CN106591448A - Kit for detecting MyD88 gene expression of odontobutis potamophila based on fluorescence RT-RCR and applications of kit - Google Patents
Kit for detecting MyD88 gene expression of odontobutis potamophila based on fluorescence RT-RCR and applications of kit Download PDFInfo
- Publication number
- CN106591448A CN106591448A CN201611146087.9A CN201611146087A CN106591448A CN 106591448 A CN106591448 A CN 106591448A CN 201611146087 A CN201611146087 A CN 201611146087A CN 106591448 A CN106591448 A CN 106591448A
- Authority
- CN
- China
- Prior art keywords
- myd88
- gene
- kit
- odontobulis mpotamophila
- myd88 gene
- 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
- 108010077432 Myeloid Differentiation Factor 88 Proteins 0.000 title claims abstract description 57
- 230000014509 gene expression Effects 0.000 title claims abstract description 31
- 102000010168 Myeloid Differentiation Factor 88 Human genes 0.000 title claims abstract description 23
- 241000969797 Odontobutis potamophila Species 0.000 title abstract description 8
- 238000001514 detection method Methods 0.000 claims abstract description 9
- 238000006243 chemical reaction Methods 0.000 claims description 18
- 108010085238 Actins Proteins 0.000 claims description 15
- 239000002299 complementary DNA Substances 0.000 claims description 13
- 208000015181 infectious disease Diseases 0.000 claims description 12
- 210000002966 serum Anatomy 0.000 claims description 10
- 244000052769 pathogen Species 0.000 claims description 6
- 230000001717 pathogenic effect Effects 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 238000003786 synthesis reaction Methods 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 230000004087 circulation Effects 0.000 claims description 4
- 102000007469 Actins Human genes 0.000 claims description 3
- 238000004364 calculation method Methods 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 238000010839 reverse transcription Methods 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 241000208340 Araliaceae Species 0.000 claims 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 claims 1
- 235000003140 Panax quinquefolius Nutrition 0.000 claims 1
- 235000008434 ginseng Nutrition 0.000 claims 1
- 241000251468 Actinopterygii Species 0.000 abstract description 8
- 201000010099 disease Diseases 0.000 abstract description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 8
- 230000007246 mechanism Effects 0.000 abstract description 4
- 238000003757 reverse transcription PCR Methods 0.000 abstract description 4
- 230000005965 immune activity Effects 0.000 abstract description 3
- 210000000987 immune system Anatomy 0.000 abstract description 3
- 208000035240 Disease Resistance Diseases 0.000 abstract description 2
- 230000002924 anti-infective effect Effects 0.000 abstract description 2
- 230000033228 biological regulation Effects 0.000 abstract description 2
- 230000019491 signal transduction Effects 0.000 abstract description 2
- 230000004071 biological effect Effects 0.000 abstract 1
- 230000008827 biological function Effects 0.000 abstract 1
- 230000008105 immune reaction Effects 0.000 abstract 1
- 238000005119 centrifugation Methods 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 8
- 235000019688 fish Nutrition 0.000 description 7
- 238000003753 real-time PCR Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241000607528 Aeromonas hydrophila Species 0.000 description 5
- 102000002689 Toll-like receptor Human genes 0.000 description 5
- 108020000411 Toll-like receptor Proteins 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 230000036039 immunity Effects 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000001179 sorption measurement Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- 102000007863 pattern recognition receptors Human genes 0.000 description 4
- 108010089193 pattern recognition receptors Proteins 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- 108700039887 Essential Genes Proteins 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 241001669656 Odontobutis obscura Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013505 freshwater Substances 0.000 description 2
- 230000002440 hepatic effect Effects 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 238000011321 prophylaxis Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241001139305 Channa asiatica Species 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 241000252212 Danio rerio Species 0.000 description 1
- 241001669679 Eleotris Species 0.000 description 1
- 101001065501 Escherichia phage MS2 Lysis protein Proteins 0.000 description 1
- 101000831496 Homo sapiens Toll-like receptor 3 Proteins 0.000 description 1
- 241001596950 Larimichthys crocea Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 241001327682 Oncorhynchus mykiss irideus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000002944 PCR assay Methods 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 241000694873 Paralichthyidae Species 0.000 description 1
- 241000277263 Salmo Species 0.000 description 1
- 241001417495 Serranidae Species 0.000 description 1
