chrysophyceae cell proportion4:Can be effective when 1
The presence for detecting chrysophyceae, testing result is shown in Table 6, and the fluorescence signal of detection is shown in Fig. 7.
The cell of the chlorella of table 6. is set as 108Fluorescent quantitative PCR result when individual
From the experiment of above example as can be seen that detection of the detection method to chrysophyceae of the present invention have high specificity,
The characteristics of high sensitivity, the plasmid control curve y=-4.0439x+45.344 obtained according to quantitative fluorescent PCR, it is thin that x represents log
The copy number of born of the same parents, y represent ct fluorescent values, and when period reaches 40, the least concentration that can be calculated to detect can reach
The presence of 21copies/ μ l chrysophyceae mitochondria CoI genes；A chrysophyceae cell CoI gene copy number is put down as known to the above results
It is 135copies, can calculates and learn that detection limit is 0.15cells/ μ l chrysophyceae cells, and can be in 100000 beads
The presence of 1 chrysophyceae cell is quickly detected in frustule, and the early monitoring that pollutant is cultivated chlorella is significant.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, some improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Claims (16)
1. chrysophyceae CoI genes are in the molecule mark as the Quantitative detection qPCR methods that chrysophyceae is endangered in being cultivated for chlorella
Application in note.
2. a kind of DNA molecular marker, its sequence is as shown in SEQ ID No.1.
3. a kind of primer, molecular labeling that it can be described in specific amplification claim 2.
4. primer according to claim 3, its sequence is as follows：
CoI-F：5’-TATATATCATCTTCGGAGCATTCTC-3’；
CoI-R：5’-TTATTTAGCCTTGGGAACG-3’.
5. molecular labeling described in claim 2 or the primer of claim 3 or 4 endanger the quick of chrysophyceae in chlorella is cultivated
Quantitatively detect the application in qPCR methods.
6. the Quantitative detection qPCR methods of chrysophyceae are endangered in a kind of chlorella culture, it is characterised in that using claim 3
Or the specific primer described in 4, fluorescence quantitative PCR detection is carried out to the genomic DNA of testing sample.
7. endangering the Quantitative detection qPCR methods of chrysophyceae in chlorella culture according to claim 6, its feature exists
In, in addition to step：Using described specific primer, by the gene cloning comprising purpose amplified fragments to plasmid PGEM-T
In, the recombinant plasmid PGEMT-CoI of structure is as the plasmid standards for quantitation for detecting chrysophyceae concentration.
8. endangering the Quantitative detection qPCR methods of chrysophyceae in chlorella culture according to claim 6, its feature exists
In the specific primer：The concentration of sense primer and anti-sense primer is 10 μm of ol/L.
9. endangering the Quantitative detection qPCR methods of chrysophyceae in chlorella culture according to claim 6, its feature exists
In for the fluorescent dye that the quantitative fluorescent PCR uses for SYBR Green dyestuffs, fluoroscopic examination wavelength is 465-510nm.
10. endangering the Quantitative detection qPCR methods of chrysophyceae in chlorella culture according to claim 6, its feature exists
In the reaction system of the quantitative fluorescent PCR is：Included in 20 μ l reaction systems：2 × the SYBR of LightCycler 480
I Master of Green 10 μ l, 10 μm of ol/L sense primer 0.5 μ l, 10 μm of ol/L anti-sense primer 0.5 μ l, the μ of DNA profiling 1
L, add water to the μ l of cumulative volume 20.
11. endangering the Quantitative detection qPCR methods of chrysophyceae in chlorella culture according to claim 6, its feature exists
In the response procedures of the quantitative fluorescent PCR are：95 DEG C of 5min, 1 circulation；95 DEG C of 10s, 62 DEG C of 20s, 72 DEG C of 30s, 40
Circulation.
12. endangering the Quantitative detection qPCR methods of chrysophyceae in the chlorella culture according to claim 6-11, it is special
Sign is, as follows with the determination methods of result：
1) the Ct values of positive control should be less than 33.0, and the Ct values of negative control should be greater than 35.0, and positive template is that plasmid is quantitatively marked
Quasi- product PGEMT-CoI, negative template are aseptic double-distilled water；
2) if Ct values are less than 33.0, and typical amplification curve is presented, then it is determined as positive findings, shows to detect and contain in sample
Chrysophyceae；
3) if Ct values are more than 35.0 or without amplified signal, it is determined as negative findings, shows to detect in sample without chrysophyceae；
If 4) Ct values are between 33.0~35.0, it is considered as suspect results, sample needs duplicate test once, if result is still in this model
In enclosing, then it is determined as negative findings；
5) sample that result is positive, the chrysophyceae content in sample is calculated according to its Ct value and the quantitation curves established；
Calculation formula is as follows：
Cell number (cells/ml)=conc/N, N of the sample containing chrysophyceae represents the copy number of CoI genes in each cell.
13. a kind of qPCR kits, it includes the specific primer described in claim 3 or 4.
14. kit according to claim 12, it is characterised in that：Including detection reagent and extracting genome DNA reagent.
15. kit according to claim 13, it is characterised in that：The detection reagent includes fluorescent dye, right will
Ask specific primer described in 3 or 4, positive criteria product.
16. kit according to claim 13, it is characterised in that：The extracting genome DNA reagent includes Roche's
High Pure Template Preparation Kit kits.
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