CN105695568A - Quantitative PCR reagent box for detecting DNA damage of cyanobacteria and application - Google Patents
Quantitative PCR reagent box for detecting DNA damage of cyanobacteria and application Download PDFInfo
- Publication number
- CN105695568A CN105695568A CN201510881486.9A CN201510881486A CN105695568A CN 105695568 A CN105695568 A CN 105695568A CN 201510881486 A CN201510881486 A CN 201510881486A CN 105695568 A CN105695568 A CN 105695568A
- Authority
- CN
- China
- Prior art keywords
- gene
- anl50
- pcr
- primer
- quantitative pcr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a quantitative PCR reagent box for detecting DNA damage of cyanobacteria and application.A common PCR reaction solution I includes a pair of specific primers designed oriented to a synechococcus anl50 gene, a common PCR reaction solution II includes a pair of specific primers designed oriented to a synechococcus CDS: ABB57481.1 gene, a quantitative PCR reaction solution I includes a pair of specific primers designed oriented to the synechococcus anl50 gene, and a quantitative PCR reaction solution II includes a pair of specific primers designed oriented to the synechococcus CDS: ABB57481.1 gene, wherein the combination position of the common PCR upstream primer of the amplification anl50 gene and the amplification anl50 gene is located at the upstream of the combination position of the quantitative PCR upstream primer of the amplification anl50 gene and the amplification anl50 gene, and the combination position of the common PCR downstream primer of the anl50 gene and the anl50 gene is located at the downstream of the combination position of the quantitative PCR downstream primer of the anl50 gene and the anl50 gene.The reagent box is high in sensitivity and specificity, a detection method is easy, convenient and fast to implement, and an experiment result is reliable.The quantitative PCR reagent box is suitable for quantitative detection of plasmid DNA copy number detection of cyanobacteria and DNA damage of cyanobacteria, and practical application value is achieved on predicating outburst and subduction of cyanobacterial bloom.
Description
Technical field
The present invention relates to molecular biology and experimental technique field, it is more particularly to the Fluorescent quantitative PCR (RealTimePolymeraseChainReaction of a kind of detection by quantitative Cells of Blue-green Algae plasmid DNA copies number and cyanophyceae DNA damage, RealTimePCR) test kit, also relates to the purposes of a kind of cyanophyceae DNA real-time fluorescence quantitative PCR test kit。
Background technology
In molecular biology, real-time fluorescence quantitative PCR (real-timequantitativePCR) is as a kind of method carrying out quantitative analysis to unknown template, owing to it is quantitatively accurate, highly sensitive, high specificity and high repeatability and other advantages have become as a kind of important tool, be widely used in the research of the aspects such as the mensuration of starting template, genotypic analysis, melting curve analysis。
Research shows, intracellular plasmid copy number relevant to the growth course of cell (E,AzzoniAR,PrazeresDM,MonteiroGA,FJ.MolBiotechnol.2007,37 (2): 120-126.)。The copy number of bioblast and the environment residing for the presence or absence of plasmid and cell and the closely related (PickettMA of compound exposure, EversonJS, PeadPJ, ClarkeIN.Microbiology.2005,151 (Pt3): 893-903)。What the plasmid copy number of escherichia coli (Escherichiacoli) in prokaryote antibacterial was studied is more, and the plasmid copy number of chlamydial (Chlamydiatrachomatis and Chlamydophilapneumoniae (N16)) not of the same race also has research。In eukaryote, Chinese hamster ovary cell (Chinesehamsterovary (CHO) cells) also has relevant report。Still lacking the plasmid copy number to prokaryote Cells of Blue-green Algae carries out quantitative method at present。Quantitative to cyanophyceae plasmid copy number, it is possible to evaluate the physiological status of Cells of Blue-green Algae, it is possible to the detection Cells of Blue-green Algae response to environment-stress, thus the risk factor detected in environment。
As a kind of sensitivity detection method, quantitative PCR has been used for the DNA damage (JungD of detection environmental pattern biology section's bony fish side edge bottom (Fundulusheteroclitus), ChoY, MeyerJN, DiGiulioRT.CompBiochemPhysiolCToxicolPharmacol.2009,149 (2): 182-6.)。By the mitochondrial DNA in supercoiled plasmid DNA and cancerous cell is carried out real-time fluorescence quantitative PCR, the conformation indicating template DNA can affect the method amplification efficiency (ChenJ to it, KadlubarFF, ChenJZ.NucleicAcidsRes.2007,35 (4): 1377-88.)。In real-time fluorescence quantitative PCR process, the DNA of relaxed configuration (open loop and/or linear) is easily available fluorescence signal compared with the DNA of superhelix；Meanwhile, the chain interruption on open loop structure DNA can cause sensitive response (ChenJ, KadlubarFF, ChenJZ.NucleicAcidsRes.2007,35 (4): the 1377-88. of the method；ChanSW, NguyenPN, AyeleD, ChevalierS, AprikianA, ChenJZ.MutatRes.2011,716 (1-2): 40-50.)。Utilize early stage detection (ChanSW, NguyenPN, the AyeleD of the method Successful utilization mitochondrial DNA damage of a small amount of prostatic cell in Male urine, ChevalierS, AprikianA, ChenJZ.MutatRes.2011,716 (1-2): 40-50.)。Still lack at present and utilize the quantifying PCR method method to Cells of Blue-green Algae DNA damage。Detection to Cells of Blue-green Algae DNA damage, it is possible to the sensitive assessment Cells of Blue-green Algae response to environment-stress, has the using value of reality in blue algae bloom prealarming。
Summary of the invention
It is an object of the invention to there are provided a kind of real-time quantitative PCR test kit by detection by quantitative Cells of Blue-green Algae plasmid and Cells of Blue-green Algae DNA damage, for evaluating the physiological status of Cells of Blue-green Algae, and the response that Cells of Blue-green Algae is to environment-stress。This test kit is applicable to all types fluorescence quantitative gene extender that presently, there are on market。The present invention can detect the plasmid copy number level of Cells of Blue-green Algae, it is judged that Cells of Blue-green Algae normally whether, and the DNA damage degree of Cells of Blue-green Algae, in Cells of Blue-green Algae is to the response of environment-stress, have good application prospect。
