CN113481221A - Coding gene SOD in blood related to milk cow ketosis and PCR detection kit thereof - Google Patents
Coding gene SOD in blood related to milk cow ketosis and PCR detection kit thereof Download PDFInfo
- Publication number
- CN113481221A CN113481221A CN202110992400.5A CN202110992400A CN113481221A CN 113481221 A CN113481221 A CN 113481221A CN 202110992400 A CN202110992400 A CN 202110992400A CN 113481221 A CN113481221 A CN 113481221A
- Authority
- CN
- China
- Prior art keywords
- sod
- ketosis
- blood
- milk cow
- pcr detection
- 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
- 210000004369 blood Anatomy 0.000 title claims abstract description 39
- 239000008280 blood Substances 0.000 title claims abstract description 39
- 208000007976 Ketosis Diseases 0.000 title claims abstract description 34
- 230000004140 ketosis Effects 0.000 title claims abstract description 34
- 238000001514 detection method Methods 0.000 title claims abstract description 32
- 235000013336 milk Nutrition 0.000 title abstract description 28
- 239000008267 milk Substances 0.000 title abstract description 28
- 210000004080 milk Anatomy 0.000 title abstract description 28
- 241000283690 Bos taurus Species 0.000 claims abstract description 68
- 101150017120 sod gene Proteins 0.000 claims abstract description 22
- 230000036542 oxidative stress Effects 0.000 claims abstract description 9
- 239000002773 nucleotide Substances 0.000 claims abstract description 6
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 6
- 235000013365 dairy product Nutrition 0.000 claims description 14
- 239000012154 double-distilled water Substances 0.000 claims description 6
- 239000013642 negative control Substances 0.000 claims description 5
- 239000013641 positive control Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 4
- 230000014509 gene expression Effects 0.000 claims description 3
- 238000007400 DNA extraction Methods 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 238000012360 testing method Methods 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 17
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 4
- 238000003771 laboratory diagnosis Methods 0.000 abstract description 3
- 108010012715 Superoxide dismutase Proteins 0.000 description 33
- 238000006243 chemical reaction Methods 0.000 description 12
- 102000019197 Superoxide Dismutase Human genes 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 5
- 150000003254 radicals Chemical class 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 201000010099 disease Diseases 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000009984 peri-natal effect Effects 0.000 description 3
- 208000015754 perinatal disease Diseases 0.000 description 3
- 102000004506 Blood Proteins Human genes 0.000 description 2
- 108010017384 Blood Proteins Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 2
- 210000000577 adipose tissue Anatomy 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 235000021588 free fatty acids Nutrition 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 150000005830 nonesterified fatty acids Chemical class 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- WHBMMWSBFZVSSR-UHFFFAOYSA-N 3-hydroxybutyric acid Chemical class CC(O)CC(O)=O WHBMMWSBFZVSSR-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000004145 Endometritis Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000031964 Other metabolic disease Diseases 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 210000003165 abomasum Anatomy 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 235000019577 caloric intake Nutrition 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007247 enzymatic mechanism Effects 0.000 description 1
- 230000012173 estrus Effects 0.000 description 1
- 210000002219 extraembryonic membrane Anatomy 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 210000003701 histiocyte Anatomy 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- -1 lipid peroxides Chemical class 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229940118019 malondialdehyde Drugs 0.000 description 1
- 208000004396 mastitis Diseases 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004089 microcirculation Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000008634 non enzymatic mechanism Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000007348 radical reaction Methods 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Images
Classifications
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
-
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to coding gene SOD related to ketosis in milk cow blood, and the nucleotide sequence is shown in SEQ ID No. 1. The invention also relates to a PCR detection kit for rapidly detecting coding gene SOD related to ketosis in cow blood and application of the kit. Through extensive and intensive research, the invention detects that SOD genes exist in the blood of ketosis cows, namely the SOD genes containing 143bp nucleotide sequences in the blood of the ketosis cows, and verifies the content of the SOD genes in different states of the cow organism by using specific primers through a PCR technology; the kit can be used for quickly and high-flux detecting the SOD gene in the milk cow blood, can be used as an auxiliary method for the traditional detection of the ketosis oxidative stress of the milk cow, and provides a new method with simplicity, rapidness and good repeatability for the detection and laboratory diagnosis of the ketosis oxidative stress of the milk cow.
Description
Technical Field
The invention belongs to the field of animal molecular biology, and relates to a coding gene SOD in blood related to dairy cow ketosis and a PCR detection kit thereof.
Background
In recent years, as the dairy cattle breeding industry in China continuously develops to scale and intensification, the incidence of metabolic diseases of dairy cattle in perinatal period is increased. The perinatal period of the dairy cattle is in the transition period, the energy intake does not meet the energy demand in the body, and the dairy cattle is in the negative energy balance state. Therefore, the cow will be subjected to body fat mobilization, and excessive body fat mobilization will generate a large amount of non-esterified fatty acids (NEFA) and beta-hydroxybutyric acids (BHBA) to cause ketosis and other metabolic diseases. The diseases not only obviously reduce the milk yield of the lactating cows, but also seriously affect the oestrus and mating of the postpartum cows, and also often induce other diseases in perinatal period such as abomasum displacement, retained afterbirth, endometritis, mastitis and the like, thereby causing great economic loss to the dairy cow breeding industry. The research finds that animals eliminate free radicals in vivo mainly through an enzymatic mechanism and a non-enzymatic mechanism, wherein superoxide dismutase (SOD) is one of the most main enzymes and can eliminate superoxide anion free radicals in the biological oxidation process(superoxide anion，O2) Considered as the first line of defense of the antioxidant system. SOD is one of the most effective antioxidants in nature to protect against oxidative attack, plays an important role in eliminating oxidative damage, and can also participate in immune reactions caused by viral infection and bacteria. When a disease occurs in a body, free radicals are increased, so that the change of SOD content in the body is initiated, and lipid peroxides are further oxidized to form Malondialdehyde (malonodialdehyde MDA) which is a final product of lipid peroxidation. MDA can cause cellular metabolic and functional disorders, and even death. When the peroxidation of lipid in vivo is enhanced and the ability of scavenging oxygen free radicals is weakened, the disorder of free radical reaction, microcirculation disturbance and blood stasis in vivo are caused, thus resulting in diseases such as retained fetal membranes. Therefore, the detection of the change of the SOD content in the milk cow serum with perinatal diseases can provide more reliable theoretical basis for further discussing the pathogenesis of the milk cow perinatal diseases and preventing the milk cow perinatal diseases.
SOD is mainly detected by traditional methods such as collecting samples such as blood or histiocyte, separating serum protein, performing biochemical identification and the like, but the process is complicated, labor-consuming, time-consuming, has a plurality of external influence factors, and the result is unstable. Therefore, an efficient and rapid detection method for detecting SOD is urgently needed, the occurrence of oxidative stress of the dairy cows is found in time, and the method has great significance for the development of the dairy cow breeding industry in China.
Disclosure of Invention
The first purpose of the invention is to provide a nucleotide sequence of coding gene SOD related to ketosis in milk cow blood.
The invention aims to provide a PCR detection kit for coding gene SOD in blood related to dairy cow ketosis, which solves the problems of tedious, labor-consuming, time-consuming, many external influence factors and unstable result of the traditional methods such as serum protein separation, biochemical identification and the like.
The third purpose of the invention is to provide the application of the PCR detection kit.
The invention is realized by the following technical scheme:
an encoding gene SOD related to ketosis in milk cow blood, the nucleotide sequence of which is shown in SEQ ID NO. 1.
Secondly, a PCR detection kit for rapidly detecting coding gene SOD related to ketosis in cow blood, wherein primer sequences in the kit are as follows:
a forward primer: TGTTCTGCGGCGTCGTTTTC (SEQ ID NO. 1);
reverse primer: CCTCGAAGTGGATGGTGCCT (SEQ ID NO. 2).
Further, the kit also contains one or more of ddH2O, 2XTaq Master Mix and a sample genomic DNA extraction reagent.
Further, the kit may further comprise one or more of a positive control or a negative control.
Further, the positive control is a DNA sample expressed by SOD genes in the blood of healthy cows.
Furthermore, the negative control is a DNA sample expressed by SOD genes in the blood of ketosis cows.
And thirdly, the PCR detection kit is applied to the detection and identification of the oxidative stress of the ketosis dairy cows.
Adopt above-mentioned technical scheme's positive effect: through extensive and intensive research, the invention detects that SOD genes exist in the blood of ketosis cows, namely the SOD genes containing 143bp nucleotide sequences in the blood of the ketosis cows, and verifies the content of the SOD genes in different states of the cow organism by using specific primers through a PCR technology; the kit can be used for quickly and high-flux detecting the SOD gene in the milk cow blood, can be used as an auxiliary method for the traditional detection of the ketosis oxidative stress of the milk cow, and provides a new method with simplicity, rapidness and good repeatability for the detection and laboratory diagnosis of the ketosis oxidative stress of the milk cow.
Drawings
FIG. 1 is a photograph showing the identification of SOD gene by PCR, wherein lane M is: DL500 Marker, lane 1 is milk cow SOD gene fragment (with target band at 143 bp);
FIG. 2 is a picture of PCR identification of SOD gene distribution and fragment size in milk cow blood; wherein lane M is: DL500 Marker; lane 1 is the SOD gene fragment of healthy cows (the band of interest at 143 bp); lane 2 shows the fragment of the SOD gene of ketosis cows (the target band at 143 bp).
Detailed Description
The technical solution of the present invention is further described with reference to the following specific examples, which should not be construed as limiting the present invention:
example 1
Example 1 illustrates the bioinformatics approach to identify the distribution of SOD genes.
Searching SOD genes in a full-genome database by using blast online comparison software (http:// blast. ncbinlm. nih. gov/blast. cgi) at NCBI, wherein the search result shows that a milk cow organism contains the SOD genes (SEQ ID NO.1) and the length of the genes is 134bp of nucleotide sequence, and the nucleotide sequence is reversely transcribed into cDNA after extracting RNA detection concentration and quantifying, wherein the expression level of the SOD genes in the blood of healthy milk cows is extremely high; the SOD gene expression level in the blood of ketosis milk cow is extremely low; both bands were visible at 143 bp. The bioinformatics identification result shows that the SOD specificity is good, and the PCR method can be used for quickly detecting the SOD in the blood of the dairy cow.
Example 2
This example illustrates the preparation of a kit.
Designing and synthesizing a primer: the primers were designed and analyzed using SOD gene as template, and the optimal detection primers were selected according to the genomic DNA sequence, whose nucleotide sequences are shown in Table 1 below:
TABLE 1 primer sequences
The primer pair can be packaged independently or can be prepared into PCR detection solution. In the PCR detection solution, the amount of the primer pair may be any conventional amount known to those skilled in the art.
That is, the kit of the present invention may contain the primer set packaged independently, or may contain a PCR detection solution containing the primer set.
Further, the kit may further contain double distilled water (ddH20), 2XTaq Master Mix, and the like.
Example 3
This example illustrates the use of the kit.
(1) Extracting sample genome RNA; and detecting the concentration of RNA in the extracted sample, and performing reverse transcription to obtain cDNA after quantification. The step of extracting the RNA of the sample comprises the following steps: (a) and adding Trizol lysate into the treated tissue, the feces supernatant and the blood for lysis. After being mixed uniformly, the liquid in a 2mL EP tube is violently shaken by a vortex oscillator, and then is placed on ice and stands for 10 min. (b) Add 200uL chloroform, shake the EP tube vigorously for 1min to better extract nucleic acid, and stand on ice for 10 min. (c) Centrifuging at 12000r/min for 15min, collecting two thirds of supernatant of transparent liquid in the test tube, adding equal amount of isopropanol, and mixing gently. Standing on ice for 10 min. (d) The centrifugation time was 12000r/min for 10min, and 75% absolute ethanol (prepared with DEPC water) was prepared. (e) The supernatant was discarded, 1mL of prepared 75% absolute ethanol was added, and the EP tube was gently shaken. (f) Centrifuging at 8000r/min at 4 deg.C for 6min, discarding supernatant, and air drying EP tube at room temperature for 10min to remove 75% anhydrous ethanol in the tube. The precipitate was dissolved in 10uL of DEPC sterile water. (e) After the concentration of the extracted RNA is measured, the uniform concentration (g) is calculated, the RNA is reversely transcribed into cDNA according to the instruction of the kit, and the cDNA is placed at the temperature of minus 20 ℃ for standby.
(2) Sample adding: respectively adding the sample genome DNA and the positive control or the negative control into a PCR tube provided with a PCR reaction system to obtain the corresponding sample reaction tube, the positive reaction tube or the negative reaction tube, wherein the PCR reaction system contains the SOD gene detection primer pair; preparing a PCR detection solution: double distilled water (ddH)2O): 9 uL; an upstream primer: 1 uL; the following primers are provided: 1 uL; DNA sample: 1.5 uL; 2XTaq Master Mix: 12.5 uL; a total of 25 uL.
(3) And (3) PCR reaction: the reaction tube is arranged on a PCR instrument, and circulation parameters are set for carrying out PCR reaction; the PCR reaction conditions were set as follows: (a) 3min at 94 ℃; (b) 30s at 94 ℃; (c) 35s at 60 ℃; (d) 35s at 72 ℃; the steps (b) to (d) are circulated for 34 times; (e) 5min at 72 ℃.
(4) After the PCR reaction was completed, the results were analyzed.
Example 4
This example illustrates the specific identification of SOD genes in milk cow blood by the kit.
The primers in the kit of example 2 are used, and the distribution characteristics of the SOD genes in the milk cow blood under different states are identified by a PCR method by using the milk cow blood groups under different physiological and pathological states as templates.
The PCR reaction system is (25 uL): 9uL double distilled water (ddH)20) 12.5uL 2XTaq Master Mix, SOD-F1uL, SOD-R1 uL, template l.5uL.
The PCR procedure was (a)94 ℃ for 3 min; (b) 30s at 94 ℃; (c) 35s at 60 ℃; (d) 35s at 72 ℃; the steps (b) to (d) are circulated for 34 times; (e) 5min at 72 ℃.
The PCR product was subjected to 2% agarose gel electrophoresis, and the results of the PCR electrophoresis showed that all lanes using the milk cow blood genome as the template had 143bp target bands (FIG. 1), indicating that the specific SOD amplification primers in the kit described in example 2 could rapidly detect SOD genes in ketosis milk cow blood by PCR.
Example 5
This example illustrates the use of the kit in a cattle farm.
Using the kit of example 2, 5 healthy cows and 5 ketosis cows were tested for blood. Can quickly and accurately detect the SOD gene. The method comprises the following specific steps:
(1) sample collection
The sample was collected from a cattle farm in Heilongjiang province.
(2) PCR method for detecting SOD gene in sample
Extracting RNA from collected blood, quantifying and then carrying out reverse transcription to obtain cDNA, wherein a PCR reaction system is (25 uL): 9uL double distilled water (ddH)20) 12.5uL 2XTaq Master Mix, SOD-F1uL, SOD-R1 uL, template l.5uL. The PCR procedure was (a)94 ℃ for 3 min; (b) 30s at 94 ℃; (c) 35s at 60 ℃; (d) 35s at 72 ℃; the steps (b) to (d) are circulated for 34 times; (e) 5min at 72 ℃. The PCR product is subjected to 2% agarose gel electrophoresis, and a 134bp SOD band can be amplified. The results show that healthy cows, ketosis cows all have the target bands, lane 1, 2 respectively (figure 2).
Through extensive and intensive research, the invention detects that SOD genes exist in the blood of ketosis cows, namely the SOD genes containing 143bp nucleotide sequences in the blood of the ketosis cows, and verifies the content of the SOD genes in different states of the cow organism by using specific primers through a PCR technology; the kit can be used for quickly and high-flux detecting the SOD gene in the milk cow blood, can be used as an auxiliary method for the traditional detection of the ketosis oxidative stress of the milk cow, and provides a new method with simplicity, rapidness and good repeatability for the detection and laboratory diagnosis of the ketosis oxidative stress of the milk cow.
Sequence listing
<110> university of eight agricultural reclamation of Heilongjiang
<120> coding gene SOD in blood related to cow ketosis and PCR detection kit thereof
<141> 2021-08-27
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 867
<212> DNA
<213> Bos taurus
<400> 1
gtgtgttctg cggcgtcgtt ttctctactt ggttggggcg tctctagcgg tccgggccgg 60
agagcctttc ccccgagtca tggcgacgaa ggccgtctgc gtgctgaagg gcgacggccc 120
ggtgcaaggc accatccact tcgaggcaaa gggagataca gtcgtggtaa ctggatccat 180
tacaggattg actgaaggtg atcatggatt ccacgtccat cagtttggag acaatacaca 240
aggctgtacc agtgcaggtc ctcactttaa tcctctgtcc aaaaaacacg gtgggccaaa 300
agatgaagag aggcatgttg gagacctggg caatgtgaca gctgacaaaa acggtgttgc 360
catcgtggat attgtagatc ctctgatttc actctcagga gaatattcca tcattggccg 420
cacgatggtg gtccatgaaa aaccagatga cttgggcaga ggtggaaatg aagaaagtac 480
aaagactggc aacgctggaa gccgtttggc ctgtggtgta attggaattg ccaagtaaac 540
atccttggga catggcttag tcctagtaac tcatctgtta tcttgctagt tgtagcaaat 600
ttaatttgat aagcatttaa cactgtaaac ttaaaaagtg taatatgtgt gactttttaa 660
aaattgcttt aagtacctgt aatgagaact gacttatgat cacttgaaag catttgtata 720
attttataaa actcatatgg caattaaaat gtgtttcatt catctctgta ttttggccag 780
acttaatcac atgtatatta tttgtctgaa tttcttcgat tctctcaagc ctgcaataaa 840
cattctctgt ggcactgtaa aaaaaaa 867
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgttctgcgg cgtcgttttc 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cctcgaagtg gatggtgcct 20
Claims (7)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110992400.5A CN113481221A (en) | 2021-08-27 | 2021-08-27 | Coding gene SOD in blood related to milk cow ketosis and PCR detection kit thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110992400.5A CN113481221A (en) | 2021-08-27 | 2021-08-27 | Coding gene SOD in blood related to milk cow ketosis and PCR detection kit thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113481221A true CN113481221A (en) | 2021-10-08 |
Family
ID=77946267
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110992400.5A Pending CN113481221A (en) | 2021-08-27 | 2021-08-27 | Coding gene SOD in blood related to milk cow ketosis and PCR detection kit thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113481221A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102657297A (en) * | 2012-05-30 | 2012-09-12 | 扬州大学 | Method for regulating and controlling oxidative stress of diary cows in perinatal period |
| US20130005693A1 (en) * | 2005-05-03 | 2013-01-03 | Accera, Inc. | Method for Reducing Levels of Disease Associated Proteins |
| CN103305480A (en) * | 2013-04-08 | 2013-09-18 | 辽宁盘谷科技发展有限公司 | Superoxide dismutase from cattle blood cells and preparation method thereof |
| CN103792230A (en) * | 2014-01-13 | 2014-05-14 | 东北农业大学 | Dairy cow ketosis diagnostic kit and detecting method thereof |
| CN104215774A (en) * | 2014-08-22 | 2014-12-17 | 黑龙江八一农垦大学 | Method for diagnosing recessive ketosis of cow by utilizing FGF21 factors |
-
2021
- 2021-08-27 CN CN202110992400.5A patent/CN113481221A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130005693A1 (en) * | 2005-05-03 | 2013-01-03 | Accera, Inc. | Method for Reducing Levels of Disease Associated Proteins |
| CN102657297A (en) * | 2012-05-30 | 2012-09-12 | 扬州大学 | Method for regulating and controlling oxidative stress of diary cows in perinatal period |
| CN103305480A (en) * | 2013-04-08 | 2013-09-18 | 辽宁盘谷科技发展有限公司 | Superoxide dismutase from cattle blood cells and preparation method thereof |
| CN103792230A (en) * | 2014-01-13 | 2014-05-14 | 东北农业大学 | Dairy cow ketosis diagnostic kit and detecting method thereof |
| CN104215774A (en) * | 2014-08-22 | 2014-12-17 | 黑龙江八一农垦大学 | Method for diagnosing recessive ketosis of cow by utilizing FGF21 factors |
Non-Patent Citations (10)
| Title |
|---|
| JAIN, SK等，: ""Ketosis (acetoacetate) can generate oxygen radicals and cause increased lipid peroxidation and growth inhibition in human endothelial cells"", 《FREE RADICAL BIOLOGY AND MEDICINE》 * |
| RAMASARMA T等，: ""Bos taurus superoxide dismutase 1 (SOD1), mRNA"", 《GENBANK DATABASE》 * |
| YUXIANG SONG等，: ""Enhanced mitochondrial dysfunction and oxidative stress in the mammary gland of cows with clinical ketosis"", 《JOURNAL OF DAIRY SCIENCE》 * |
| 刘健男等，: ""奶牛酮病与肝功和氧化应激之间的关系"", 《黑龙江畜牧兽医》 * |
| 孙应祥等，: ""奶牛围产期疾病对血液中SOD及MDA含量的影响"", 《安徽农业科学》 * |
| 宝华等，: ""围产后期亚临床酮病对奶牛氧化应激、免疫功能和生产性能的影响"", 《饲料工业》 * |
| 徐闯等，: ""围产后期奶牛的血液生化指标特征"", 《中国兽医学报》 * |
| 李玉等: ""亚临床酮病奶牛的氧化抗氧化状态和乳品质"", 《中国兽医学报》 * |
| 肖立群等，: ""奶牛酮病对抗氧化系统影响的研究"", 《中国畜牧兽医文摘》 * |
| 谷娟等: ""氧化应激与奶牛围产期疾病的关系及其作用机制"", 《中国奶牛》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111471756B (en) | Specific DNA fragment SSM1 for sturgeon gender identification and application | |
| CN107523633B (en) | A method for developing novel molecular markers based on porcine SINE transposon insertion polymorphism | |
| CN107354219B (en) | Method and kit for identifying penguin sex | |
| CN109680070B (en) | SNP (Single nucleotide polymorphism) marker and molecular marker remarkably related to Australia white sheep hoof color and application | |
| CN110904243A (en) | A molecular marker method for predicting and identifying chicken abdominal fat mass and its application | |
| CN108384859A (en) | With the relevant SNP marker of tail type character of broadtail type sheep and application | |
| CN102719426A (en) | Method for extracting genomic DNA (deoxyribonucleic acid) in milk appropriate for large-scale genotype identification | |
| JP6466725B2 (en) | Assisting identification of eel species | |
| CN110724735B (en) | SNP locus and primer for rapidly identifying individual sex of fugu obscurus and method thereof | |
| Medrano et al. | Comparative analysis of bovine milk and mammary gland transcriptome using RNA-Seq | |
| CN111154852A (en) | Specific DNA Fragments and Applications for Sex Identification of Loach | |
| JP7090357B2 (en) | Use of primer sets, kits, and microRNA serum markers and primer sets to identify the sex of sturgeon | |
| CN111118174B (en) | Macrobrachium rosenbergii sex identification method based on PCR and sequencing technology | |
| CN113481221A (en) | Coding gene SOD in blood related to milk cow ketosis and PCR detection kit thereof | |
| CN118652986A (en) | Primer pairs for SNP markers related to porcine chromosome 2 and small intestine length and their application | |
| CN104988240B (en) | Differentiate the method for bee colony Higher production royal jelly character using SNP marker rs16287910 | |
| CN118166123A (en) | Yangtze river dolphin environment DNA primer and application thereof | |
| CN108384845A (en) | Duck sex identification RT-PCR primer, kit and identification method | |
| CN103290113B (en) | A kind of RFLP method of fast detecting ox SIRT1 gene SNP | |
| CN113481220A (en) | Blood coding gene CYP1A1 related to dairy cow ketosis and PCR detection kit thereof | |
| CN106521000B (en) | Molecular markers associated with piglet weaning weight and resistance to diarrheal disease and their application in pig breeding | |
| CN108486122A (en) | A kind of the DNA bar code standard sequence and its method for identifying molecules of South Mountain Storehouse midge | |
| CN107988232A (en) | The DNA bar code standard sequence and its method for identifying molecules of a kind of open country Storehouse midge | |
| CN109355379B (en) | Kit for detecting autosomal dominant hereditary deafness family-induced deafness gene mutation | |
| CN109112190B (en) | Corbicula fluminea mitochondrial genome enrichment extraction and identification method |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20211008 |