CN113774116A - Method for quantitatively detecting phycocyanin and special standard product thereof - Google Patents
Method for quantitatively detecting phycocyanin and special standard product thereof Download PDFInfo
- Publication number
- CN113774116A CN113774116A CN202111066846.1A CN202111066846A CN113774116A CN 113774116 A CN113774116 A CN 113774116A CN 202111066846 A CN202111066846 A CN 202111066846A CN 113774116 A CN113774116 A CN 113774116A
- Authority
- CN
- China
- Prior art keywords
- standard
- phycocyanin
- quantitative pcr
- quantitatively detecting
- copies
- 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.)
- Pending
Links
- 108010053210 Phycocyanin Proteins 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 28
- 239000013612 plasmid Substances 0.000 claims abstract description 34
- 238000011529 RT qPCR Methods 0.000 claims abstract description 26
- 230000003321 amplification Effects 0.000 claims abstract description 24
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 24
- 101150107228 cpcB gene Proteins 0.000 claims abstract description 18
- 239000000126 substance Substances 0.000 claims abstract description 14
- 238000012408 PCR amplification Methods 0.000 claims abstract description 7
- 238000012417 linear regression Methods 0.000 claims abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims description 18
- 238000011144 upstream manufacturing Methods 0.000 claims description 6
- 238000000137 annealing Methods 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 22
- 238000001514 detection method Methods 0.000 description 15
- 238000003753 real-time PCR Methods 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 238000004090 dissolution Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000012851 eutrophication Methods 0.000 description 4
- 241000195493 Cryptophyta Species 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 229930192334 Auxin Natural products 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010010522 Phycobilisomes Proteins 0.000 description 1
- 241000206572 Rhodophyta Species 0.000 description 1
- 101100352021 Synechococcus sp. (strain ATCC 27144 / PCC 6301 / SAUG 1402/1) cpcB1 gene Proteins 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000013211 curve analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 210000002306 phycobilisome Anatomy 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 101150031932 rpcB gene Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
Images
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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the field of molecular biology, and discloses a method for quantitatively detecting phycocyanin, which comprises the following steps: step one, taking a recombinant plasmid as a standard template, performing PCR amplification by using real-time quantitative PCR, and establishing a unary linear regression curve corresponding to the concentration of the standard and the critical cycle number to obtain a standard curve; and step two, replacing the standard substance in the step one with a sample to be detected, carrying out real-time quantitative PCR amplification, and obtaining the copy number of the cpcB gene in the sample to be detected according to the critical cycle number of the amplification result and a standard curve, so as to obtain the content of the phycocyanin in the sample to be detected.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a method for quantitatively detecting phycocyanin and a special standard substance thereof.
Background
More than 70% of lake and reservoir water bodies in China are in eutrophication or super-eutrophication states, so that the water body functions are seriously damaged. The most common result of water eutrophication is that algae will multiply to form cyanobacterial bloom. When the cyanobacterial bloom is burst, a layer of green scum can be formed on the water surface, the water body gives off peculiar smell, the water quality and the landscape effect of the water body are reduced, and part of the cyanobacterial bloom can release various natural toxins, thereby seriously threatening the safety of an ecological system.
Phycocyanin is one of the important auxins for photosynthesis of algae, and is mainly present in phycobilisomes of blue-green algae, cryptophyceae, and some red algae. In environmental monitoring, especially in water areas where cyanobacterial bloom frequently occurs, the phycocyanin concentration can effectively represent the biomass of cyanobacterial bloom, and is a key concern index for water eutrophication and cyanobacterial bloom. Therefore, the method has important significance for rapidly and accurately characterizing the phycocyanin in the water body and monitoring the cyanobacterial bloom.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for quantitatively detecting phycocyanin and a special standard product thereof so as to realize the rapid analysis of the content of phycocyanin in water by using a cpcB gene.
In order to achieve the purpose, the invention provides a special standard substance for quantitatively detecting phycocyanin, wherein the standard substance is a recombinant plasmid containing a cpcB gene, and the sequence of the cpcB gene is the sequence in a sequence table 1.
Preferably, the starting plasmid in the recombinant plasmid is pMD-18T.
In order to achieve the above object, the present invention further provides a method for quantitatively detecting phycocyanin, comprising the following steps:
step one, taking a recombinant plasmid as a standard template, performing PCR amplification by using real-time quantitative PCR, and establishing a unary linear regression curve corresponding to the concentration of the standard and the critical cycle number to obtain a standard curve;
and step two, replacing the standard substance in the step one with a sample to be detected, carrying out real-time quantitative PCR amplification, and obtaining the copy number of the cpcB gene in the sample to be detected according to the critical cycle number of the amplification result and a standard curve, so as to obtain the content of the phycocyanin in the sample to be detected.
Preferably, the real-time quantitative PCR amplification system consists of a real-time quantitative PCR amplification buffer solution, a primer pair and a template; the concentration of the upstream primer and the downstream primer in the reaction system is 0.2 mu M/L, and the concentration of the upstream primer is 5'-ATGTTACCTACGCTACCTTCTCTGG-3', and the concentration of the downstream primer is 5'-GCGGCTTCTTTCATTTTGCT-3'.
Preferably, the concentration of the standard template in the real-time quantitative PCR amplification system is 4.65X 102copies/μL、4.65×103copies/μL、4.65×104copies/μL、4.65×105copies/μL、4.65×106copies/. mu.L and 4.65X 107copies/. mu.L.
Preferably, the annealing temperature in the PCR reaction is 60 ℃.
Compared with the prior art, the invention designs specific primers aiming at the cpcB gene, quantitatively detects the phycocyanin in the water environment by adopting a real-time quantitative PCR method, and the key technology is to prepare an external standard substance and optimize the real-time quantitative PCR reaction conditions. The standard substance for quantitatively detecting phycocyanin has the characteristics of combination of broad-spectrum identification and specificity, high sensitivity, simple and convenient preparation method, long-term storage, good purity and wide linear detection range, and can be used for quickly and quantitatively detecting phycocyanin in a water environment sample.
Drawings
FIG. 1 is a flow chart showing the steps of a method for quantitatively detecting phycocyanin according to the present invention;
FIG. 2 is an amplification curve of a quantitative PCR standard in an embodiment of the present invention;
FIG. 3 is a PCR dissolution curve of a standard in an embodiment of the present invention.
Detailed Description
Other advantages and capabilities of the present invention will be readily apparent to those skilled in the art from the present disclosure by describing the embodiments of the present invention with specific embodiments thereof in conjunction with the accompanying drawings. The invention is capable of other and different embodiments and its several details are capable of modification in various other respects, all without departing from the spirit and scope of the present invention.
In a first embodiment of the invention, the invention provides a special standard for quantitatively detecting phycocyanin, wherein the standard is a recombinant plasmid containing a cpcB gene; the sequence of the cpCB gene is the sequence in the sequence table 1.
Preferably, the starting plasmid in the recombinant plasmid is pMD-18T.
In a second embodiment of the present invention, a method for quantitatively detecting phycocyanin according to the present invention is shown in FIG. 1, and the flow chart of the steps of the method for quantitatively detecting phycocyanin according to the present invention comprises the following steps:
step 101, taking the recombinant plasmid as a standard template, performing PCR amplification by using real-time quantitative PCR, and establishing a unary linear regression curve corresponding to the concentration of the standard and the critical cycle number to obtain a standard curve;
and 102, replacing the standard substance in the first step with the sample to be detected, performing real-time quantitative PCR amplification, and obtaining the copy number of the cpcB gene in the sample to be detected according to the critical cycle number of the amplification result and a standard curve, so as to obtain the content of the phycocyanin in the sample to be detected.
Preferably, the real-time quantitative PCR amplification system consists of a real-time quantitative PCR amplification buffer solution, a primer pair and a template; the concentration of the upstream primer and the downstream primer in the reaction system is 0.2 mu M/L, and the concentration of the upstream primer is 5'-ATGTTACCTACGCTACCTTCTCTGG-3', and the concentration of the downstream primer is 5'-GCGGCTTCTTTCATTTTGCT-3'.
Preferably, the concentration of the standard template in the real-time quantitative PCR amplification system is 4.65X 102copies/μL、4.65×103copies/μL、4.65×104copies/μL、4.65×105copies/μL、4.65×106copies/. mu.L and 4.65X 107copies/. mu.L.
Preferably, the annealing temperature in the PCR reaction is 60 ℃.
Preferably, the recombinant plasmid is a recombinant plasmid containing a cpcB gene; the sequence of the cpcB gene is the sequence in a sequence table 1, and the sequence 1 in the sequence table 1 consists of 139 deoxyribonucleotides.
Preferably, the starting plasmid in the recombinant plasmid is pMD-18T.
The invention will be further illustrated by the following specific examples:
example one, preparation of plasmid standard for quantitative determination of phycocyanin
1. Primers specific for design of cpcB Gene
The sequence of the cpcB gene is downloaded in GenBank, homology comparison analysis is carried out, a specific primer is designed and synthesized, and the length of a PCR product is 139 bp. The primer sequences are as follows: the forward primer was 5'-ATGTTACCTACGCTACCTTCTCTGG-3' and the reverse primer was 5'-GCGGCTTCTTTCATTTTGCT-3'. The primers were prepared into 100. mu.M stock solutions with sterile double distilled water, subpackaged, and stored at-20 ℃.
2. Extraction of Total genomic DNA from samples
And (4) extracting and purifying the total DNA by using a DNA extraction kit to obtain a DNA sample.
3. Preparation of plasmid Standard
1) Preparation of the fragment of interest
And D, performing PCR amplification by using the DNA sample in the step two as a template, wherein the reaction system is 50 mu L. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5min, followed by 95 ℃ for 15s, annealing temperature 60 ℃ for 45s and 72 ℃ extension for 40s, for 45 cycles, and final extension at 72 ℃ for 10 min. The product was confirmed by 1.5% (wt/vol) agarose gel electrophoresis.
2) PCR product purification
Purifying the target gene: the DNA column type gel recovery kit of Axygen is used for gel recovery, the target gene is purified, and the success of recovery is confirmed by agarose gel electrophoresis of 1.5% (wt/vol).
3) Ligation of PCR products with plasmids
The purified PCR product is connected with a plasmid vector pMD18-T, and is further transformed into DH5 alpha competent Escherichia coli solution, and then is coated on an LB solid culture medium added with Amp resistance, and white colonies transformed with plasmids are selected through blue-white screening.
4) PCR identification of Positive clones and sequencing analysis
And (3) selecting a full single colony to be inoculated into a liquid LB culture solution containing Amp, carrying out shaking culture at 37 ℃ and 200 r/min overnight, and carrying out PCR identification and sequencing analysis on the bacterial liquid.
5) Mass acquisition of plasmid standards
Inoculating the transformed plasmid into a liquid LB culture solution containing Amp, culturing overnight, and extracting the plasmid by using a plasmid extraction kit to obtain a large amount of plasmid standard products.
6) Detection of plasmids
The large amount of plasmid standards obtained above was diluted with a 1 XTE buffer solution having a pH of 8.0 in a gradient, and the concentration of the plasmid standards was measured by a ultramicro nucleic acid protein measuring instrument (NanoDropND-2000C, USA) to calculate the copy number of the plasmid. The copy number of the plasmid standard was determined to be 4.65X 10 after calculation8copies/μL。
EXAMPLE II establishment of quantitative PCR detection method
1. Optimization of quantitative PCR reaction conditions
The DNA sample extracted in example 1 was used as a template. According to
PremixExTaqTMThe kit is indicated for operation, reagents required by real-time quantitative PCR are added, and the reaction system is 20 mu L of 2 mu LDNA sample. And setting a negative control and a positive control.
The real-time quantitative PCR reaction is carried out by using a SteponeplusDetectionSystem of ABI company, and the PCR amplification is carried out by adopting a two-step method, wherein the reaction conditions are as follows: pre-denaturation, 1 cycle, 15s at 95 ℃; PCR reaction, 45 cycles, 95 ℃ 5s, 60 ℃ 45 s.
Experimental results show that the optimal annealing temperature of the gene cpCB primer is 60 ℃, the optimal primer concentration is 0.2 mu M/L, and the amplification effect is best.
2. Quantitative PCR detection limit, quantitative interval and standard curve
The plasmid standard obtained in example one was diluted in 10-fold gradient to obtain a quantitative PCR template DNA. The PCR reaction system is 20. mu.L, 2. mu.L of plasmid standard is added as a reaction template, and the final concentration is 4.65X 101～4.65×108copies/. mu.L. The quantitative PCR reaction was performed under the optimized conditions. Deionized water was used as a negative control. Establishing real-time quantitative PCR by taking the logarithmic value of the dilution gradient of the standard as an abscissa and the critical cycle (Ct) as an ordinateA standard curve.
The PCR amplification curve of the standard product is shown in FIG. 2, the exponential amplification stage and the plateau stage of the target gene are very obvious, the fluorescence quantitative kinetic curve is smooth, and the amplification curve is smooth, which shows that the PCR method of the research has good amplification effect and can be used for quantitative analysis.
The plasmid standard substance is detected by using quantitative PCR reaction conditions, and the minimum detection limit is 4.65 multiplied by 101copies/mu L, quantitative detection interval of 4.65X 102～4.65×107copies/μ L, standard curve slope of standard curve equation of-3.56, R20.9995, and the amplification efficiency E was 91%.
The results show that the real-time quantitative PCR method for the research has high amplification efficiency of the standard product, good linear relation and accurate and feasible detection method, and meets the requirement of preparing a real-time quantitative PCR standard curve.
3. Specificity analysis of quantitative PCR detection method
The PCR dissolution curve of the standard product is shown in figure 3, and the dissolution curve analysis shows that the dissolution curve of the target gene presents a single peak, the dissolution temperature is 85.31 +/-1 ℃, which indicates that the gene cpcB can be specifically amplified, and the PCR method has good specificity and reliable real-time quantitative PCR result.
Example three, detection of phycocyanin in practical environmental water body by quantitative PCR method
1. Actual water sample collection and concentration
And taking a surface water sample at a position 0.5m below the water surface in 8 months. After a water sample is filtered by a 0.22 mu m micropore, the filter membrane is preserved to-20 ℃ for subsequent experimental analysis.
2. Extraction of DNA
After the experiment is finished, the total DNA of the samples is extracted and purified according to the kit operation instructions, and finally 50 mu L of DNA samples are obtained from each sample.
3. Quantitative PCR detection
Taking 2 μ L of the obtained DNA sample as a template, carrying out quantitative PCR detection, wherein the quantitative PCR detection system and method refer to example two 1.
Table 1 shows the final results of the copy number detection of the cpcB gene in water by quantitative PCR.
Therefore, the method for quantitatively detecting phycocyanin and the special standard product thereof are disclosed, a specific primer is designed aiming at a cpcB gene, and the phycocyanin in a water environment is quantitatively detected by adopting a real-time quantitative PCR method.
Compared with the prior art, the invention has the advantages that:
the standard substance and the detection method for quantitatively detecting phycocyanin have the characteristics of combination of broad spectrum identification and specificity, high sensitivity, simple and convenient preparation method, long-term storage, good purity and wide linear detection range, and can be used for quickly and quantitatively detecting phycocyanin in a water environment sample.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Modifications and variations can be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the present invention. Therefore, the scope of the invention should be determined from the following claims.
Sequence listing
<110> Shanghai City Water resources exploitation and utilization national engineering center Limited
<120> method for quantitatively detecting phycocyanin and special standard substance thereof
<130> WISE-1798I-CN
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 139
<212> DNA
<213> Artificial Synthesis (Artificial sequence)
<400> 1
atgttaccta cgctaccttc tctggcgacg gcagtgttct cgatgatcgt tgcttaaatg 60
gtctgcgcga aacctatgta gctttgggag taccaggagc ttccgtagct gctggcgtaa 120
gcaaaatgaa agaagccgc 139
Claims (6)
1. A special standard substance for quantitatively detecting phycocyanin is characterized in that:
the standard substance is a recombinant plasmid containing a cpcB gene, and the sequence of the cpcB gene is the sequence in the sequence table 1.
2. The special standard for quantitatively detecting phycocyanin according to claim 1, wherein: the starting plasmid in the recombinant plasmid is pMD-18T.
3. The method for quantitatively detecting phycocyanin according to claim 1, comprising the steps of:
step one, taking a recombinant plasmid as a standard template, performing PCR amplification by using real-time quantitative PCR, and establishing a unary linear regression curve corresponding to the concentration of the standard and the critical cycle number to obtain a standard curve;
and step two, replacing the standard substance in the step one with a sample to be detected, carrying out real-time quantitative PCR amplification, and obtaining the copy number of the cpcB gene in the sample to be detected according to the critical cycle number of the amplification result and a standard curve, so as to obtain the content of the phycocyanin in the sample to be detected.
4. The method for quantitatively detecting phycocyanin according to claim 3, wherein the method comprises the steps of: the real-time quantitative PCR amplification system consists of a real-time quantitative PCR amplification buffer solution, a primer pair and a template; the concentration of the upstream primer and the downstream primer in the reaction system is 0.2 mu M/L, and the concentration of the upstream primer is 5'-ATGTTACCTACGCTACCTTCTCTGG-3', and the concentration of the downstream primer is 5'-GCGGCTTCTTTCATTTTGCT-3'.
5. The method for quantitatively detecting phycocyanin according to claim 3, wherein the method comprises the steps of: the concentration of the standard template in the real-time quantitative PCR amplification system is 4.65 multiplied by 102copies/μL、4.65×103copies/μL、4.65×104copies/μL、4.65×105copies/μL、4.65×106copies/. mu.L and 4.65X 107copies/. mu.L.
6. The method for quantitatively detecting phycocyanin according to claim 3, wherein the method comprises the steps of: the annealing temperature in the PCR reaction was 60 ℃.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111066846.1A CN113774116A (en) | 2021-09-13 | 2021-09-13 | Method for quantitatively detecting phycocyanin and special standard product thereof |
| PCT/CN2021/120093 WO2023035334A1 (en) | 2021-09-13 | 2021-09-24 | Method for quantitatively measuring phycocyanin and special standard product therefor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111066846.1A CN113774116A (en) | 2021-09-13 | 2021-09-13 | Method for quantitatively detecting phycocyanin and special standard product thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113774116A true CN113774116A (en) | 2021-12-10 |
Family
ID=78842804
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111066846.1A Pending CN113774116A (en) | 2021-09-13 | 2021-09-13 | Method for quantitatively detecting phycocyanin and special standard product thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN113774116A (en) |
| WO (1) | WO2023035334A1 (en) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974632B (en) * | 2010-10-25 | 2012-05-16 | 清华大学 | Method for quantitatively detecting toxigenic microcystis and special standard substance thereof |
| CN101974633B (en) * | 2010-10-25 | 2012-05-16 | 清华大学 | Method for quantitatively detecting microcystin and specific standard product thereof |
| CN107502678A (en) * | 2017-10-24 | 2017-12-22 | 上海城市水资源开发利用国家工程中心有限公司 | A kind of method and device for detecting blue algae producing microcystic toxins |
| CN110863033A (en) * | 2019-11-27 | 2020-03-06 | 上海城市水资源开发利用国家工程中心有限公司 | Standard substance for quantitatively detecting microcystis and preparation and detection methods thereof |
-
2021
- 2021-09-13 CN CN202111066846.1A patent/CN113774116A/en active Pending
- 2021-09-24 WO PCT/CN2021/120093 patent/WO2023035334A1/en active Application Filing
Non-Patent Citations (3)
| Title |
|---|
| CHEN LIE等: ""Changes in metabolites, antioxidant system, and gene expression in Microcystis aeruginosa under sodium chloride stress"", 《ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY》 * |
| FORMIGHIERI, CINZIA等: "A phycocyanin· phellandrene synthase fusion enhances recombinant protein expression and β-phellandrene (monoterpene) hydrocarbons production in Synechocystis (cyanobacteria)", 《METABOLIC ENGINEERING》 * |
| WU，XIAN-JUN等: "Biosynthesis of fluorescent β subunits of c-phycocyanin from spirulina subsalsa in escherichia coli, and their antioxidant properties", 《MOLECULES》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2023035334A1 (en) | 2023-03-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2020258531A1 (en) | Detection primer, kit and quantitative measurement method for cable bacteria candidatus electronema | |
| CN102925581A (en) | Method for quantitating anaerobic ammonia oxidizing bacteria in sediment of aquiculture environment | |
| CN114561374A (en) | Novel thermophilic endonuclease mutant and preparation method and application thereof | |
| CN110863033A (en) | Standard substance for quantitatively detecting microcystis and preparation and detection methods thereof | |
| CN111471776A (en) | Shrimp liver enterocytozoon detection kit and detection method thereof | |
| CN114196786A (en) | Poultry adenovirus type 4 and 8 dual fluorescent quantitative PCR rapid detection kit and method | |
| CN107130030B (en) | DNA Marker for detecting coal geology microorganism archaea species and preparation method and application thereof | |
| CN106702008B (en) | Method for detecting six functional genes of Cr (VI) reduction complex flora by multiplex real-time fluorescent PCR | |
| CN110819699A (en) | Quantitative detection method for human excrement indicator in water environment | |
| CN113774116A (en) | Method for quantitatively detecting phycocyanin and special standard product thereof | |
| CN111647605A (en) | crRNA for detecting African swine fever virus and kit | |
| CN103966244A (en) | DNA polymerase coding DNA, enzyme coded thereby, and application and preparation method of DNA polymerase | |
| CN108004334B (en) | Quadruple fluorescent PCR primer group, probe group, kit and method for detecting four pathogenic bacteria in drinking water | |
| CN113817856B (en) | Primer pair, kit and method for detecting archaea Candidatus Methanoliparum | |
| AU2021104733A4 (en) | Method for identifying and detecting pythium pathogen of red rot disease of laver | |
| CN115725756A (en) | RPA primer group, reagent and kit for detecting treponema pallidum and application of RPA primer group, reagent and kit | |
| CN114622041A (en) | Primer and TaqMan probe for detecting canine torque teno virus and application thereof | |
| CN114032293A (en) | Quantitative detection method and application of nitrifying bacillus bacteria in sludge | |
| CN111378724B (en) | RNA amplification detection method and detection kit | |
| CN112322707A (en) | Detection method of resistance gene intl1 in offshore sea area | |
| CN101709334B (en) | DNA molecular mark and application thereof of SXI inbred line mouse | |
| CN111996269A (en) | Fluorescence PCR detection method for bacillus amyloliquefaciens, bacillus subtilis and bacillus licheniformis | |
| CN112063759A (en) | RT-LAMP primer, kit and detection method for simultaneously detecting multiple viruses of banana | |
| CN114107539B (en) | LAMP primer composition for detecting pythium delicicola and application thereof | |
| CN116179722B (en) | Primer pair, kit and method for detecting archaea Hadesarchaea |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20211210 |
|
| WD01 | Invention patent application deemed withdrawn after publication |