It is another object of the present invention to there are provided a kind of quantitative PCR kit detecting cyanophyceae DNA damage Cells of Blue-green Algae plasmid copy number quantitatively and DNA damage detect in application, the sensitivity of this test kit and specificity are high, detection method is easy, quick, and experimental result is reliable。
In order to realize above-mentioned purpose, the present invention adopts techniques below measure:
A kind of quantitative PCR kit detecting cyanophyceae DNA damage, this test kit, including: comprise 2 × TaqPCRMasterMix of the common PCR reaction YeI{Duo Ge company of the primer of Synechococcus (Synechococcussp.) anl50 gene, such as the 2xPCRReagent (KT207) that sky root is biochemical }, comprise 2 × TaqPCRMasterMix of the common PCR reaction YeII{Duo Ge company of the primer of Synechococcus (Synechococcussp.) CDS:ABB57481.1 gene, such as the 2xPCRReagent (KT207) that sky root is biochemical }, blank product (aseptic ultra-pure water), comprise 2 × realtimePCRMix of the quantitative PCR reactant liquor I{ Duo Ge company of the quantification PCR primer of Synechococcus (Synechococcussp.) anl50 gene, QuantiTectPrimerAssay (Productno.249900) such as Qiagen } and comprise the 2 × realtimePCRMix of quantitative PCR reactant liquor II{ Duo Ge company of quantification PCR primer of Synechococcus (Synechococcussp.) CDS:ABB57481.1 gene, QuantiTectPrimerAssay (Productno.249900) such as Qiagen }, it is characterized in that: common PCR reaction liquid I comprises a pair specific primer for Synechococcus (Synechococcussp.) anl50 gene design, common PCR reaction liquid II comprises a pair specific primer for Synechococcus (Synechococcussp.) CDS:ABB57481.1 gene design, quantitative PCR reactant liquor I comprises a pair specific primer for Synechococcus (Synechococcussp.) anl50 gene design, quantitative PCR reactant liquor II comprises a pair specific primer for Synechococcus (Synechococcussp.) CDS:ABB57481.1 gene design。Wherein expanding the regular-PCR forward primer of anl50 gene and the binding site of this gene in the upstream of its quantitative PCR forward primer Yu this gene binding site, the regular-PCR downstream primer of anl50 gene and the binding site of this gene are in the downstream of its quantitative PCR downstream primer Yu this gene binding site。CDS:ABB57481.1 gene is designed primer by same method。
In a preferred version of the present invention, common PCR reaction liquid I is by the primer pair of amplification Synechococcus (Synechococcussp.) anl50 gene, 2xPCRReagent (KT207, sky root is biochemical) and aseptic ultra-pure water composition。In a concrete scheme of the present invention, the regular-PCR forward primer (SEQIDNO:1) of Synechococcus (Synechococcussp.) anl50 gene is 5 '-CGATTCGCCATGCCGAAGAGG-3 ', and reverse primer (SEQIDNO:2) is 5 '-ACTGTCTCGGCAGCAATTT-3 '。
In a preferred version of the present invention, common PCR reaction liquid II is the primer pair of the PCR by amplification Synechococcus (Synechococcussp.) CDS:ABB57481.1 gene, 2xPCRReagent (KT207, sky root is biochemical) and aseptic ultra-pure water composition。In a concrete scheme of the present invention, the regular-PCR forward primer (SEQIDNO:3) of Synechococcus (Synechococcussp.) CDS:ABB57481.1 gene is 5 '-CGTCCTACCTGCTCGTTTACA-3 ', and reverse primer (SEQIDNO:4) is 5 '-AGCCCTCGTTAGTCCCAGTA-3 '。
In a preferred version of the present invention, quantitative PCR reactant liquor I is made up of anl50 gene quantification PCR primer pair, QuantiTectPrimerAssay (249900, Qiagen) and sterilized water。In a concrete scheme of the present invention, the quantitative PCR forward primer (SEQIDNO:5) of anl50 gene is 5 '-CATCGCTGGCTTACAACTG-3 ', and reverse primer (SEQIDNO:6) is 5 '-GCTCTGGCTGCTGATACG-3 '。
In a preferred version of the present invention, quantitative PCR reactant liquor II is made up of Synechococcus (Synechococcussp.) CDS:ABB57481.1 gene quantification PCR primer pair, QuantiTectPrimerAssay (249900, Qiagen) and sterilized water。In a concrete scheme of the present invention, the quantitative PCR forward primer (SEQIDNO:7) of Synechococcus (Synechococcussp.) CDS:ABB57481.1 gene is 5 '-ATCTACGGTCGGGTCTACG-3 ', and reverse primer (SEQIDNO:8) is 5 '-TTCCTCAACCTCACCTAAGC-3 '。
In a preferred version of the present invention, standard substance I is 1401 nucleotide fragments containing Synechococcus (Synechococcussp.) anl50 gene。The standard substance I (SEQIDNO:9) of described anl50 gene sees appendix sequence table。Standard substance I Nanodrop carries out quantitatively, and storing concentration is 105Copy/μ L, 106Copy/μ L, 107Copy/μ L, 108Copy/μ L, 109L5 Concentraton gradient of copy/μ。
In a preferred version of the present invention, standard substance II is 1414 nucleotide fragments containing Synechococcus (Synechococcussp.) CDS:ABB57481.1 gene。The standard substance II (SEQIDNO:10) of described CDS:ABB57481.1 gene sees appendix sequence table。Standard substance II Nanodrop carries out quantitatively, and storing concentration is 104Copy/μ L, 105Copy/μ L, 106Copy/μ L, 107Copy/μ L, 108L5 Concentraton gradient of copy/μ。
In a preferred version of the present invention, blank product are aseptic ultra-pure water。
Composition described above in test kit has following function and effect: common PCR reaction liquid I is used for expanding anl50 genetic fragment, and then is used for preparing standard substance I。Common PCR reaction liquid II is used for expanding CDS:ABB57481.1 genetic fragment, and then is used for preparing standard substance II。Standard substance I and standard substance II is respectively used to make the standard curve of quantitative PCR。Quantitative PCR reactant liquor I for carrying out quantitative analysis to anl50 gene, and anl50 gene relative amplification difference under varying environment is coerced is carried out quantitative analysis。Quantitative PCR reactant liquor II for carrying out quantitative analysis to CDS:ABB57481.1 gene, and CDS:ABB57481.1 gene relative amplification difference under varying environment is coerced is carried out quantitative analysis。
In a preferred version of the present invention, additionally provide a kind of copy number by detection by quantitative anl50 gene, the application in the detection by quantitative of Cells of Blue-green Algae plasmid of a kind of quantitative PCR kit detecting cyanophyceae DNA damage, comprise the following steps:
1) with genome DNA extracting reagent kit, cyanophyceae STb gene is extracted；
2) cyanophyceae STb gene is separately added into common PCR reaction liquid I and common PCR reaction liquid II, respectively anl50 genetic fragment and CDS:ABB57481.1 genetic fragment is expanded with PCR detector；
3) PCR primer being purified recovery, Nanodrop carries out concentration mensuration, according to formula: copy number/mL=(6.02 × 1023Copy number/mol) × [concentration (g/mL)/molecular weight (g/mol)] calculate the copy number of purification DNA。Standard substance I and standard substance II is carried out serial dilution according to 10 times of dilution methods；
4) standard substance I and testing sample are added quantitative PCR reactant liquor I, be used for detecting anl50 gene；Standard substance II and testing sample are added quantitative PCR reactant liquor II, is used for detecting CDS:ABB57481.1 gene。Detect with quantitative PCR detector；
5) by comparing the circulation thresholding of testing sample and standard substance, the initial anl50 gene of testing sample and the copy number of CDS:ABB57481.1 gene are calculated according to standard curve。Cells of Blue-green Algae under environment-stress, the anl50 gene of process group Cells of Blue-green Algae and the copy number of CDS:ABB57481.1 gene compared with matched group by change (increase or reduce)。
In a preferred version of the present invention, additionally provide and a kind of changed by the amplification efficiency of detection by quantitative anl50 gene and CDS:ABB57481.1 gene, the application in detection by quantitative Cells of Blue-green Algae DNA damage of a kind of quantitative PCR kit detecting cyanophyceae DNA damage。The detection method of cyanophyceae DNA damage is comprised the following steps: by quantitative PCR kit
1) with genome DNA extracting reagent kit, cyanophyceae STb gene is extracted；
2) cyanophyceae STb gene is separately added into common PCR reaction liquid I and common PCR reaction liquid II, respectively anl50 genetic fragment and CDS:ABB57481.1 genetic fragment is expanded with PCR detector；
3) PCR primer being purified recovery, Nanodrop carries out concentration mensuration, according to formula: copy number/mL=(6.02 × 1023Copy number/mol) × [concentration (g/mL)/molecular weight (g/mol)] calculate the copy number of purification DNA。Standard substance I and standard substance II is carried out serial dilution according to 10 times of dilution methods。
4) standard substance I and testing sample are added quantitative PCR reactant liquor I, be used for detecting anl50 gene；Standard substance II and testing sample are added quantitative PCR reactant liquor II, is used for detecting CDS:ABB57481.1 gene。Detect with quantitative PCR detector；
5) by comparing the circulation thresholding of testing sample and standard substance, compare the amplification efficiency of the tripe systems quantitative PCR as DNA, characterize the level of cyanophyceae DNA damage。Under normal circumstances, the DNA of damage is owing to there occurs uncoiling, and the amplification efficiency of its quantitative PCR is higher, less than the quantitative PCR cycle threshold of matched group cell DNA。Utilize the relative amplification difference value with matched group, can indirectly represent the DNA damage degree of Cells of Blue-green Algae under environment-stress。
The present invention compared with prior art, has the following advantages and effect:
1) specificity is high。Take specific primer and the relevant PCR reaction of cyanophyceae, cyanophyceae specific gene has been expanded, and has thus carried out the formulation of standard curve as template so that the specificity of detection is high。
2) detection method is simple, and detection speed is fast。Only using the Cells of Blue-green Algae DNA of extraction as template, need to add in the test kit of the present invention, according to the step provided, the plasmid copy number of Cells of Blue-green Algae and Cells of Blue-green Algae DNA damage level can be obtained in 2 hours。Traditional plasmid copy number assay method, if the minute of CsCl-ethidium bromide (CsCl-EB) equilibrium gradient centrifugation method was more than 24 hours, detection process needs isotopic participation；The accuracy of hybridizing method (such as Dot-blot and Southern-blot) is higher, and detection process needs also exist for isotopic participation, detects respectively 20 hours and more than 48 hours time。
3) result of plasmids detection represents with copy number, and quantitative result is accurately and reliably。CsCl-ethidium bromide (CsCl-EB) equilibrium gradient centrifugation method accuracy is poor；Although living in indirect determination plasmid copy number required time short (10 minutes) by measuring the enzyme of plasmid expression, but its poor accuracy。
4) testing result of DNA damage represents with the amplification efficiency difference of quantitative PCR, and quantitative result is accurately and reliably。And utilizing TUNEL method detection cyanophyceae DNA damage is a kind of semiquantitative method。
5) present invention has filled up the blank of cyanophyceae plasmid DNA copies number detection, has filled up the blank of the DNA damage of quantitative PCR method detection Cells of Blue-green Algae simultaneously。The method cannot be only used for evaluating the physiological status of Cells of Blue-green Algae, also can detect Cells of Blue-green Algae response under environment-stress。
Utilize this test kit detection Synechococcus cell under normal physiological status, along with the prolongation of growth time, the situation of change that plasmid copy number (calculates with anl50 gene copy number)。As shown in table 1。Inoculating the 0th～6 day, along with the prolongation of growth time, plasmid copy number increases, and the plasmid copy number inoculating the 6th day is maximum, is 62 ± 22/cell；In 3 days subsequently, plasmid copy number reduces。
The relation of table 1 cyanophyceae plasmid copy number and trophophase
| Inoculation natural law | Plasmid copy number |
| 0 | 32±6 |
| 1 | 43±8 4 --> |
