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 PDF

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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
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尹绍武
张宏叶
汪亚媛
赵诚
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Nanjing Normal University
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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

Fluorescence RT-RCR detection Odontobulis mpotamophila MyD88 gene expressions kit and its Using
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.
CN201611146087.9A 2016-12-13 2016-12-13 Kit for detecting MyD88 gene expression of odontobutis potamophila by fluorescent RT-RCR and application of kit Active CN106591448B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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》 *

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