CN110468216A - Digital pcr detects the primer and probe of long oyster interleukins 17-5 gene expression amount - Google Patents
Digital pcr detects the primer and probe of long oyster interleukins 17-5 gene expression amount Download PDFInfo
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- 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
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- 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
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- 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
Abstract
The present invention discloses the primer and probe that a kind of PCR detects long oyster interleukins 17-5 gene expression amount, and the primer and probe nucleotide sequence are as follows: forward primer: 5 '-tccctggtccaccatcatg-3 '；Reverse primer: 5 '-ggaacaccactgaggacttgg-3 '；Probe: 5 '-cgcgtcagctacc-3 '；The fluorophor of the probe addition is 6- Fluoresceincarboxylic acid.The accuracy and specificity of primer and probe are high, therefore the timing variations of long oyster interleukins 17-5 gene expression amount can be detected using digital pcr technology, the index of long oyster health status is cultivated in this, as evaluation, the appraisement system established has potential using value in cultivated shellfish disease early-warning and predicting.Setting control group is not needed, without reference gene (Internal control), breaches the application limitation of conventional fluorescent real-time quantitative PCR detection method.
Description
Technical field
The invention belongs to molecular biology and technological field of biochemistry more particularly to a kind of digital pcr to detect long oyster
The primer and probe of -5 gene expression amount of IL-17.
Background technique
IL-17 (Interleukin17, IL17) played an important role in the system of defense of body.
IL17 can be by inducing downstream cytokine (such as chemotactic factor (CF) or IL8) indirectly-acting in neutrophil leucocyte, to promote its increasing
It grows and migrates；It can also act synergistically with other cell factors (IL1, IL6 and TNF etc.), activate the neutral grain infiltrated in tissue
Cell clearance cause of disease；In addition, IL17 can also promote a variety of expression for participating in the effector molecule that cause of disease is removed in downstream, such as enteron aisle
In IL17 can be enhanced sealing PROTEIN C laudin and tight junction protein Zona occludens-1 synthesis, to make
Connection between chrotoplast is even closer, promotes being isolated for body and intestinal contents and pathogenic microorganism.
Recent studies have found that co-existing in 10 IL17 family members in long oyster genome, it is named asCgIL17-2~CgIL17-10, whereinCgIL17-5 plays vital effect in body inflammatory reaction and environment-stress adapt to.Such as when
After long oyster is by the secondary stimulus of Vibrio splindidus, in blood lymphocyteCgThe expression quantity of IL17-5 is bright after swashing compared with first time bacteria thorn
It is aobvious to reduce；Under the conditions of dry dew,CgAfter IL17-5 gene is disturbed, antibacterial peptideCgDefensin and the pro-apoptotic factorCgDFFA base
Because expression quantity is compared with control group decrease to some degree, inhibit antiapoptotic factorsCgIAP expression quantity increases, and shows height under the conditions of dry dew
Expression quantityCgThe tune that IL17-5 can promote cell is died, and plays important adjustment effect to long oyster system of defense.CgIL17-
5 also can be combined in long oyster blood lymphocyte surface and then activate immune associated transcription factor, and induce downstream effect molecule
Expression.The studies above showsCgIL17-5 is a kind of important long oyster proinflammatory cytokine, may participate in the immune anti-of long oyster
Imperial and environment-stress response process, plays an important role to the maintenance of body homeostasis, can be used as the long oyster health of cultivation
The important indication molecule of state is of great significance to the building of cultivated shellfish disease early-warning and predicting system.
Fluorescence real-time quantitative PCR (Quantitative real-time PCR, abbreviation qPCR) is for detecting gene
The classical way of mRNA expression.But the method is only used for the relative expression levels of detection gene, needs that control group is arranged
And suitable house-keeping gene is chosen as internal reference, therefore the application of qPCR technology has significant limitations.Relatively, digital pcr
It is a kind of nucleic acid molecules absolute quantitation technology, can directly detects the number of DNA molecular, is the absolute quantitation to initial sample,
Meet the needs of field investigation.But so far, long oyster interleukins 17-5 gene is not detected about digital pcr
The primer of expression quantity and the relevant report of probe, so that there is no detect the long oyster leucocyte of long oyster based on digital pcr to be situated between
The timing variations of plain 17-5 gene expression amount and the index that long oyster health status is cultivated in this, as evaluation.
Summary of the invention
The present invention is that it is male to provide a kind of digital pcr detection length in order to solve above-mentioned technical problem present in the prior art
The primer and probe of -5 gene expression amount of oyster IL-17.
The technical solution of the invention is as follows: a kind of long oyster interleukins 17-5 gene expression amount of digital pcr detection
Primer and probe, it is characterised in that the primer and probe nucleotide sequence are as follows:
Forward primer: 5 '-TCCCTGGTCCACCATCATG -3 '；
Reverse primer: 5 '-GGAACACCACTGAGGACTTGG-3 '；
Probe: 5 '-CGCGTCAGCTACC-3 '；
The fluorophor of the probe addition is 6- Fluoresceincarboxylic acid.
The digital pcr that the present invention designs detects the primer of long oyster interleukins 17-5 gene expression amount and the standard of probe
True property and specificity are high, therefore the timing that long oyster interleukins 17-5 gene expression amount can be detected using digital pcr technology is become
Change, the index of long oyster health status is cultivated in this, as evaluation, the appraisement system established is pre- in cultivated shellfish disease early warning
There is potential using value in report.Setting control group is not needed to breach without reference gene (Internal control)
The application limitation of conventional fluorescent real-time quantitative PCR detection method.
Detailed description of the invention
Fig. 1 is long oyster in the embodiment of the present inventionCgThe qPCR amplification curve and response procedures figure of IL17-5.
Fig. 2 is long oyster in the embodiment of the present inventionCgThe quality control chart of the digital pcr reaction of IL17-5.
Fig. 3 different times of the embodiment of the present invention cultivate in long oyster blood lymphocyteCgThe absolute expression quantity of IL17-5 gene
Figure.
Specific embodiment
1. CgThe screening of IL17-5 sequence and design of primers
Identification obtains IL-17-5(from long oyster genomeCgIL17-5, CGI_10004922) full length sequence,
Search its ORF.
2. CgThe digital pcr primer and probe of IL17-5 designs
Using ThermoFisher company Primer Express V3.0 software according toCgThe ORF sequence design number of IL17-5
Word PCR primer and probe.Primer and probe sequence information is as follows:
Forward primer: 5 '-TCCCTGGTCCACCATCATG -3 '；
Reverse primer: 5 '-GGAACACCACTGAGGACTTGG -3 '；
Probe: 5 '-CGCGTCAGCTACC -3 '；
The forward primer of digital pcr long 19 bp, 59.0 °C of Tm value；Reverse primer grow 21 bp, 57.0 °C of Tm value；Probe long 13
68.0 °C of bp, Tm value, preparation method is to be prepared using HPLC, and the fluorophor of probe addition is 6- Fluoresceincarboxylic acid (6-FAM).
Experimental example 1: in long oyster blood lymphocyteCgThe detection of the absolute expression quantity of IL17-5 gene
1. sample collection
After prying open long oyster shell with oyster knife, blood sinus is punctured with 10 mL syringes and extracts hemolymph；500 mesh thin,tough silk mistakes
It is added after filter in 1.5 mL EP pipes, 800g10 min are centrifuged, blood lymphocyte is collected.
2. Total RNAs extraction and cDNA library building
1 mL Trizol reagent is added into each EP pipe for filling blood lymphocyte.200 μ are added to every 1 mL sample
The chloroform (- 20 DEG C preservation) of L pre-cooling, acutely shakes 3 min, and 4 DEG C, 12,000gIt is centrifuged 15 min.Careful absorption upper layer is transparent
400 μ L of liquid is added the pre- cold isopropanol (- 20 DEG C of preservations) of same volume, mixes well and be placed on -80 DEG C of ultra low temperature freezers
Middle natural subsidence.After 10 min, taking-up sample, 4 DEG C, 12,000gIt is centrifuged 15 min.Liquid in pipe is discarded, 1 mL 75% is added
Sterile alcohol；It has flicked tube bottom with finger to precipitate, 4 DEG C, 12,000gIt is centrifuged 5 min.Discard liquid in pipe, 4 DEG C, 12,000g
30 s are centrifuged, carefully blot tube bottom residual liquid with 1 mL syringe.2 ~ 3 min of standing of uncapping makes ethyl alcohol sufficiently volatilize completely.
Every pipe is added 20 μ L DEPC water and dissolves RNA.Micro-example is taken to carry out nucleic acid electrophoresis to detect RNA mass.Use dnase digestion
The DNA being mixed in RNA synthesizes first chain of cDNA library using two-step method.
3. the validity of qPCR method verifying digital pcr primer and probe
Using the digital pcr primer of the embodiment of the present invention, tested according to following reaction system (14.5 μ L):
| Reagent | Volume |
| QuantStudio™ 3D Digital PCR Master Mix v2 | 7.3 μL |
| Probe (200 nM of final concentration) | 0.7 μL |
| Forward primer (900 nM of final concentration) | 0.7 μL |
| Reverse primer (900 nM of final concentration) | 0.7 μL |
| CDNA template | 1.4 μL |
| DEPC water | 3.7 μL |
QPCR response procedures: 50 DEG C of 2 min；95 DEG C of 10 min, 95 DEG C 15,50 circulations；60℃ 1 min.
Using 7500 analysis softwares, Ct value is obtained.Utilize 2-ΔΔCtAlgorithm obtains relative expression levels' value of gene.With
The softwares such as Excel and SPSS carry out statistical analysis.
Experimental result is as shown in Figure 1.A is long oyster in Fig. 1CgThe qPCR amplification curve of IL17-5, B are response procedures figure.
The result shows that: reaction detects the fluorescence signal of FAM group when proceeding to the about the 27th wheel, and reaches when reaction progress to the about the 45th wheel
To plateau.The result illustrates designed for detectingCgThe digital pcr primer and probe of the absolute expression quantity of IL17-5 is effective,
Meet subsequent experimental requirement.
Experimental example 2: digital pcr method detectionCgThe absolute expression levels of IL17-5 gene
Using the digital pcr primer and probe of the embodiment of the present invention, tested according to following reaction system (14.5 μ L):
| Reagent | Volume |
| QuantStudio™ 3D Digital PCR Master Mix v2 | 7.3 μL |
| Probe (200 nM of final concentration) | 0.7 μL |
| Forward primer (900 nM of final concentration) | 0.7 μL |
| Reverse primer (900 nM of final concentration) | 0.7 μL |
| CDNA template | 1.4 μL |
| DEPC water | 3.7 μL |
By ProFlex 2x Flat PCR System (or Dual Flat Block GeneAmp PCR System
9700) system, according to following reaction condition: 96 DEG C of 10 min(denaturation), 60 DEG C of 2 min(anneals and extends), 98 DEG C 30
S, 39 circulations；60 DEG C of 2 min, 10 DEG C of ∞ are detected in long oyster blood lymphocyteCgThe absolute copy number of IL17-5 gene.
Data are read using QuantStudio 3D Digital PCR Instrument system, are used
QuantStudio 3D AnalysisSuite Software carries out data analysis.Utilize the softwares pair such as Excel and SPSS
Data carry out statistical analysis.
By carrying out Analysis of quality control to initial data, as a result as shown in Figure 2.It was found that being only able to detect the FAM letter of specificity
Number it is unimodal, and FAM positive signal and ROX negative control signal can obviously be divided into two groups.The result shows that the present invention is for detectingCgThe digital pcr primer and probe of the absolute expression quantity of IL17-5 has high degree of specificity.
Experimental example 3: different times cultivate in long oyster blood lymphocyteCgThe absolute expression quantity of IL17-5 gene
Using digital pcr primer and probe of the invention between in May, 2019 into the long oyster blood lymphocyte AugustCgThe mRNA expression of IL17-5 is detected, as a result as shown in Figure 3.Wherein, in long oyster blood lymphocyte in MayCgThe absolute magnitude of IL17-5 be 265.79 copies/microlitre, in July duration oyster blood lymphocyteCgThe absolute magnitude of IL17-5
For 261.60 copy/microlitre, with May level it is close.And in August part duration oyster blood lymphocyteCgIL17-5's is exhausted
To amount for 133.95 copy/microlitre, substantially less than May and July level (p< 0.05).August part is long oyster breeding
Crucial season, and the surface temperature in long oyster culture sea area reaches average annual peak, pair of physiology and environment near Dalian at this time
Ghost image pilot causes immunocompetence of the long oyster in August to be remarkably decreased, and is embodied as the expression quantity of the inflammatory factors such as IL17-5
It is substantially reduced.The above results explanation can be detected effectively in the long Oysters of cultivation using digital pcrCgThe absolute table of IL17-5 gene
Up to amount, so that reflection cultivates the immune defense ability of long oyster indirectly, correlative study result is pre- in the long oyster disease early warning of cultivation
There is potential using value in report.
Sequence table
<110>Dalian Ocean University
<120>digital pcr detects the primer and probe of long oyster interleukins 17-5 gene expression dose
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>artificial sequence (Artificial)
<220>
<221> misc_feature
<222> (1)..(19)
<400> 1
tccctggtcc accatcatg 19
<210> 2
<211> 21
<212> DNA
<213>artificial sequence (Artificial)
<220>
<221> misc_feature
<222> (1)..(21)
<400> 2
ggaacaccac tgaggacttg g 21
<210> 3
<211> 13
<212> DNA
<213>artificial sequence (Artificial)
<220>
<221> misc_feature
<222> (1)..(13)
<400> 3
cgcgtcagct acc 13
Claims (1)
1. primer and probe that a kind of digital pcr detects long oyster interleukins 17-5 gene expression amount, it is characterised in that institute
It states primer and probe nucleotide sequence is as follows:
Forward primer: 5 '-TCCCTGGTCCACCATCATG -3 '；
Reverse primer: 5 '-GGAACACCACTGAGGACTTGG-3 '；
Probe: 5 '-CGCGTCAGCTACC-3 '；
The fluorophor of the probe addition is 6- Fluoresceincarboxylic acid.
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| CN201910813289.1A CN110468216A (en) | 2019-08-30 | 2019-08-30 | Digital pcr detects the primer and probe of long oyster interleukins 17-5 gene expression amount |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110845594A (en) * | 2019-12-02 | 2020-02-28 | 大连海洋大学 | Recombinant serum amyloid protein A capable of enhancing immune response of crassostrea gigas and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104402987A (en) * | 2014-11-25 | 2015-03-11 | 中国科学院海洋研究所 | Crassostrea gigas interleukin IL17N recombination protein as well as preparation and application thereof |
| US20180105864A1 (en) * | 2015-04-17 | 2018-04-19 | The Translational Genomics Research Institute | Quality assessment of circulating cell-free dna using multiplexed droplet digital pcr |
-
2019
- 2019-08-30 CN CN201910813289.1A patent/CN110468216A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104402987A (en) * | 2014-11-25 | 2015-03-11 | 中国科学院海洋研究所 | Crassostrea gigas interleukin IL17N recombination protein as well as preparation and application thereof |
| US20180105864A1 (en) * | 2015-04-17 | 2018-04-19 | The Translational Genomics Research Institute | Quality assessment of circulating cell-free dna using multiplexed droplet digital pcr |
Non-Patent Citations (2)
| Title |
|---|
| JUNLI等: "Genomic characterization and expression analysis of five novel IL-17 genes in the Pacific oyster, Crassostrea gigas", 《FISH & SHELLFISH IMMUNOLOGY》 * |
| 李伟等: "《分子诊断学》", 30 September 2015, 中国医药科技出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110845594A (en) * | 2019-12-02 | 2020-02-28 | 大连海洋大学 | Recombinant serum amyloid protein A capable of enhancing immune response of crassostrea gigas and preparation method thereof |
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Application publication date: 20191119 |