| 2 | 44±21 |
| 3 | 45±13 |
| 4 | 47±18 |
| 5 | 46±23 |
| 6 | 62±22 |
| 7 | 59±23 |
| 8 | 55±12 |
| 9 | 40±11 |
Utilize this test kit detection H2O2Plasmid copy number level in Synechococcus cell under exposure。It is found that variable concentrations H from table 22O2Exposing Synechococcus cell, the copy number of plasmid, compared with matched group, there occurs change。Specifically, 1～3mMH2O2Expose, along with H2O2The rising of exposure concentrations, plasmid copy number reduces, and is below matched group。4～5mMH2O2Exposing Cells of Blue-green Algae, plasmid copy number increases, and is above matched group。
Table 2 variable concentrations H2O2The cyanophyceae plasmid copy number level exposed
| H2O2Exposure concentrations mM | Plasmid copy number |
| 0 | 33±7 |
| 1 | 28±5 |
| 2 | 26±7 |
| 3 | 23±5 |
| 4 | 42±8 |
| 5 | 44±7 |
Cyanophyceae DNA damage is detected by the test kit utilizing the present invention, as shown in table 3, at variable concentrations H2O2The anl50 gene of Synechococcus cell DNA and the relative amplification difference of CDS:ABB57481.1 gene that expose increase along with the increase of exposure concentrations, present obvious dose-effect relationship。Describe reliability and the high sensitivity of this test kit。
Table 3 variable concentrations H2O2The cyanophyceae DNA damage (in gene relative amplification difference) exposed
| H2O2Exposure concentrations (mM) | Anl50 gene 2ΔCt | CDS:ABB57481.1 gene 2ΔCt |
| 1 | 0.83±0.12 | 1.28±0.19 |
| 2 | 1.05±0.16 | 1.75±0.26 |
| 3 | 2.81±0.42 | 5.02±0.75 |
| 4 | 4.13±0.62 | 10.03±1.50 |
Accompanying drawing explanation
Figure 1A is the amplification curve of a kind of standard substance I。
Figure 1B is the standard curve of a kind of standard substance I。
Fig. 2 A is the amplification curve of a kind of standard substance II。
Fig. 2 B is the standard curve of a kind of standard substance II。
Fig. 3 is the absolute quantitation amplification curve of a kind of normal Cells of Blue-green Algae anl50 gene。
Fig. 4 is a kind of H2O2Expose the absolute quantitation amplification curve of lower cyanophyceae anl50 gene。
Fig. 5 is the amplification curve of a kind of blank product。
Fig. 6 is the absolute quantitation amplification curve of a kind of normal Cells of Blue-green Algae CDS:ABB57481.1 gene。
Fig. 7 is a kind of H2O2Expose the absolute quantitation amplification curve of lower cyanophyceae CDS:ABB57481.1 gene。
Fig. 8 is a kind of H2O2Expose the relative quantification amplification curve of lower cyanophyceae anl50 gene。
Fig. 9 is a kind of H2O2Expose the relative quantification amplification curve of lower cyanophyceae CDS:ABB57481.1 gene。
Figure 1A is the amplification curve of standard substance I, and Figure 1B is the standard curve obtained according to this curve。It can be seen that the R of standard curve from Figure 1B2Value is 0.9998。Fig. 2 A is the amplification curve of standard substance II, and Fig. 2 B is the standard curve obtained according to this curve。It can be seen that the R of standard curve from Fig. 2 B2Value is 0.9995。Standard substance I and standard substance II meets the requirement as standard substance。Fig. 3 is the amplification curve of normal Cells of Blue-green Algae anl50 gene, and surveyed Ct value is between 15～25, and within the range of linearity of standard substance, quantitative result is 8 × 107～9 × 108Copy/μ about L。Fig. 4 is H2O2Expose the absolute quantitation amplification curve of lower cyanophyceae anl50 gene, compared with normal Cells of Blue-green Algae, H2O2Expose and make the copy number of cyanophyceae plasmid change (substantially increase or reduce)。Fig. 5 is the amplification curve of blank product, and result is without amplified signal。Fig. 6 is the absolute quantitation amplification curve of normal Cells of Blue-green Algae CDS:ABB57481.1 gene, and institute's test sample Ct value originally is between 14～17, and within the range of linearity of standard substance, quantitative result is the copy number of CDS:ABB57481.1 gene is 3 × 106～5 × 107Copy/μ about L。Fig. 7 is H2O2Expose the absolute quantitation amplification curve of lower cyanophyceae CDS:ABB57481.1 gene, H2O2Exposing makes the copy number of cyanophyceae CDS:ABB57481.1 gene change。Fig. 8 is that Cells of Blue-green Algae is at H2O2Exposing the relative quantification pcr amplification curve of lower anl50 gene, surveyed Ct value is between 14～27。Fig. 9 is that Cells of Blue-green Algae is at H2O2Exposing the relative quantification PCR curve of lower CDS:ABB57481.1 gene, surveyed Ct value is between 23～29。The relative amplification difference of Cells of Blue-green Algae anl50 gene and CDS:ABB57481.1 gene is as shown in table 9, along with H2O2The increase of exposure concentrations, the relative amplification difference of anl50 gene and CDS:ABB57481.1 gene increases。Illustrate that the detection method that this test kit provides is very sensitive, reliable。
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further。Should be appreciated that these embodiments are merely to illustrate the present invention rather than restriction the scope of protection of present invention, unreceipted specific experiment condition and method in the following example, generally conventionally condition such as Sambrook, J.；Russell, D.W.inMolecularCloning:ALaboratoryManual.3rded.；ColdSpringHarborPress:ColdSpringHarbor, NewYork, 2001 or according to manufacturer it is proposed that condition。
Below in conjunction with drawings and the embodiments, the present invention is further detailed explanation:
Embodiment 1:
The preparation method of a kind of quantitative PCR kit detecting cyanophyceae DNA damage, the steps include:
1. the design of primer and synthesis:
Anl50 and CDS:ABB57481.1 gene order is inquired about according to NCBI website (http://www.ncbi.nlm.nih.gov/), DNAMAN is utilized to design the upstream and downstream primer of regular-PCR and the upstream and downstream primer of quantitative PCR in anl50 gene order, wherein the regular-PCR forward primer of anl50 gene and the binding site of this gene are in the upstream of its quantitative PCR forward primer Yu this gene binding site, and the regular-PCR downstream primer of anl50 gene and the binding site of this gene are in the downstream of its quantitative PCR downstream primer Yu this gene binding site。CDS:ABB57481.1 gene is designed primer by same method。Selected primer is combined with good specificity for gene, has higher pcr amplification efficiency。Primer student on commission's work bio-engineering corporation (Shanghai) synthesizes, and wherein primer is HAP purification。Primer sequence is table 4 such as。
Table 4 specific primer sequence
2. the preparation of cyanophyceae STb gene template:
1) 10000g, obtains Cells of Blue-green Algae sample to be measured and matched group in centrifugal 1 minute。
2) the method extraction of test kit (sky root is biochemical) is extracted according to bacteria total DNA。
3) Nanodrop measures STb gene concentration。
4) dilution STb gene is respectively to 5ng/ μ L and 10ng/ μ L, standby。
3. common PCR reaction liquid I composition, such as table 5。
Table 5 common PCR reaction liquid I forms
| Formula | Concentration or amount |
| 2x PCR Reagent | 1× |
| Anl50 common PCR primers | 500nM |
| DNA profiling | 10μL |
| ddH2O | Surplus |
| Cumulative volume | 200μL |
4. common PCR reaction liquid II composition, such as table 6。
Table 6 common PCR reaction liquid II forms
| Formula | Concentration or amount |
| 2x PCR Reagent | 1× 7 --> |
| CDS:ABB57481.1 gene common PCR primers | 500nM |
| DNA profiling | 10μL |
| ddH2O | Surplus |
| Cumulative volume | 200μL |
5. the preparation of standard substance I and standard substance II:
By anl50 gene and CDS:ABB57481.1 gene through regular-PCR primer amplified product, it is purified recovery, the DNA of acquisition is carried out concentration and purity testing。According to formula: copy number/mL=(6.02 × 1023Copy number/mol) × [concentration (g/mL)/molecular weight (g/mol)] calculate the copy number of purification DNA, and carry out gradient dilution according to the copy number measured, it is thus achieved that 5 Concentraton gradient of standard substance I: 105Copy/μ L, 106Copy/μ L, 107Copy/μ L, 108Copy/μ L, 109Copy/μ L；Obtain 5 Concentraton gradient of standard substance II: 104Copy/μ L, 105Copy/μ L, 106Copy/μ L, 107Copy/μ L, 108Copy/μ L。-20 DEG C of preservations。
1) regular-PCR:
Taking the cyanophyceae STb gene of step 2 extraction as template, be configured to common PCR reaction system according to the composition of common PCR reaction liquid, each main component of system is as follows:
A) common PCR reaction liquid I190 μ L, STb gene template 10 μ L, cumulative volume 200 μ L；
B) common PCR reaction liquid II190 μ L, STb gene template 10 μ L, cumulative volume 200 μ L；
Common PCR reaction program such as table 7:
Table 7 common PCR reaction program
2) purification of standard substance I and standard substance II reclaims:
A) take step 1) PCR primer carry out agarose gel electrophoresis, deposition condition is 0.7% (mass ratio) agarose gel, and electrophoretic buffer is 1 × TBE, and voltage is 100V, and electrophoresis time is 40 minutes。
B) utilize plain agar sugar gel to reclaim test kit (sky root is biochemical) and PCR primer is purified recovery。
3) copy number of standard substance I and standard substance II calculates:
According to formula: copy number/mL=(6.02 × 1023Copy number/mol) × [concentration (g/mL)/molecular weight (g/mol)] calculate the copy number of purification DNA。
4) gradient dilution of standard substance I and standard substance II:
According to determination step 3) DNA carries out gradient dilution by the copy number that calculates, it is thus achieved that 5 Concentraton gradient of standard substance I: 105Copy/μ L, 106Copy/μ L, 107Copy/μ L, 108Copy/μ L, 109Copy/μ L；Obtain 5 Concentraton gradient of standard substance II: 104Copy/μ L, 105Copy/μ L, 106Copy/μ L, 107Copy/μ L, 108Copy/μ L。
6. the preparation of blank product:
Blank product are through 121 DEG C of high pressure moist heat sterilizations Milli-Q water of 15 minutes。
7. quantitative PCR reactant liquor I composition, such as table 8。
Table 8 quantitative PCR reactant liquor I forms
| Formula | Concentration or amount |
| QuantiTect Primer Assay | 1× |
| Anl50 quantification PCR primer | 500nM |
| DNA profiling | 1μL |
| ddH2O | Surplus |
| Cumulative volume | 20μL |
8. Fluorescence PCR liquid II composition, such as table 9。
Table 9 quantitative PCR reactant liquor II forms
| Formula | Concentration or amount |
| QuantiTect Primer Assay | 1× |
| CDS:ABB57481.1 gene quantification PCR primer | 500nM |
| DNA profiling | 1μL |
| ddH2O | Surplus |
| Cumulative volume | 20μL |
Utilize common PCR reaction liquid I and common PCR reaction liquid II can prepare standard substance I standard substance II respectively。Utilize quantitative PCR reactant liquor I and the quantitative PCR reactant liquor II can the plasmid copy number of the different cyanophyceae sample of detection by quantitative。Quantitative PCR reactant liquor I and quantitative PCR reactant liquor II detection by quantitative Cells of Blue-green Algae DNA damage can also be utilized。The test kit of the present invention can evaluate the plasmid copy number level of Cells of Blue-green Algae, it is judged that normally whether Cells of Blue-green Algae, it is possible to the DNA damage degree of detection Cells of Blue-green Algae, has good application prospect in Cells of Blue-green Algae is to the response of environment-stress。
Embodiment 2:
The application in detection cyanophyceae plasmid copy number of a kind of quantitative PCR kit detecting cyanophyceae DNA damage, the steps include:
1. the copy number of quantitative PCR detection anl50 gene:
1) forming according to quantitative PCR reactant liquor I, prepare quantitative PCR reaction system, take cyanophyceae STb gene, 5 standard substance of standard substance I and each 1 μ L of blank product, each main component of system is as follows:
Quantitative PCR reactant liquor I19 μ L, DNA profiling 1 μ L；Cumulative volume 20 μ L。
2) arranging the fluorescence detection channel collecting SYBRGreen fluorescence signal, reaction tube is put into quantitative PCR apparatus (ABIHT7900) and starts amplification, response procedures is table 10 such as:
Table 10 absolute quantitation PCR response procedures
2. the copy number of quantitative PCR detection CDS:ABB57481.1 gene:
1) form according to quantitative PCR reactant liquor II, prepare quantitative PCR reaction system, take the STb gene of cyanophyceae, 5 standard substance of standard substance II and each 1 μ L of blank product, each main component of system is as follows:
Quantitative PCR reactant liquor II19 μ L, DNA profiling 1 μ L；Cumulative volume 20 μ L。
2) fluorescence detection channel collecting SYBRGreen fluorescence signal is set, reaction tube is put into quantitative PCR apparatus (ABI7900HT) and starts amplification, response procedures and step 1) identical。
3) result judges:
The Ct value (period) of baseline range is automatically selected (such as SDS2.4.1, ABI company) by software, sets threshold value and exceedes the peak of random amplification curve。Fluorescent PCR instrument is different, and the Ct value of gained baseline range is different。Fig. 1 be standard substance I amplification curve, and according to the standard curve that this curve obtains。Fig. 2 is the amplification curve of standard substance II, and according to the standard curve that this curve obtains。
4) interpretation of result:
Fig. 3 is the amplification curve of normal Cells of Blue-green Algae anl50 gene, and surveyed Ct value is between 15～25, and within the range of linearity of standard substance, quantitative result is 8 × 107～9 × 108Copy/μ about L。Fig. 4 is H2O2Expose the amplification curve of lower cyanophyceae anl50 gene, compared with normal Cells of Blue-green Algae, H2O2Expose and make the copy number of cyanophyceae plasmid change (substantially increase or reduce)。Fig. 5 is the amplification curve of blank product, and result is without amplified signal。Fig. 6 is the absolute quantitation amplification curve of normal Cells of Blue-green Algae CDS:ABB57481.1 gene, and institute's test sample Ct value originally is between 14～17, and within the range of linearity of standard substance, quantitative result is the copy number of CDS:ABB57481.1 gene is 3 × 106～5 × 107Copy/μ about L。Fig. 7 is H2O2Expose the absolute quantitation amplification curve of lower cyanophyceae CDS:ABB57481.1 gene, H2O2Exposing makes the copy number of cyanophyceae CDS:ABB57481.1 gene change。
Utilize this test kit detection Synechococcus cell under normal physiological status, along with the prolongation of growth time, the situation of change that plasmid copy number (calculates with anl50 gene copy number)。As shown in table 1。Inoculating the 0th～6 day, along with the prolongation of growth time, plasmid copy number increases, and the plasmid copy number inoculating the 6th day is maximum, is 62 ± 22/cell；In 3 days subsequently, plasmid copy number reduces。
Utilize this test kit detection H2O2Plasmid copy number level in Synechococcus cell under exposure。As can be found from Table 2, variable concentrations H2O2Exposing Synechococcus cell, the copy number of plasmid, compared with matched group, there occurs change。Specifically, 1～3mMH2O2Expose, along with H2O2The rising of exposure concentrations, plasmid copy number reduces, and is below matched group。4～5mMH2O2Exposing Cells of Blue-green Algae, plasmid copy number increases, and is above matched group。
This test kit is consistent with traditional OD value detection Cells of Blue-green Algae response under environment-stress, the reliability of this method is described, and highly sensitive, favorable reproducibility, provide strong detection method for the physiological status and detection Cells of Blue-green Algae response under environment-stress evaluating Cells of Blue-green Algae。
Embodiment 3:
The application in detection cyanophyceae DNA damage of a kind of quantitative PCR kit detecting cyanophyceae DNA damage, the steps include:
1. relative quantification PCR detects the amplification difference of anl50 gene
Forming according to quantitative PCR reactant liquor I, prepare quantitative PCR reaction system, take each 1 μ L of process group cyanophyceae STb gene, matched group cyanophyceae STb gene and blank product, each main component of system is as follows:
Quantitative PCR reactant liquor I19 μ L, DNA profiling 1 μ L；Cumulative volume 20 μ L。
Arranging the fluorescence detection channel collecting SYBRGreen fluorescence signal, reaction tube is put into quantitative PCR apparatus (ABI7900HT) and starts amplification, response procedures is table 11 such as:
Table 11 relative quantification PCR response procedures
2. relative quantification PCR detects the amplification difference of CDS:ABB57481.1 gene:
Forming according to quantitative PCR reactant liquor II, prepare quantitative PCR reaction system, take each 1 μ L of process group cyanophyceae STb gene, matched group cyanophyceae STb gene and blank product, each main component of system is as follows:
Quantitative PCR reactant liquor II19 μ L, DNA profiling 1 μ L；Cumulative volume 20 μ L。
Arranging the fluorescence detection channel collecting SYBRGreen fluorescence signal, reaction tube is put into quantitative PCR apparatus (ABI7900HT) and starts amplification, response procedures is identical with step 1.。
3. result judges:
The Ct value (period) of baseline range is automatically selected (such as SDS2.4.1, ABI company) by software, sets threshold value and exceedes the peak of random amplification curve。Fluorescent PCR instrument is different, and the Ct value of gained baseline range is different。
4. interpretation of result:
Fig. 8 is that Cells of Blue-green Algae is at H2O2Exposing the relative quantification pcr amplification curve of lower anl50 gene, surveyed Ct value is between 14～27。Fig. 9 is that Cells of Blue-green Algae is at H2O2Exposing the relative quantification PCR curve of lower CDS:ABB57481.1 gene, surveyed Ct value is between 23～29。The cyanophyceae DNA damage utilizing the test kit of the present invention detects, and the relative amplification difference of Cells of Blue-green Algae anl50 gene and CDS:ABB57481.1 gene is as shown in table 3, along with H2O2The increase of exposure concentrations, the relative amplification difference of anl50 gene and CDS:ABB57481.1 gene increases, and presents obvious dose-effect relationship。Illustrate that the detection method that this test kit provides is very sensitive, reliable。
SEQUENCELISTING
<110>Inst. of Hydrobiology, Chinese Academy of Sciences
<120>a kind of quantitative PCR kit detecting cyanophyceae DNA damage and application
<130>a kind of quantitative PCR kit detecting cyanophyceae DNA damage and application
<160>10
<170>PatentInversion3.1
<210>1
<211>21
<212>DNA
<213>artificial sequence
<400>1
cgattcgccatgccgaagagg21
<210>2
<211>19
<212>DNA
<213>artificial sequence
<400>2
actgtctcggcagcaattt19
<210>3
<211>21
<212>DNA
<213>artificial sequence
<400>3
cgtcctacctgctcgtttaca21
<210>4
<211>20
<212>DNA
<213>artificial sequence
<400>4
agccctcgttagtcccagta20
<210>5
<211>19
<212>DNA
<213>artificial sequence
<400>5
catcgctggcttacaactg19
<210>6
<211>18
<212>DNA
<213>artificial sequence
<400>6
gctctggctgctgatacg18
<210>7
<211>19
<212>DNA
<213>artificial sequence
<400>7
atctacggtcgggtctacg19
<210>8
<211>20
<212>DNA
<213>artificial sequence
<400>8
ttcctcaacctcacctaagc20
<210>9
<211>1401
<212>DNA
<213>gene order
<400>9
cgattcgccatgccgaagagggcttcgcgatcgcccctcatgtagcttactggtggcgtt60
tttacgccccaaaaacagccccaaccgtcggccattggcaacgcaatccagaactggccc120
agagtctccgccaaatcgcagacagtcacggcgaagcgttttatcgcggcgacctagcgc180
agcaagcgatcgccgccgctcaggcagccgggggtagtcaaaccctcgccgattgggccg240
agcatcaagtggaatgggtggagcctttttcagtcagctacagcggggcagaggtttggg300
ccttgccacccaacagccaaggcatgatcacgctgattgcgttggaactcctcaagcgtt360
ggcacgggccagagtctgccagtgaaagtggcgcgaatcagcatcgtctgctggaagtgg420
tgcgggctgctctcgatctgggctatcgagaacttggcgatctggcctggatgccactaa480
cgccagcagaacttctcgactccaagcggctcgatcgcctggctgcgacgctgaagccga540
ccactgcagcgatttacagaccccaagtcacaccgactggcggcaccgtttatctctgca600
ctgccgatgcccaaggacgaatggtgtccttgattcagtccaactatcagggttttgggg660
cacgcgttgaggtgccaggaacagggctgaccttcaataatcgcggtgcctgctttgtcc720
tcacccccgaacatcccaatcaagtagggccacagaagcgaccctttcataccttgacgc780
cgggttttttgactcgtaatggtcaagcgctcgccgcttttggcatcatgggcggcccgc840
tccaatcccaagcccaggtgcagttactgcatcgcctcctcgatcgtggcgagaatgtcc900
aagcagcgatcgaagctccccgctgggaagcccaaagccacggcagagtacggattgagc960
cagggtttccggccacaacctatgccagcttgacccgtcgcggtcatcgccttgaacggc1020
tgacagaacccagactgggtggcgggcagatcatttggcgacagcctcagggttatgttg1080
ctgcttccgatccccgcaaagatggccaagcctcaggtttctaggactctagccggcctg1140
aatcaggatgagccctacgatcgctttctaaccccagtcagtggctatagaaacttgtat1200
ctggcagcacttgttgactgtcccctcgcccgactagcgtaaacatcgaaatatcaatcg1260
gtttatcggtaattctccgcagggctgtctagttcccgaattacagacagcctgccgata1320
aaccattggtctgaccctaggagtggatatgccgctgtatctcgttgaggcaaccctgcc1380
cgaaattgctgccgagacagt1401
<210>10
<211>1414
<212>DNA
<213>gene order
<400>10
cgtcctacctgctcgtttacagggctatcgcggcaatctggaagcgctggtgcgggaggt60
gttgaccattgttgaactgccagaaaatttagacctccagcaagcggcccgccattgttc120
accagagacccaactccgcctctgtctggccagggcgatcgccatggaacctgacattct180
gctcctcgatgagccctgtctgccactggattcagcagcgacccttgcctttgaagaact240
gctgctgcggctctgcgatcgccacactatccttctgatcaccaacaatttggcccaagc300
cggacgctgcgccacctacacagccctgctccatcctgtcctgatcgacgaacagaaact360
gcccgtgaccaagctaattgagtttgcaccgaccagccaaatctttcgccagccccagca420
tccgatcaccgatgactttgtctgtggtcgacgctaaagcgtcgcaataatagaaaagct480
gcttgcgaaggaaatcactatgaaaatctggcgatcgctcagtctcttgctcgtactgat540
tggcggtctgattttcggcgcgggcccagcgttggctatcactcagctcaatgtcaccac600
cgttgactatcaacagtgcccggctgaactcaattcaggagctgtcaccagtggcggtag660
tagtcgcccagccacctgctacttggtggtgggcacgctcaacaaccccagcaacaaaac720
cgtctacgacgtcgatatctacggtcgggtctacgacgcgaacggcgactcagtgatgca780
aaatcgcacgcgcttaggtgaggttgaggaagtgccgccgggcgatcatcctttcgaaat840
tcggatcagcgttccggccagccaaccagaaccactgcaacttaagcaattcaaagcatc900
gggcttcagcgccacggtccggacccaactgctagaagaaccggctgaatctcgtttcta960
agaacgatcgctgctcgggggggcttcttgcagcgctagaccgatcgcatcaaccaagcg1020
gctgaccggcaccagttccagatccgactcgcagcgctcctgtcccttgggtaagagcgc1080
tcgttgaaagccgagcttggcagcctccttgaggcgcagttcaacctgactgacagggcg1140
cacttgtccccctaagccgatttcaccaatcagtaccgtatcgggccgcaggcagcgatc1200
gcggaaactggcgacaagagcgatcgcaattcccaaatccgcagcgggttcagccacatt1260
cagcccgccagccgaggcgacgtaggcatcgaattttgacagcggcaaacccaagcgttt1320
ttctaagaccgccaaaatttgcagcaagcggttgtactcaattcccgtaccagcacggcg1380
cggtgaactatagctactgggactaacgagggct1414
Claims (3)
1. the quantitative PCR kit detecting cyanophyceae DNA damage, this test kit includes: common PCR reaction liquid I, common PCR reaction liquid II, comprise the standard substance I of Synechococcus anl50 gene inserts, comprise the standard substance II of the CDS:ABB57481.1 gene inserts of Synechococcus, blank product, the quantitative PCR reactant liquor I of the quantification PCR primer comprising Synechococcus anl50 gene and comprise the quantitative PCR reactant liquor II of quantification PCR primer of Synechococcus CDS:ABB57481.1 gene, it is characterized in that: common PCR reaction liquid I comprises a pair specific primer for Synechococcus anl50 gene design, common PCR reaction liquid II comprises a pair specific primer for Synechococcus CDS:ABB57481.1 gene design, quantitative PCR reactant liquor I comprises a pair specific primer for poly-ball anl50 gene design, quantitative PCR reactant liquor II comprises a pair specific primer for Synechococcus CDS:ABB57481.1 gene design, wherein expand the regular-PCR forward primer of anl50 gene and the binding site of this gene in the upstream of its quantitative PCR forward primer Yu this gene binding site, the regular-PCR downstream primer of anl50 gene and the binding site of this gene are in the downstream of its quantitative PCR downstream primer Yu this gene binding site；The design of primers of CDS:ABB57481.1 gene adopts the same manner；
Described common PCR reaction liquid I is that the regular-PCR forward primer of anl50 gene is SEQIDNO:1, and reverse primer is SEQIDNO:2 by the primer pair of amplification Synechococcus anl50 gene, 2 × TaqPCRMasterMix and aseptic ultra-pure water composition；
Described common PCR reaction liquid II is the primer pair of the PCR by amplification Synechococcus CDS:ABB57481.1,2 × TaqPCRMasterMix and aseptic ultra-pure water composition, the regular-PCR forward primer of Synechococcus CDS:ABB57481.1 is SEQIDNO:3, and reverse primer is SEQIDNO:4；
Described quantitative PCR reactant liquor I is made up of anl50 gene quantification PCR primer pair, 2 × realtimePCRMix and sterilized water, and the quantitative PCR forward primer of anl50 gene is SEQIDNO:5, and reverse primer is SEQIDNO:6；
Described quantitative PCR reactant liquor II is made up of Synechococcus CDS:ABB57481.1 gene quantification PCR primer pair, 2 × realtimePCRMix and sterilized water, the quantitative PCR forward primer of Synechococcus CDS:ABB57481.1 gene is SEQIDNO:7, and reverse primer is SEQIDNO:8；
Described standard substance I is 1401 nucleotide fragments containing Synechococcus anl50 gene, and the standard substance I of described anl50 gene is SEQIDNO:9, and standard substance I Nanodrop carries out quantitatively, and storing concentration is 105Copy/μ L, 106Copy/μ L, 107Copy/μ L, 108Copy/μ L, 109L5 Concentraton gradient of copy/μ；
Described standard substance II is 1414 nucleotide fragments containing Synechococcus CDS:ABB57481.1 gene, and the standard substance II of described CDS:ABB57481.1 gene is SEQIDNO:10, and standard substance II Nanodrop carries out quantitatively, and storing concentration is 104Copy/μ L, 105Copy/μ L, 106Copy/μ L, 107Copy/μ L, 108L5 Concentraton gradient of copy/μ；
Described blank product are aseptic ultra-pure water。
2. a kind of described in claim 1 detects the application in detection cyanophyceae plasmid copy number of the quantitative PCR kit of cyanophyceae DNA damage。
3. a kind of described in claim 1 detects the application in detection by quantitative Cells of Blue-green Algae DNA damage of the quantitative PCR kit of cyanophyceae DNA damage。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510881486.9A CN105695568B (en) | 2015-12-03 | 2015-12-03 | It is a kind of detect cyanobacteria DNA damage quantitative PCR kit and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510881486.9A CN105695568B (en) | 2015-12-03 | 2015-12-03 | It is a kind of detect cyanobacteria DNA damage quantitative PCR kit and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105695568A true CN105695568A (en) | 2016-06-22 |
| CN105695568B CN105695568B (en) | 2019-02-05 |
Family
ID=56228193
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510881486.9A Active CN105695568B (en) | 2015-12-03 | 2015-12-03 | It is a kind of detect cyanobacteria DNA damage quantitative PCR kit and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105695568B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112342279A (en) * | 2020-11-06 | 2021-02-09 | 中国科学院城市环境研究所 | Kit and method for simultaneously detecting specific genes of cyanobacteria bloom Ralstonia and prototheca toxin |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004275050A (en) * | 2003-03-14 | 2004-10-07 | Japan Science & Technology Agency | Gene-deleted synechococcus |
| CN104561281A (en) * | 2014-12-24 | 2015-04-29 | 中国环境科学研究院 | Brown tide algae species detection method and kit |
| CN105368950A (en) * | 2015-12-03 | 2016-03-02 | 中国科学院水生生物研究所 | Quantitative PCR (polymerase chain reaction) reagent kit for detecting blue-green algae plasmids and application of quantitative PCR reagent kit |
-
2015
- 2015-12-03 CN CN201510881486.9A patent/CN105695568B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004275050A (en) * | 2003-03-14 | 2004-10-07 | Japan Science & Technology Agency | Gene-deleted synechococcus |
| CN104561281A (en) * | 2014-12-24 | 2015-04-29 | 中国环境科学研究院 | Brown tide algae species detection method and kit |
| CN105368950A (en) * | 2015-12-03 | 2016-03-02 | 中国科学院水生生物研究所 | Quantitative PCR (polymerase chain reaction) reagent kit for detecting blue-green algae plasmids and application of quantitative PCR reagent kit |
Non-Patent Citations (5)
| Title |
|---|
| YOU CHEN 等: "The complete sequence and functional analysis of pANL, the large plasmid of the unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942", 《PLASMID》 * |
| 业勤 译: "蓝藻质粒", 《遗传》 * |
| 孙雪梅 等: "焦性没食子酸对铜绿微囊藻的氧化胁迫效应研究", 《中国环境科学》 * |
| 彭宇科 等: "应用RT-PCR技术定量检测湖泊水体中蓝藻方法的比较", 《微生物学通报》 * |
| 沈淞海 等: "一种蓝藻质粒DNA的快速检测法", 《水生生物学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112342279A (en) * | 2020-11-06 | 2021-02-09 | 中国科学院城市环境研究所 | Kit and method for simultaneously detecting specific genes of cyanobacteria bloom Ralstonia and prototheca toxin |
| CN112342279B (en) * | 2020-11-06 | 2022-10-14 | 中国科学院城市环境研究所 | Kit and method for simultaneously detecting specific genes of cyanobacteria bloom Ralstonia and prototheca toxin |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105695568B (en) | 2019-02-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bhatia et al. | Distinct bacterial communities exist beneath a high Arctic polythermal glacier | |
| JP2011504102A5 (en) | ||
| CN103725796B (en) | Mark parting fluorescence PCR detection kit and preparation thereof in a kind of bovine viral diarrhea virus | |
| CN101624621B (en) | Kit for quantitatively detecting BCR/ABL mRNA level | |
| CN105385780A (en) | Kit for rapidly detecting methicillin-resistant staphylococcus aureus and application thereof | |
| CN105368950B (en) | It is a kind of detection cyanobacteria plasmid quantitative PCR kit and application | |
| CN104480202A (en) | Towel gourd reference gene and application thereof | |
| CN103468806B (en) | The method for quick of scallop pathogenic Vibrio splindidus Vibrio splendidus | |
| CN104745689A (en) | Primers, probe and kit used for detecting bordetella pertussis | |
| CN110358816A (en) | A kind of primer sets, kit and application for chicken derived cell PCR detection | |
| CN102634605B (en) | Method for detecting egg drop syndrome viruses and kit for method | |
| CN109207634A (en) | Fluorescent quantitation reference gene and its primer special and application under clerodendron trichotomum drought stress | |
| CN105695568A (en) | Quantitative PCR reagent box for detecting DNA damage of cyanobacteria and application | |
| CN104673885A (en) | Detection kit and detection method for I-form legionella pneumophila | |
| CN105018637A (en) | Application of tetA gene in detection of soil antibiotic resistance pollution level and method for detecting soil antibiotic resistance pollution level through tetA gene | |
| CN101985652B (en) | High-throughput molecular detection of Fusarium graminearum on carbendazim medicament-resistant gene frequency | |
| Zhao et al. | Application of the Ludox-QPS method for estimating ciliate diversity in soil and comparison with direct count and DNA fingerprinting | |
| CN105907874A (en) | Method for quick combinative monitoring of total coliform and escherichia coli | |
| CN105177156A (en) | Human EGFR gene mutation detection kit and application thereof | |
| CN106399512A (en) | Detecting kit for aeromonas hydrophila and detecting method of detecting kit | |
| CN104673927A (en) | Pseudomonas aeruginosa PCR (polymerase chain reaction) detection kit and detection method thereof | |
| CN102899390A (en) | Small cell lung cancer markers and their detection | |
| CN111926095A (en) | LAMP primer based on Brucella BvrR sequence and using method thereof | |
| CN107641644A (en) | A kind of method for detecting yellow rice wine putrefactive microorganisms | |
| CN105087794A (en) | Kit for tuberculosis detection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |