CN108103205A - A kind of the real time fluorescent quantitative primer and its detection method of Larimichthys crocea reference gene β-Actin - Google Patents
A kind of the real time fluorescent quantitative primer and its detection method of Larimichthys crocea reference gene β-Actin Download PDFInfo
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- CN108103205A CN108103205A CN201711353658.0A CN201711353658A CN108103205A CN 108103205 A CN108103205 A CN 108103205A CN 201711353658 A CN201711353658 A CN 201711353658A CN 108103205 A CN108103205 A CN 108103205A
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Abstract
The present invention relates to gene engineering technology fields.A kind of real time fluorescent quantitative primer of Larimichthys crocea reference gene β Actin of the present invention, primer sequence are：Sense primer：5’‑TC CTC GGT ATG GAA TCT TGC‑3’；Anti-sense primer：5’‑GA GTA TTT ACG CTC AGG TGG G‑3’.The detection method of the real time fluorescent quantitative primer of Larimichthys crocea reference gene β Actin, comprises the following steps：（1）Using designed primer, primer extension product segment is prepared, then standard PCR amplification is carried out using the primer extension product segment, obtains PCR reaction products；（2）By the PCR reaction products with 2 ~ 3% agarose gel electrophoresis 25 ~ 35 minutes.The primer of the present invention has the characteristics of at low cost, accuracy is high, and detection method of the invention has the high advantage of accuracy easy to operate.
Description
Technical field
The present invention relates to gene engineering technology field, in particular to a kind of reality of Larimichthys crocea reference gene β-Actin
When fluorescent quantitation primer and its detection method.
Background technology
Using β-Actin as the real time fluorescent quantitative method of reference gene, it is immunized in research Larimichthys crocea, the related genes such as develops
Expression regulation in terms of be widely used.The genome sequence of Larimichthys crocea β-Actin genes and open reading frame sequence length phase
Seemingly, therefore respectively the common real time fluorescent quantitative primer on two different extrons is easy to and the residual in cDNA templates
DNA is combined, and amplifies the slightly longer product of a segment length, so as to influence the accuracy of real time fluorescent quantitative result.In order to avoid
This problem, conventional method need to carry out the DNA in total serum IgE using DNA enzymatic kit digestion before reverse transcription cDNA
It removes.But this single stepping is not only time-consuming, laborious, DNA enzymatic kit is expensive, and a large amount of losses of total serum IgE can be caused.
Therefore a kind of at low cost, real time fluorescent quantitative of the high Larimichthys crocea reference gene β-Actin of accuracy is needed at present
Primer.
The content of the invention
Real time fluorescent quantitative primer and its inspection of a kind of Larimichthys crocea reference gene β-Actin is provided to solve the above-mentioned problems
Survey method, the present invention use following technical scheme：
A kind of real time fluorescent quantitative primer of Larimichthys crocea reference gene β-Actin, the sequence of primer are：
Sense primer：5’-TC CTC GGT ATG GAA TCT TGC-3’；
Anti-sense primer：5’-GA GTA TTT ACG CTC AGG TGG G-3’.
Present invention design is a kind of while across the β-Actin real time fluorescent quantitative primers of two exon regions, i.e. primer
A part of sequence be located at a upper extron and another part sequence is located at next extron, then can prevent primer and cDNA
Residual DNA in template combines and wrong amplification occurs, and then DNA is avoided to remain the influence to real time fluorescent quantitative result, inspection
The remaining influences of DNA can be excluded completely by surveying the length of amplified production segment and illustrating the primer, be obtained by this method big
Yellow croaker reference gene β-Actin real time fluorescent quantitatives primer has amplified production fragment length suitable, and amplification efficiency is high, can be complete
It is complete to exclude the features such as DNA remains the influence to experimental result.
The detection method of the real time fluorescent quantitative primer of Larimichthys crocea reference gene β-Actin, comprises the following steps：
（1）Using designed primer, prepare primer extension product segment, then using the primer extension product segment into
Row standard PCR amplification obtains PCR reaction products；
（2）By the PCR reaction products with 2 ~ 3% agarose gel electrophoresis 25 ~ 35 minutes.
The present invention can exactly be detected primer with the above method, improve the testing result of the present invention
Accuracy.
Preferably, mispairing, dimer and hairpin structure are not present in the primer extension product segment.
Preferably, in step（1）PCR amplification during, selected template be Larimichthys crocea tissue cDNA.
Preferably, the reaction density of the Larimichthys crocea tissue cDNA for 40 ~ 50 nanograms/microlitre.
After the present invention has carried out real time fluorescent quantitative experiment, using log cDNA concentration values as abscissa, Larimichthys crocea internal reference base
Because the reaction Ct values of β-Actin are ordinate, coordinate system is established, obtains R2For 0.999 X+ of linear equation Y=- 3.128
24.68.Show that the amplification efficiency of Larimichthys crocea reference gene β-Actin primers is linearly closed with cDNA template concentrations in extremely significant
System.There is good reaction result under the concentration.
Preferably, the standard PCR amplification reaction system is 20 microlitres of reaction systems, sense primer dosage is 0.6-
0.8 microlitre, anti-sense primer dosage is 0.6-0.8 microlitres, and template consumption is 0.6-0.8 microlitres.
Preferably, the PCR response procedures described in the standard PCR amplification reaction process are：95 DEG C of pre-degenerations 5
Minute, 32 Xun Huans（95 DEG C are denatured 30 seconds, and 60 DEG C are annealed 30 seconds, and 72 DEG C extend 15 seconds）, 72 DEG C finally extend 10 minutes.
The present invention can exactly be detected primer under the testing conditions, improve the testing result of the present invention
Accuracy.
The beneficial effects of the present invention are：The present invention has at low cost, the high advantage of accuracy.
Specific embodiment
The present invention is further explained with reference to specific implementation case：
Embodiment 1
A kind of real time fluorescent quantitative primer of Larimichthys crocea reference gene β-Actin, primer sequence are：
Sense primer：5’-TC CTC GGT ATG GAA TCT TGC-3’；
Anti-sense primer：5’-GA GTA TTT ACG CTC AGG TGG G-3’.
The detection method of the real time fluorescent quantitative primer of Larimichthys crocea reference gene β-Actin, comprises the following steps：
（1）Using designed primer, prepare primer extension product segment, then using the primer extension product segment into
Row standard PCR amplification obtains PCR reaction products；
（2）By the PCR reaction products with 2% agarose gel electrophoresis 25 minutes.
Wherein, mispairing, dimer and hairpin structure are not present in the primer extension product segment.
Wherein, in step（1）PCR amplification during, selected template be Larimichthys crocea tissue cDNA.
Wherein, the reaction density of the Larimichthys crocea tissue cDNA for 40 nanograms/microlitre.
The standard PCR amplification reaction system is 20 microlitres of reaction systems, and sense primer dosage is 0.8 microlitre, downstream
Primer dosage is 0.6 microlitre, and template consumption is 0.8 microlitre.
Wherein, the PCR response procedures described in the standard PCR amplification reaction process are：95 DEG C of pre-degenerations 5 are divided
Clock, 32 Xun Huans（95 DEG C are denatured 30 seconds, and 60 DEG C are annealed 30 seconds, and 72 DEG C extend 15 seconds）, 72 DEG C finally extend 10 minutes.
Embodiment 2
A kind of real time fluorescent quantitative primer of Larimichthys crocea reference gene β-Actin, primer sequence are：
Sense primer：5’-TC CTC GGT ATG GAA TCT TGC-3’；
Anti-sense primer：5’-GA GTA TTT ACG CTC AGG TGG G-3’.
The detection method of the real time fluorescent quantitative primer of Larimichthys crocea reference gene β-Actin, comprises the following steps：
（1）Using designed primer, prepare primer extension product segment, then using the primer extension product segment into
Row standard PCR amplification obtains PCR reaction products；
（2）By the PCR reaction products with 3% agarose gel electrophoresis 35 minutes.
Wherein, mispairing, dimer and hairpin structure are not present in the primer extension product segment.
Wherein, in step（1）PCR amplification during, selected template be Larimichthys crocea tissue cDNA.
Wherein, the reaction density of the Larimichthys crocea tissue cDNA for 50 nanograms/microlitre.
The standard PCR amplification reaction system is 20 microlitres of reaction systems, and sense primer dosage is 0.8 microlitre, downstream
Primer dosage is 0.6 microlitre, and template consumption is 0.8 microlitre.
Wherein, the PCR response procedures described in the standard PCR amplification reaction process are：95 DEG C of pre-degenerations 5 are divided
Clock, 32 Xun Huans（95 DEG C are denatured 30 seconds, and 60 DEG C are annealed 30 seconds, and 72 DEG C extend 15 seconds）, 72 DEG C finally extend 10 minutes.
Embodiment 3
A kind of real time fluorescent quantitative primer of Larimichthys crocea reference gene β-Actin, primer sequence are：
Sense primer：5’-TC CTC GGT ATG GAA TCT TGC-3’；
Anti-sense primer：5’-GA GTA TTT ACG CTC AGG TGG G-3’.
The detection method of the real time fluorescent quantitative primer of Larimichthys crocea reference gene β-Actin, comprises the following steps：
（1）Using designed primer, prepare primer extension product segment, then using the primer extension product segment into
Row standard PCR amplification obtains PCR reaction products；
（2）By the PCR reaction products with 2 ~ 3% agarose gel electrophoresis 25 ~ 35 minutes.
Wherein, mispairing, dimer and hairpin structure are not present in the primer extension product segment.
Wherein, in step（1）PCR amplification during, selected template be Larimichthys crocea tissue cDNA.
Wherein, the reaction density of the Larimichthys crocea tissue cDNA for 40 ~ 50 nanograms/microlitre.
Wherein, the standard PCR amplification reaction system is 20 microlitres of reaction systems, and sense primer dosage is 0.8 microlitre,
Anti-sense primer dosage is 0.6 microlitre, and template consumption is 0.8 microlitre.
Wherein, the PCR response procedures described in the standard PCR amplification reaction process are：95 DEG C of pre-degenerations 5 are divided
Clock, 32 Xun Huans（95 DEG C are denatured 30 seconds, and 60 DEG C are annealed 30 seconds, and 72 DEG C extend 15 seconds）, 72 DEG C finally extend 10 minutes.
Using TaKaRa DL2000 DNA Marker as reference in the result of above example, seen using gel imager
Electrophoresis result is examined to find：It is then obtained near 200 bp, bright and single amplification using the liver cDNA of Larimichthys crocea as template
Band, then proving the primer of the present invention has good effect.
Claims (7)
1. a kind of real time fluorescent quantitative primer of Larimichthys crocea reference gene β-Actin, which is characterized in that primer sequence is：
Sense primer：5’-TC CTC GGT ATG GAA TCT TGC-3’；
Anti-sense primer：5’-GA GTA TTT ACG CTC AGG TGG G-3’.
2. a kind of detection side of the real time fluorescent quantitative primer of Larimichthys crocea reference gene β-Actin according to claim 1
Method, it is characterised in that comprise the following steps：
（1）Using designed primer, prepare primer extension product segment, then using the primer extension product segment into
Row standard PCR amplification obtains PCR reaction products；
（2）By the PCR reaction products with 2 ~ 3% agarose gel electrophoresis 25 ~ 35 minutes.
3. a kind of detection side of the real time fluorescent quantitative primer of Larimichthys crocea reference gene β-Actin according to claim 1
Method, it is characterised in that：Mispairing, dimer and hairpin structure are not present in the primer extension product segment.
4. a kind of detection side of the real time fluorescent quantitative primer of Larimichthys crocea reference gene β-Actin according to claim 1
Method, it is characterised in that：In step（1）PCR amplification during, selected template be Larimichthys crocea tissue cDNA.
5. a kind of detection side of the real time fluorescent quantitative primer of Larimichthys crocea reference gene β-Actin according to claim 1
Method, it is characterised in that：The reaction density of the Larimichthys crocea tissue cDNA for 40 ~ 50 nanograms/microlitre.
6. a kind of detection side of the real time fluorescent quantitative primer of Larimichthys crocea reference gene β-Actin according to claim 1
Method, it is characterised in that：The standard PCR amplification reaction system is 20 microlitres of reaction systems, and sense primer dosage is 0.6-0.8
Microlitre, anti-sense primer dosage is 0.6-0.8 microlitres, and template consumption is 0.6-0.8 microlitres.
7. a kind of detection side of the real time fluorescent quantitative primer of Larimichthys crocea reference gene β-Actin according to claim 1
Method, which is characterized in that the PCR response procedures described in the standard PCR amplification reaction process are：95 DEG C of pre-degenerations 5 are divided
Clock, 32 Xun Huans（95 DEG C are denatured 30 seconds, and 60 DEG C are annealed 30 seconds, and 72 DEG C extend 15 seconds）, 72 DEG C finally extend 10 minutes.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5543509A (en) * | 1992-08-14 | 1996-08-06 | The United States Of America As Represented By The Department Of Health And Human Services | Method for quantifying laminin and β-actin messenger RNA |
| CN102643799A (en) * | 2012-04-23 | 2012-08-22 | 中国水产科学研究院淡水渔业研究中心 | Method for cloning intron-containing partial sequence of GAPDH (glyceraldehyde-phosphate dehydrogenase) gene of Cyprinus carpio var. Jian |
| CN103301162A (en) * | 2013-04-17 | 2013-09-18 | 苏州大学 | Method for establishing fish inflammatory bowel disease model and established model thereof |
| CN106520769A (en) * | 2016-11-23 | 2017-03-22 | 中国水产科学研究院珠江水产研究所 | Infectious spleen and kidney necrosis virus (ISKNV) gene intron and its use in distinguishing of live and inactivated ISKNVs |
-
2017
- 2017-12-15 CN CN201711353658.0A patent/CN108103205A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5543509A (en) * | 1992-08-14 | 1996-08-06 | The United States Of America As Represented By The Department Of Health And Human Services | Method for quantifying laminin and β-actin messenger RNA |
| CN102643799A (en) * | 2012-04-23 | 2012-08-22 | 中国水产科学研究院淡水渔业研究中心 | Method for cloning intron-containing partial sequence of GAPDH (glyceraldehyde-phosphate dehydrogenase) gene of Cyprinus carpio var. Jian |
| CN103301162A (en) * | 2013-04-17 | 2013-09-18 | 苏州大学 | Method for establishing fish inflammatory bowel disease model and established model thereof |
| CN106520769A (en) * | 2016-11-23 | 2017-03-22 | 中国水产科学研究院珠江水产研究所 | Infectious spleen and kidney necrosis virus (ISKNV) gene intron and its use in distinguishing of live and inactivated ISKNVs |
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Application publication date: 20180601 |