- 102100024324 Toll-like receptor 3 Human genes 0.000 description 1
- 208000034817 Waterborne disease Diseases 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 235000021167 banquet Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000001364 causal effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 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
- 239000012074 organic phase Substances 0.000 description 1
- 238000003909 pattern recognition Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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
- 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)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit for detecting MyD88 gene expression of odontobutis potamophila based on fluorescence RT-RCR and applications of the kit. According to the kit and the applications of the kit, due to the establishment of the odontobutis potamophila MyD88 gene real-time fluorescence RT-PCR detection method, a theoretical basis is laid for researching the evolutionary status of the MyD88 gene in the immune system of odontobutis potamophila and the relevance of the MyD88 gene with disease resistance, further researching the biological activity, functions and expression regulation of the MyD88 gene and disclosing the immune anti-infection mechanism of odontobutis potamophila. MyD88 mediates the most of TLR signal transduction, and plays the important role in the inherent immune reaction, the expressive abundance of the MyD88 gene reflects the immune system activity of odontobutis potamophila to a certain degree, and can be used for judging that whether fish bodies are infected with diseases or not before the fish bodies show disease features as the important detection index for preventing and treating the diseases of odontobutis potamophila, so that effective preventing measures can be taken in advance, and the great financial loss caused by diseases can be prevented.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to a kind of fluorescence RT-RCR detects Odontobulis mpotamophila
The kit of MyD88 gene expressions and its application.
Background technology
Fish resist the infringement of external causal organism, the elder generation of body by specific immunity and non-specific immune systems
The limited pattern recognition receptors of nature immune dependent go to recognize pathogen associated molecular pattern.Certified pattern-recognition at present is received
Body mainly has two big class：Plasmotype pattern recognition receptors (Cytosolic PRRs) and film combination Toll-like receptor (Membrane-
bound Toll-like receptors,TLRs)。
Toll-like receptor is the pattern recognition receptors of most study, and wherein MyD88 has mediated most TLR signal transductions
(in addition to TLR3), is one of of paramount importance linkers, is played an important role in innate immune response.At present with regard to
The research of MyD88 is concentrated mainly on mammal, less with regard to the result of study of fish, only in zebra fish, lefteye flounder, rainbow trout, tiltedly
Have been reported that in band grouper, Atlantic salmon, large yellow croaker etc..
Real-Time Fluorescent Quantitative PCR Technique is the nucleic acid quantitation technique grown up on the basis of Standard PCR technology.In real time
Fluorescent PCR not only realizes leap of the Standard PCR from qualitative to quantitative, and compared with Standard PCR, real-time fluorescence PCR technology
Have the advantages that sensitivity is high, specificity is high, can quantitatively, the pollution problem of effectively solving PCR and high degree of automation, extensively answer
For fields such as molecular biology and medical researches.With the further exploitation of real-time fluorescent PCR reagent case, in recent years, in real time
Fluorescence PCR assay is also widely used in animal epidemic diagnosis.
Odontobulis mpotamophila (Odontobutis potamophila) is dwelt carnivorous economy for the distinctive small-sized fresh water bottom of China
Fish, in China the water systems such as the middle and lower reach of Yangtze River, the Qiantang River, the Min River are distributed widely in.The fish dwells small-sized carnivorous fish for fresh water bottom
Class, fine and tender taste is entertained distinguished guests and uses fish for 2011 as Shanghai World's Fair state banquet, is known as " fish of Expo first ", is got in recent years
More to be liked by people, 2012-2015 market prices have reached more than 160 yuan/kilogram, with higher Development volue, but
It is that bacillary, fungal disease easily occurs when propagating artificially, causes serious economic loss.Research to Odontobulis mpotamophila at present
The aspect such as select index and cultivation is concentrated mainly on, report is had no to its gene involved in immunity and disease-resistant factor research.Therefore, carry out
Gene involved in immunity (factor) research of Odontobulis mpotamophila, tentatively understands its immune response mechanism, anti-to Odontobulis mpotamophila disease
Control and open up Odontobulis mpotamophila breeding for disease resistance new way significant.
The content of the invention
The technical problem of solution：It is an object of the invention to provide a kind of fluorescence RT-RCR detects Odontobulis mpotamophila MyD88 bases
Kit and its application because of expression, is the evolutionary degree and and disease for studying MyD88 genes in Odontobulis mpotamophila immune system
The correlation of sick resistance, is further to study their BA, function and expression regulation and disclose Odontobulis mpotamophila
The anti-infective mechanism based theoretical of immunity.
Technical scheme：A kind of fluorescence RT-RCR detects the kit of Odontobulis mpotamophila MyD88 gene expressions, including MyD88
Gene specific primer to, reference gene β-actin primer pairs and PCR reaction reagents, wherein,
MyD88 gene-specific primers are to sequence：
MyD88-F：5’-TCCAAGGGTATCCCAACCCTC-3’(SEQIDNO.1)
MyD88-R：5’-CGTGGAGGACCTTCGTGCTT-3’(SEQIDNO.2)
Reference gene β-actin primer pair sequences are：：
β-actinF：5’-TGGACTTCGAGCAGGAGATGAG-3’(SEQIDNO.3)
β-actinR：5’-AAGGAAAGATGGCTGGAAGAGG-3’(SEQIDNO.4).
The invention further relates to application of the described kit in detection Odontobulis mpotamophila MyD88 gene expressions.
Specifically, the application process is as follows：
(1) extraction and purification of total serum IgE：Collection test serum, extracts total serum IgE；
(2) synthesis of the chains of cDNA first：With Odontobulis mpotamophila sample tissue total serum IgE as template, reverse transcription reaction is carried out, obtained
To the chains of cDNA first；
(3) chains of cDNA first for being obtained with step 2 as template, add MyD88 gene specific primers to, reference gene β-
Actin primer pairs and PCR reaction reagents, carry out quantitative fluorescent PCR reaction；
(4) the Ct values be given according to quantitative fluorescent PCR instrument, calculate the Δ under Odontobulis mpotamophila pathogen infection each time period
Ct and 2–ΔΔCtValue, calculation is as follows：
Δ Ct=Ct (MyD88)-Ct (β-actin)
Δ Δ Ct=Δ Ct (MyD88)-Δ Ct0 (healthy Odontobulis mpotamophila when 0)
With 2–(ΔΔCt)Numerical value represents table of the Odontobulis mpotamophila MyD88 genes relative to healthy Odontobulis mpotamophila MyD88 genes
Up to amount.
Further, the PCR reaction conditions are 95 DEG C of 10min, and 1 circulates；95 DEG C of 10s, 55 DEG C of 30s, 40 circulations.
Further, the concentration of the chains of cDNA first is 5ng/ μ L in the PCR reactions.
Further, in the PCR reaction MyD88 gene specific primers pair and reference gene β-actin primer pairs it is dense
Degree is 2mol/ μ L.
Beneficial effect：
(1) compared with Standard PCR, real-time fluorescence PCR technology is by automatic for the real-time fluorescence RT-PCR technology that the present invention is adopted
Change instrument and collect fluorescence signal, it is to avoid the subjectivity that naked eyes judge, can further improve sensitivity；Real-time fluorescence PCR technology
Totally-enclosed reaction, without PCR post processings, it is to avoid pollution, it is ensured that the reproducibility and reliability of result.Real-time fluorescence RT-PCR skill
Art also eliminates the follow-up tedious steps such as electrophoresis, quantitative scanning in Standard PCR, substantially reduces experimental period.
(2) MyD88 gene expression amounts reflect to a certain extent the immune system activity of Odontobulis mpotamophila, work as MyD88
When gene relative expression quantity increases, Odontobulis mpotamophila is likely to be at pathogen infection state, and (Odontobulis mpotamophila was not applied at no distant date
Vaccine), and now can't have any infection illness from being observed visually on Odontobulis mpotamophila body surface.Therefore by monitoring rivers and creeks
Odontobutis obscura principal immune organizes the change of MyD88 gene expression amounts, can learn whether Odontobulis mpotamophila infects pathogeny ahead of time, adopts in time
Take prophylactic treatment measure, it is to avoid situation severe development causes irremediable loss.
Description of the drawings
Fig. 1 is Odontobulis mpotamophila MyD88 gene real-time fluorescence PCR product electrophoretograms, is successively from left to right DL500DNA
Marker, MyD88 gene (151bp)；
Fig. 2 is Odontobulis mpotamophila spleen, kidney, hepatic tissue of the MyD88 genes in lumbar injection Aeromonas hydrophila in embodiment 1
The expression of middle different periods compares, and block diagram is shown as mean+SD (n=3), and * represents significant difference (P<
0.05), * * represent pole significant difference (P<0.01).
Specific embodiment
With reference to the embodiment explanation present invention, the scheme of embodiment described here does not limit the present invention, this area it is special
Industry personnel can make improvements and change according to the spirit of the present invention, and described such modifications and variations are regarded as at this
In the range of invention, the scope of the present invention and essence are defined by the claims；Wherein agents useful for same is commercially available.
Embodiment 1
Odontobulis mpotamophila infection experiment：
Aeromonas hydrophila (Aeromonas hydrophila) is provided by Jiangsu Province water-borne diseases prevention and control center, in 28 DEG C
Under the conditions of cultivate to exponential phase in LB liquid medium, thalline is collected by centrifugation, the SPSSs of Jing 0.85% are washed simultaneously
It is configured to bacterial suspension.It is 1.5 × 10 that bacterial concentration is adjusted Jing after trial test8cfu·mL-1。
One age Odontobulis mpotamophila, average weight 30g is temporarily supported 1 week in laboratory, it is determined that health nothing starts after being ill test.Abdomen
Aeromonas hydrophila is injected in chamber, is 0.2mL per tail injection dosage.Control group penetrates the physiological saline of same dose per endnote.Infection
Afterwards, in 0,4,24,48,72, each spleens for taking 3 tail fishes at random of 96h, kidney, hepatic tissue next step experiment is carried out respectively.
The real-time fluorescence quantitative PCR detection of MyD88 genes
MyD88 gene specific primers pair：
MyD88-F：5’-TCCAAGGGTATCCCAACCCTC-3’(SEQIDNO.1)
MyD88-R：5’-CGTGGAGGACCTTCGTGCTT-3’(SEQIDNO.2)
Reference gene β-actin primer pairs：
β-actinF：5’-TGGACTTCGAGCAGGAGATGAG-3’(SEQIDNO.3)
β-actinR：5’-AAGGAAAGATGGCTGGAAGAGG-3’(SEQIDNO.4)
As shown in figure 1, the PCR primer estimated length 151bp of Odontobulis mpotamophila MyD88 genes, agarose gel electrophoresis knot
Fruit shows that the product length after PCR amplifications is consistent with estimated length, is single band, illustrates that designed primer is applied to real-time
Fluorescence quantitative PCR detection.
1. the extraction of total serum IgE and purifying：
(1) homogenized：Acquired tissue sample is stirred evenly with homogenizer, is organized per 50-100mg and is first added in homogenizer
It is homogenized after the lysate of 1mL.The 10% of tissue sample volume no more than lysate volume；
(2) homogenised sample is acutely shaken mixing, 5min is incubated under the conditions of 15-30 DEG C so that ribosome divides completely
Solution；
12,000rpm centrifugations 10min under conditions of (3) 4 DEG C, supernatant is proceeded in the centrifuge tube of new RNase-free；
(4) 0.2mL chloroforms are added per 1mL lysates.Sample tube cover is covered tightly, 15s is acutely shaken and is at room temperature incubated it
3min；
(5) in 4 DEG C of 12,000rpm centrifugation 10min, sample can be divided into three layers：Lower floor's organic phase, intermediate layer and upper strata it is colourless
Water phase, RNA is present in water phase.The capacity of aqueous layer is about the 60% of added lysate volume, and water is mutually transferred to newly
RNase-free centrifuge tube in, carry out next step operation；
(6) 1 times of ethanol of volume 70% is added, overturns and mix.The solution and possible precipitation for obtaining is transferred to together adsorption column
In (adsorption column is enclosed within collecting pipe)；
(7) 10,000rpm are centrifuged 45s, discard waste liquid, and adsorption column is recovered collecting pipe again；
(8) 500 μ L protein liquid removals, 12,000rpm centrifugation 45s are added to discard waste liquid；
(9) 700 μ L rinsing liquids, 12,000rpm centrifugation 60s are added to discard waste liquid；
(10) 500 μ L rinsing liquids, 12,000rpm centrifugation 60s are added to discard waste liquid；
(11) adsorption column is put back in sky collecting pipe, 12,000rpm centrifugation 2min remove rinsing liquid as far as possible, in order to avoid rinsing
Residual ethanol suppresses downstream reaction in liquid；
(12) adsorption column is taken out, in being put into a RNase-free centrifuge tube, according to expected RNA yield in adsorbed film
Between position add 50-80 μ LRNase-free water (in advance heating effect is more preferable in 65-70 DEG C of water-bath), room temperature is placed
2min, 12,000rpm centrifugation 1min.If necessary to more RNA, the solution for obtaining can be rejoined in centrifugal adsorbing column,
Centrifugation 1min, or add 30 μ LRNase-free water centrifugation 1min again in addition, merge eluent twice；RNA is in -80 DEG C of guarantors
Deposit.
(13) absorption value and OD260/OD280 ratio of the spectrophotometric determination sample in 260nm and 280nm, it is desirable to sample
Product OD260/OD280 ratios can be used between 1.8～2.0, otherwise be extracted again.
The synthesis of the chains of 2.cDNA first：
To extract the Odontobulis mpotamophila total serum IgE for obtaining as template, according to HiScriptTM 1ST strand cDNA
The explanation of Synthesis kit carries out reverse transcription, synthesizes the chains of cDNA first.CDNA products can be stored or be immediately available at -20 DEG C
RT-PCR reacts.
3. real-time fluorescence quantitative PCR
With MyD88 gene specific primers to, reference gene β-actin primer pairs and the chains of cDNA first of synthesis as template,
Carry out real-time fluorescence quantitative PCR amplified reaction, each sample arrange 3 it is parallel to reduce error.
Real-time fluorescence quantitative PCR amplification system is as follows：
| Component | Volume/μ L |
| Faststart Universal SYBR Green Master | 10 |
| CDNA templates | 4 |
| Forward primer | 3 |
| Reverse primer | 3 |
| Cumulative volume | 20 |
Reaction condition is as follows：95 DEG C of 10min, 1 circulation；95 DEG C of 10s, 55 DEG C of 30s, 40 circulations, 4 DEG C of preservations.
Wherein every primer is configured to the working solution that concentration is 2mol/ μ L, and cDNA template concentrations are 5ng/ μ L.
4. result of the test data use 2–ΔΔCtMethod is calculating the relative expression quantity of genes of interest
After the completion of real-time fluorescence PCR, the Δ Ct and 2 under Odontobulis mpotamophila pathogen infection each time period is calculated according to Ct values–ΔΔCtValue, calculation is as follows：
Δ Ct=Ct (MyD88)-Ct (β-actin)
Δ Δ Ct=Δ Ct (MyD88)-Δ Ct0 (healthy Odontobulis mpotamophila when 0)
With 2-(ΔΔCt)Numerical value represents table of the Odontobulis mpotamophila MyD88 genes relative to healthy Odontobulis mpotamophila MyD88 genes
Up to amount.
2-(ΔCt)Numerical value represents expression multiple of the MyD88 genes relative to β-actin genes.2-(ΔΔCt)Numerical value is represented
Expression multiple of the MyD88 genes relative to healthy Odontobulis mpotamophila MyD88 genes.
β-actin genes are house-keeping genes, are all expressed in all types of cells, and the expression of house-keeping gene is only received
The impact that initiating sequence or promoter interact with RNA polymerase, and do not adjusted by other mechanism, by such environmental effects very
Little, expression is more constant.Therefore, house-keeping gene be often used as determine target gene expression when reference gene.This reality
When 2 in testing-(ΔCt)Or 2-(ΔΔCt)Numerical value is bigger, shows that the expression of MyD88 genes is higher.
After artificial challenge Aeromonas hydrophila, to carrying out different periods expression in 3 kinds of principal immune tissues of Odontobulis mpotamophila
The detection of amount change, as a result as shown in Fig. 2 expression trend of the MyD88 genes in spleen and liver tissue is consistent, 4-96h after infection
Expression before infection more than, it is high during simply rising degree is than liver in spleen；And in nephridial tissue, 4-24h expression after infection
Amount is slightly decreased, and rises and maintain higher level afterwards.
Result above is pointed out when MyD88 gene relative expression quantities increase, that is, show Odontobulis mpotamophila already at cause of disease
Infection Status, the inherent immunity system in Odontobulis mpotamophila body has been started up, although now can't be from being observed visually rivers and creeks
There are any infection illness on Odontobutis obscura body surface.
Therefore, MyD88 gene expression amounts reflect to a certain extent the immune activity of Odontobulis mpotamophila, by monitoring river
The change of Chuansha sleeper immuning tissue MyD88 gene relative expression quantities, can judge whether Odontobulis mpotamophila infects pathogeny ahead of time,
Prophylactic treatment measure and further Pathogen identification are taken in time, it is to avoid situation severe development causes irremediable loss.
SEQUENCE LISTING
<110>Nanjing Normal University
<120>The kit of fluorescence RT-RCR detection Odontobulis mpotamophila MyD88 gene expressions and its application
<130> 20161208
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
tccaagggta tcccaaccct c 21
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
cgtggaggac cttcgtgctt 20
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence
<400> 3
tggacttcga gcaggagatg ag 22
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence
<400> 4
aaggaaagat ggctggaaga gg 22
Claims (6)
1. a kind of fluorescence RT-RCR detects the kit of Odontobulis mpotamophila MyD88 gene expressions, it is characterised in that：Including MyD88
Gene specific primer to, reference gene β-actin primer pairs and PCR reaction reagents, wherein,
MyD88 gene-specific primers are to sequence：
MyD88-F：5 '-TCCAAGGGTATCCCAACCCTC-3 ',
MyD88-R：5 '-CGTGGAGGACCTTCGTGCTT-3 ',
Reference gene β-actin primer pair sequences are：
β-actinF：5 '-TGGACTTCGAGCAGGAGATGAG-3 ',
β-actinR：5’- AAGGAAAGATGGCTGGAAGAGG-3’.
2. application of the kit described in claim 1 in detection Odontobulis mpotamophila MyD88 gene expressions.
3. application according to claim 2, it is characterised in that：The application process is as follows：
（1）The extraction and purification of total serum IgE：Collection test serum, extracts total serum IgE；
（2）The synthesis of the chains of cDNA first：With Odontobulis mpotamophila sample tissue total serum IgE as template, reverse transcription reaction is carried out, obtained
The chains of cDNA first；
（3）The chains of cDNA first obtained with step 2 add MyD88 gene specific primers to, reference gene β-actin as template
Primer pair and PCR reaction reagents, carry out quantitative fluorescent PCR reaction；
（4）According to the Ct values that quantitative fluorescent PCR instrument is given, calculate △ Ct under Odontobulis mpotamophila pathogen infection each time period and
2–△△CtValue, calculation is as follows：
△Ct = Ct（MyD88）- Ct（β-actin）
△△Ct = △Ct（MyD88）- △ Ct0（Healthy Odontobulis mpotamophila when 0）
With 2-(△△Ct)Numerical value represents expression of the Odontobulis mpotamophila MyD88 genes relative to healthy Odontobulis mpotamophila MyD88 genes
Amount.
4. application according to claim 3, it is characterised in that：The PCR reaction conditions are 95 DEG C of 10min, and 1 circulates；
95 DEG C of 10s, 55 DEG C of 30s, 40 circulations.
5. application according to claim 3, it is characterised in that：The concentration of the chains of cDNA first is 5ng/ μ in the PCR reactions
L。
6. application according to claim 3, it is characterised in that：MyD88 gene specific primers pair and interior in PCR reaction
The concentration of ginseng gene β-actin primer pairs is 2mol/ μ L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611146087.9A CN106591448B (en) | 2016-12-13 | 2016-12-13 | Kit for detecting MyD88 gene expression of odontobutis potamophila by fluorescent RT-RCR and application of kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611146087.9A CN106591448B (en) | 2016-12-13 | 2016-12-13 | Kit for detecting MyD88 gene expression of odontobutis potamophila by fluorescent RT-RCR and application of kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106591448A true CN106591448A (en) | 2017-04-26 |
| CN106591448B CN106591448B (en) | 2020-03-31 |
Family
ID=58802189
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611146087.9A Active CN106591448B (en) | 2016-12-13 | 2016-12-13 | Kit for detecting MyD88 gene expression of odontobutis potamophila by fluorescent RT-RCR and application of kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106591448B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667499A (en) * | 2013-12-26 | 2014-03-26 | 南京师范大学 | Primer and method for identifying odontobutis potamophila, o. yaluensis and o. sinensis |
| CN104450886A (en) * | 2014-11-03 | 2015-03-25 | 青岛大学附属医院 | PCR reagent for detecting NF-kappa B signaling pathways in cells and application of PCR reagent |
-
2016
- 2016-12-13 CN CN201611146087.9A patent/CN106591448B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667499A (en) * | 2013-12-26 | 2014-03-26 | 南京师范大学 | Primer and method for identifying odontobutis potamophila, o. yaluensis and o. sinensis |
| CN104450886A (en) * | 2014-11-03 | 2015-03-25 | 青岛大学附属医院 | PCR reagent for detecting NF-kappa B signaling pathways in cells and application of PCR reagent |
Non-Patent Citations (2)
| Title |
|---|
| FENFEI LIANG等: "Molecular characterization and expression analysis of major histocompatibility complex class IIA and IIB genes of Odontobutis potamophila", 《FISH SCI》 * |
| 于兴达等: "河川沙塘鳢TLR2基因的克隆及表达分析", 《基因组学与应用生物学》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106591448B (en) | 2020-03-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Pollock et al. | The madness of microbiome: attempting to find consensus “best practice” for 16S microbiome studies | |
| CN102666878B (en) | For mark, method, biochip and test kit that milk quality detects | |
| CN101955939B (en) | Aptamer for grouping different subtype non-small cell lung cancers and screening method thereof | |
| CN101358246A (en) | LAMP kit for detecting hogcholera virus and preparation method thereof | |
| CN105112559A (en) | Kit for detecting coronavirus and application of kit | |
| CN103602757A (en) | Development and application of multiple fluorescence RT-PCR detection method for foot-and-mouth disease, vesicular stomatitis and swine vesicular disease | |
| RU2714752C2 (en) | Method of measuring change in individual's immune repertoire | |
| CN107699617A (en) | One kind early diagnosis septicopyemia triggers acute injury of kidney molecular marked compound miR 452, kit and application | |
| CN104017806A (en) | MicroRNA (Ribose Nucleic Acid) and application thereof in preparation of active tuberculosis detection reagent | |
| CN103468806A (en) | Quick detection method for scallop pathogenic vibrio splendidus | |
| CN106591448A (en) | Kit for detecting MyD88 gene expression of odontobutis potamophila based on fluorescence RT-RCR and applications of kit | |
| CN108624680B (en) | The application of RAE1 gene or albumen as the biomarker of diagnosing myocardial infarction | |
| CN104630214B (en) | Combination of SRY gene primer pair and probe and SRY multi-site detection kit | |
| CN110819726B (en) | Method for detecting mycobacterium and kit thereof | |
| CN103882095A (en) | Application of small molecule RNA as tuberculosis marker | |
| CN109136373A (en) | It is a kind of for early diagnosing the lncRNA detection kit and its application of lung cancer metastasis | |
| CN105420347A (en) | Gene diagnosis reagent kit for systemic lupus erythematosus | |
| CN110195117A (en) | Detect the method and its kit of mycobacteria | |
| CN107583052A (en) | Applications of the 5p of miR 6734 in Luminal type breast cancer diagnosis instruments are prepared | |
| CN104313163B (en) | Method and kit for triple PCR detection of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6 and application of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6 | |
| CN106544440A (en) | The application of 7 family miRNA of hsa let and its target gene in EBOV Infect And Diagnoses and treatment | |
| CN101363064B (en) | Standard substance for detecting influenza a virus load and detection method thereof | |
| CN105648060B (en) | Method for detecting drug-resistant genes of non-diagnostic human pathogenic microorganisms | |
| CN104789683B (en) | Biomarker and application thereof for diagnosis and treatment metastasis of cancer | |
| CN108796067A (en) | The diagnosis new function of MAEA genes in blood |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |