CN106755595A - A kind of Eva Green fluorescent quantificationally PCR detecting kits for detecting salmon Alphavirus and its application - Google Patents
A kind of Eva Green fluorescent quantificationally PCR detecting kits for detecting salmon Alphavirus and its application Download PDFInfo
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Abstract
The invention discloses a kind of Eva Green PCR kit for fluorescence quantitative for detecting salmon Alphavirus and its application.Present invention firstly provides the real-time fluorescence quantitative PCR primer pair for detecting salmon Alphavirus, its sequence is respectively shown in SEQ ID NO.2 and SEQ ID NO.3.Present invention also offers the Eva Green PCR kit for fluorescence quantitative containing above-mentioned primer pair and its method for detection salmon Alphavirus.Research shows, salmon Alphavirus is detected using primer of the invention or kit, have the advantages that easy, quick, accurate, sensitivity is high, specific good, can be used for six detections of genotype of salmon Alphavirus, a kind of proposition of the invention effective technological means for the detection of salmon Alphavirus is provided.
Description
Technical field
Primer and kit the present invention relates to be used for Viral diagnosis, more particularly to for detecting the spy of salmon Alphavirus
The fluorescence quantification PCR primer of the opposite sex, the invention further relates to carry out salmon Alphavirus using the primer and Eva Green fluorescent dyes
The method and kit of detection.The invention belongs to virus detection techniques field.
Background technology
Salmon Alphavirus (salmonid alphavirus, SAV) is under the jurisdiction of Togaviridae (Togaviridae), onychonosus
Poison category (Alphavirus), main infection Atlantic salmon (salmo salar), rainbow trout (Oncorhynchus mykiss) and brown
The salmon fishes such as trout (Salmo trutta L.) cause the symptoms such as pancreas disease, myocardial inflammation and lethargic sleep, fatal rate up to 1%~
48%, and have outburst in each stage of aquaculture.Nineteen ninety-five Nelson etc. Ireland first from suffer from pancreas disease it is big
Isolate the virus in Western salmon and rainbow trout body, Castric in 1997 etc. is again in France from the Atlantic salmon and rainbow for suffering from difussa
SAV is separated in trout body, the current disease is widely current in England, Scotland, Norway, France, Poland, Italy and Spain
Deng European countries, 90% is increased up to year by year from nineteen ninety-five to 2007 annual morbidities according to statistics, thus epidemic disease causes huge every year
Economic loss.Salmon Alphavirus infection in 2013 is put into OIE aquatic animal epidemic disease registers.Although China is at present not yet
There is document report to detect the virus, but, with the increase that China introduces to salmon fishes import and salmon fishes ovum in recent years,
The risk of the sick incoming China also increasingly increases.Therefore, it is urgent task that China sets up SAV quick diagnosis and surveys detecting method.
Have now been found that six genotype (SAV 1, SAV 2, SAV 3, SAV 4, SAV5, SAV 6).Wherein, SAV 1,
SAV 3, SAV 4, SAV 5, SAV 6 can infect Atlantic salmon and cause Pancreas Disease (Salmon pancreas disease, PD)；
SAV 2 can infect rainbow trout to be caused and sleeps disease (Sleeping disease, SD).SAV virion has cyst membrane, and size is 65nm.
Single strand plus RNA virus, gene group leader 11-12kb, and contain two ORFs (ORF).Second ORF of genome is compiled
Code 26S subbases because mRNA, it is final to produce 5 structural proteins, i.e. capsid protein, E3, E2,6K and E1 to together constitute virus
Membrane glycoprotein.E2 is connected in prematurity with E3, constitutes the precursor of E2, the signal peptide for functioning as E2 of E3.Work as E2
When ripe, E2 is separated with E3, and E2 is transported to virion surface.
In recent years, some places of China, particularly northern area, extensive development cold water fish is (such as：Rainbow trout) cultivation,
And achieve huge economic benefit.Although China not yet finds the virus at present, as China in recent years is to salmon fishes
The increase of import, the risk of the sick incoming China also increasingly increases, it is therefore necessary to set up quickly clever to salmon Alphavirus as early as possible
Quick detection method, prevents the sick incoming China, and effective forecast or diagnosis of SAV are the problems that we must solve.
The features such as nucleic acid detection technique is because with quick, sensitive, high specificity, it has also become salmon Alphavirus is predominantly detected
Technology, is widely used in detection and the Molecule Epidemiology Investigation of clinical sample.
The content of the invention
Salmon Alphavirus can easy, quick, sensitive, special and be efficiently detected it is an object of the invention to provide one kind
Eva Green PCR kit for fluorescence quantitative.
It is another object of the present invention to provide the fluorescence quantifying PCR method of detection salmon Alphavirus.The method is detected
Covering salmon six genotype of Alphavirus, are a kind of high specificity, the detection method that accuracy is high, sensitivity is high.
In order to realize the object of the invention, present invention employs following technological means：
The present invention compares to known salmon Alphavirus genomes sequence, filters out six guarantors of genotype of salmon Alphavirus
One section of conserved sequence (9471-9668bp) in defending zone sequence, i.e. E2 GFPs.For sequence inventor through anti-
Secondary screening is selected and verified, obtains a pair for detecting six fluorescence quantification PCR primers of different genotype of salmon Alphavirus, expands piece
Duan great little is 197bp (shown in SEQ ID NO.1), and the sequence of the primer is as follows：
Sense primer (TS1)：5’-GCAACATTGCCGATCTGAGGTGTGG-3’(SEQ ID NO.2)
Anti-sense primer (TS2)：5’-CGTAACATGCAACGACAGCCAGTGC-3’(SEQ ID NO.3)
Further, the invention allows for a kind of Eva Green quantitative fluorescent PCRs examination for detecting salmon Alphavirus
Agent box, containing for detecting that the fluorescence quantification PCR primer pair and Eva Green fluorescent quantitative PCRs of salmon Alphavirus delay
Fliud flushing, wherein described primer pair is made up of sense primer and anti-sense primer, the sequence such as SEQ ID NO.2 of the sense primer
Shown, the sequence of the anti-sense primer is as shown in SEQ ID NO.3.
In kit of the present invention, it is preferred that the Eva Green fluorescent quantitative PCR buffer solutions include
dNTPs、Mg2+, Eva Green, archaeal dna polymerase and RNase inhibitor.
In kit of the present invention, it is preferred that the Eva Green fluorescent quantitative PCR buffer solutions are
Golden HS EVA Green qPCR Mix。
In kit of the present invention, it is preferred that also including positive control and negative control, described positive control
It is the recombinant plasmid standard items containing salmon Alphavirus raq gene, described negative control is deionized water.
Further, examined the invention allows for using Eva Green PCR kit for fluorescence quantitative of the present invention
When surveying salmon Alphavirus, comprise the following steps：
(1) sample to be tested is homogenized, is centrifuged, taking supernatant carries out Total RNAs extraction；
(2) as template, reverse transcription synthesizes total cDNA to the total serum IgE for being obtained with step (1), total cDNA Sample storages to -80 DEG C,
It is standby；
(3) total cDNA, positive reference substance and the negative controls respectively template obtained with step (2), in fluorescent quantitation
The enterprising performing PCR amplification of PCR instrument；
(4) after PCR reactions terminate, the Ct values and initial DNA copy number of sample are recorded.
Wherein, it is preferred that pcr amplification reaction system is：5 × Golden HS EVA Green qPCR Mix 4 μ l, 50
The μ l of × ROX Reference Dye 0.4, concentration is 0.3 μM of sense primer and each 0.5 μ l of anti-sense primer, the μ l of template 1, finally
Mended to 20 μ l with the deionized water of sterilizing.
Wherein, it is preferred that the response procedures of real-time fluorescence quantitative PCR are：(1) 95 DEG C of predegeneration 15min；(2) 95 DEG C of changes
Property 10s, 60 DEG C annealing 30s, totally 40 circulation.
Wherein, it is preferred that when two kinds of controls are effectively to expand in the detection, sample results criterion is as follows：Ct values≤
Sample result is the positive when 40.0；The sample result of Ct values ＞ 40.0 is feminine gender.
Further, detection salmon is being prepared the invention allows for described Eva Green PCR kit for fluorescence quantitative
Application in the reagent of fish Alphavirus.
Wherein, described salmon Alphavirus includes following six genotype：SAV 1、SAV 2、SAV 3、SAV 4、SAV5
And SAV 6.
Compared to prior art, the beneficial effects of the invention are as follows：
1st, the present invention refers to various six genotype strains of salmon Alphavirus, and based on raq gene, design synthesis can be used for
The specific primer of Eva Green fluorescence quantitative PCR detections；
2nd, the present invention has been successfully established Eva Green fluorescent quantitative PCR detection methods, it is preferred to use Golden HS EVA
Green is fluorometric reagent, and low cost, quantitative PCR reaction solution prepares simple, can obtain good in broad quantification area
Standard curve, accurate quantitative analysis, qualitative detection are carried out to target gene, reproducible, with a high credibility, are applicable to each fluorescent quantitation
PCR amplification instrument；
3rd, quantitative detecting method of the present invention has preferably specificity, sensitiveness and repeatability, can be used for all salmon first
Viral six quick detections of genotype strain.
Brief description of the drawings
Fig. 1 is salmon Alphavirus Eva Green fluorescent quantitative PCR kinetic curves；
Fig. 2 is salmon Alphavirus Eva Green quantitative fluorescent PCR standard curves；
Fig. 3 is salmon Alphavirus Eva Green fluorescent quantitative PCR product solubility curves；
Fig. 4 is the sensitivity nucleic acid electrophoresis figure that the regular-PCR method detection present invention sets up primer；
Fig. 5 is the sensitivity nucleic acid electrophoresis for detecting salmon Alphavirus primer of regular-PCR method detection OIE approvals
Figure；
Fig. 6 is the specific test amplification kinetic curve of salmon Alphavirus Eva Green quantitative fluorescent PCRs.
Specific embodiment
Following examples further illustrate present disclosure, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit of the invention and essence, the modification or replacement made to the inventive method, step or condition belong to the present invention
Scope.
If not specializing, the conventional meanses that technological means used is well known to those skilled in the art in embodiment.
Embodiment 1 salmon Alphavirus, six designs of Serotype-dependent primer
According to the salmon Alphavirus (SAV) raq gene sequence (JX163854) delivered in Genebank, compare SAV 6
The raq gene sequence of genotype difference strain, filters out one section of conserved sequence (9471-9668bp).During design primer, note keeping away
The catastrophe point of sequence is opened, through repeated screening and checking, a pair is obtained for detecting six Eva of different genotype of salmon Alphavirus
Green fluorescence quantification PCR primers：
Sense primer (TS1)：5’-GCAACATTGCCGATCTGAGGTGTGG-3’(SEQ ID NO.2)
Anti-sense primer (TS2)：5’-CGTAACATGCAACGACAGCCAGTGC-3’(SEQ ID NO.3)
Amplification salmon six raq genes of genotype of Alphavirus, clip size is 197bp, its nucleotide sequence such as SEQ ID
Shown in NO.1.
The foundation of the Eva Green fluorescent quantitative PCR detection methods of the detection salmon Alphavirus of embodiment 2
1st, the preparation of salmon Alphavirus raq gene recombinant plasmid standard items：
The total serum IgE of salmon Alphavirus SAV 4640 is extracted according to Total RNAs extraction specification, with reference to TOYOBO Reverse Transcriptions
Box specification carries out reverse transcription, and reverse transcription gained cDNA is carried out into routine with the primer of amplification salmon Alphavirus E2 full-length genes
PCR is expanded, and the primer sequence is：
Upstream primer sequence：PF:5’-AGCTTCTTGCCGTCACCACCT-3’(SEQ ID NO.4)；
Downstream primer sequence：PR:5’-GCGGCCGCTTGGTCCACAAGTAG-3’(SEQ ID NO.5)
PCR primer detects that amplification E2 total lengths are 1375bp, are returned with glue reclaim kit through 1.0% agarose gel electrophoresis
Purpose product is received, fragment will be reclaimed and be connected with pET32a carriers, transformed competence colibacillus cell extracts matter with plasmid extraction kit
Grain, is sequenced by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Correct positive plasmid will be sequenced and be named as pET 32a-
SAV E2, and as DNA standard items, be serially diluted with carrying out 10 times after spectrophotometric determination concentration, it is bent for building standard
Line.And be analyzed according to the concentration of standard plasmid, the plasmid copy contained in every microlitre of plasmid liquid is calculated according to formula
Number.
Be computed pET32a-SAV E2 concentration be 119ng/ μ l, according to the result for obtaining, recombinant plasmid is carried out 10 times
Doubling dilution, the concentration for obtaining is 1.5 × 1010、1.5×109、1.5×108、1.5×107、1.5×106、1.5×105、1.5
×104、1.5×103, 1.5 × 102、1.5×101The recombinant plasmid of copies/ μ l, as standard items template.Sample is all
Set up 1 positive control sample and negative control sample.Positive control sample is above-mentioned recombinant plasmid；Negative control sample is to go
Ionized water.
2nd, Eva Green fluorescence quantitative PCR detections
It is 1.5 × 10 to take concentration respectively10、1.5×109、1.5×108、1.5×107、1.5×106The weight of copies/ μ l
Group plasmid pET32a-SAV E2 carry out the amplification of Eva Green quantitative fluorescent PCRs for template, obtain amplification kinetic curve and
Standard curve, as shown in Figures 1 and 2, the coefficient correlation of standard curve is 0.99, and slope is -3.1, it can thus be seen that linear close
System is good.
Wherein pcr amplification reaction system is：5 × Golden HS EVA Green qPCR Mix 4 μ l, 50 × ROX
The μ l of Reference Dye 0.4, concentration is 0.3 μM of each 0.5 μ l of primer TS1, TS2, the μ l of template 1, finally with the deionization of sterilizing
Water is mended to 20 μ l.
Pcr amplification reaction program is：
(1) 95 DEG C of predegeneration 15min；
(2) 95 DEG C of denaturation 10s, 60 DEG C of annealing 30s, this step is a circulation, totally 40 circulations.
After reaction terminates, the solubility curve of quantitative fluorescent PCR is confirmed, as shown in figure 3, only a single set of peak, primer spy
It is different in nature strong, amplification efficiency highest, the solution temperature of standard items is all at 87.2 DEG C.
4th, after reaction terminates, show that picture judges testing result according to quantitative real time PCR Instrument：
Negative sample does not have amplification curve to occur, and positive has obvious amplification curve, and Ct values are less than 25.It is now negative
Control and positive control are all set up, and can play control to testing sample；If testing sample amplification curve Ct values are less than or wait
In 40, then illustrate that sample is positive SAV nucleic acid；If testing sample is more than 40 without amplification curve, or amplification curve Ct values, then sample
For SAV nucleic acid is negative.
The evaluating characteristics of the salmon Alphavirus Eva Green fluorescent quantitative PCR detection methods of embodiment 3
1st, sensitivity evaluation, the i.e. LDL of real-time PCR.
Positive reference DNA original liquid concentrations are 1.5 × 1010copies/μl.First by its 10 times of gradient dilutions to 1.5 ×
101Copies/ μ l, then respectively the solution with each dilution factor as template, using the embodiment of the present invention 2 set up fluorescent quantitation
PCR method is expanded, and determines the minimum template amount that can be detected of the method.According to testing result, the quantitative fluorescent PCR side
The minimum detectable amount of method is 1.5 × 101Copies/ μ l, illustrate that detection method sensitivity is high.
For relatively Eva Green fluorescence quantification PCR primers of foundation of the present invention with OIE accreditations for detecting salmon first
The sensitivity of viral conventional RT-PCR primer (E2F/E2R), same template is detected with regular-PCR to evaluate the lowest detection of primer
Limit.OIE ratifies for detecting that the common PCR primers sequence of SAV is：
E2F:5’-CCGTTGCGGCCACACTGGATG-3’(SEQ ID NO.6)
E2R:5’-CCTCATAGGTGATCGACGGCAG-3’(SEQ ID NO.7)
The primer specificity expands salmon Alphavirus raq gene, and amplification purpose fragment size is 516bp.Therefore this hair is selected
The standard items plasmid pET32a-SAV E2 of bright foundation carry out 10 times of gradient dilutions with the deionized water of sterilizing, dilute plasmid concentration
It is 1.5 × 1010Copies/ μ l to 1.5 × 101Copies/ μ l, then with the solution of each dilution factor as template, respectively with originally grinding
Study carefully foundation Eva Green fluorescence quantification PCR primers and OIE accreditation identification salmon Alphavirus primer (E2F/E2R) carry out it is general
Logical PCR, with deionized water as negative control, detects same template to evaluate the LDL of primer.
Detect that the common PCR reaction system of salmon Alphavirus is shown in Table 1：
Table 1 detects salmon Alphavirus common PCR reaction system
The PCR conditions of fluorescent quantitation primer that the present invention sets up are：
95℃5min；94 DEG C of 30s, 58.8 DEG C of 30s, 72 DEG C of 30s, carry out 30 circulations altogether；72℃10m in.
The PCR conditions of primer E2F/E2R are：
95℃5min；94 DEG C of 30s, 57.5 DEG C of 30s, 72 DEG C of 30s, carry out 30 circulations altogether；72℃10m in.
The PCR primer that will be obtained through 2.5% nucleic acid gel electrophoresis, as shown in Figure 4 and Figure 5.Fig. 4 display present invention sets up
The minimum plasmid concentrations of primer designed by Eva Green fluorescent quantitations are 1.5 × 106Copies/ μ l, amplified fragments size
It is 197bp；The minimum plasmids detection concentration of E2F/E2R primers is 1.5 × 107Copies/ μ l, amplified fragments size is 516bp (figures
5).The primer sensitiveness of the Eva Green quantitative fluorescent PCRs that the present invention sets up is the identification salmon first that OIE assert as can be seen here
10 times of common PCR primers used by virus；The minimum detectable amount of fluorescence quantifying PCR method that the present invention sets up for 1.5 ×
101Copies/ μ l, its sensitivity is significantly larger than regular-PCR method detection salmon Alphavirus, illustrates that the invention detection method is sensitive
Degree is high.
2nd, specificity evaluation
The EVA Green fluorescent quantitative PCR detection methods set up using the present invention, have been separately added into infectiousness in system
Hematopoietic Necrosis' disease virus (IHNV), infectivity pancreatic necrosis virus (IPNV), SVCV (SVCV), fowl pass
Metachromia bursal disease virus (IBDV), bovine rota (BRV), transmissible gastro-enteritis virus (TGEV) nucleic acid are right as the positive
According to while using sterile deionized water as negative control, verifying the specificity of the method.Result of the test such as Fig. 6 shows, positive right
According to and negative control without S type curves, the PCR amplifications only with recombinant plasmid pET32a-SAV E2 as template have S type curves,
Illustrate that there is specificity well.
3rd, reproducibility
Using the positive plasmid pET32a-SAV E2 templates for building, 3 dilution factors are chosen, respectively as template same
Expanded Deng under the conditions of, each dilution factor does 3 parallel tests, calculated the coefficient of variation of Cq values；Meanwhile, by above-mentioned sample used
Product are preserved, every weekly check 1 time, continuous detection 2 weeks, the coefficient of variation of Cq values between calculating different batches.Result shows variation within batch
1% or so, interassay coefficient of variation illustrates that the method has preferably repeatability (see the table below 2) to coefficient within 1%；
The replica test result of the present invention of table 2
Embodiment 4 detects the clinical practice and sensitivity evaluation of salmon Alphavirus
Not yet find the infection of salmon Alphavirus at present due to China, thus using artificial challenge SAV fry as treating
Pathological material of disease is examined to evaluate the Eva Green fluorescence quantifying PCR methods of the detection SAV of present invention foundation.Using SAV1 (V4640) and
The rainbow trout parr of SAV2 (V4583) cell culture and virus infection health prepares pathological material of disease and is detected.
The total serum IgE that each group fry organizes pathological material of disease is extracted according to Total RNAs extraction specification, with reference to TOYOBO reverse transcription reagent box
Specification carries out reverse transcription, obtains cDNA and extremely -80 DEG C of preservation is standby.In order to be directed to E1 bases with the same detection SAV for having delivered
Because the sensitivity of the fluorescent quantitative PCR detection methods of SYBR Green I for designing is contrasted, by going that the cDNA of acquisition sterilizes
Ionized water carries out 10 doubling dilutions, detects same template with both approaches respectively to evaluate the LDL of primer, every group
Sample do three it is parallel.
1st, the system of SYBR Green I and the reaction of EVA Green quantitative fluorescent PCRs is carried out respectively using the primer delivered
And program
Delivered for E1 genes design the fluorescence quantification PCR primer sequences of SYBR Green I be：
227-F：5’-GACTGGCCTCCTTACGGGG-3’(SEQ ID NO.8)
227-R：5’-TTACAACCGTGCGGTGCTGT-3’(SEQ ID NO.9)
The fluorescent quantitative PCR reaction systems of SYBR Green I are 20 μ l, and reaction system is as follows：
THUNDERBIRDQPCR Mix10 μ l, each μ l of 1 μ l, cDNA template 3 of upstream and downstream primer (0.3 μM),
ddH2O 5μl。
Pcr amplification reaction program is：
(1) 95 DEG C of predegeneration 30s；
(2) 95 DEG C of denaturation 3s, 60 DEG C of annealing 30s, totally 40 circulations；
EVA Green fluorescent quantitative PCRs reaction system is 20 μ l, and reaction system is as follows：
The μ l of 5 × Golden HS EVA Green qPCR Mix, 4 μ l, 50 × ROX Reference Dye 0.4, concentration
It is 0.3 μM of each 0.5 μ l of primer TS1, TS2, the μ l of template 1, is finally mended to 20 μ l with the deionized water of sterilizing.
Pcr amplification reaction program is：
(1) 95 DEG C of predegeneration 15min；
(2) 95 DEG C of denaturation 10s, 60 DEG C of annealing 30s, this step is a circulation, totally 40 circulations；
2nd, the system and response procedures for being detected using primer of the invention
Primer sequence is respectively as shown in SEQ ID NO.2 and SEQ ID NO.3.
Pcr amplification reaction system is 20 μ l, and reaction system is as follows：
The μ l of 5 × Golden HS EVA Green qPCR Mix, 4 μ l, 50 × ROX Reference Dye 0.4, concentration
It is 0.3 μM of each 0.5 μ l of primer TS1, TS2, the μ l of template 1, is finally mended to 20 μ l with the deionized water of sterilizing.
Pcr amplification reaction program is：
(1) 95 DEG C of predegeneration 15min；
(2) 95 DEG C of denaturation 10s, 60 DEG C of annealing 30s, this step is a circulation, totally 40 circulations；
As shown in Table 3, the present invention is detectable minimum dilute to infecting the cDNA of SAV1 (V4640) tissue pathological material of disease for result of the test
Degree of releasing is 10-6, its sensitivity is to have delivered primer to carry out 1000 times of Eva green fluorescence quantitative detecting methods, is to have delivered
Primer carries out 100 times of the fluorescence quantitative detecting methods of SYBR Green I；CDNA to infecting SAV2 (V4583) tissue pathological material of disease can
The minimum dilution factor of detection is 10-5, its sensitivity is the primer delivered with two kinds the 10 of fluorescence quantitative detecting method times.Thus
It can be seen that, the Eva Green fluorescent quantitative PCR detection methods for SAV that the present invention sets up are for salmon Alphavirus clinical sample
Detection have sensitivity higher.
The two methods of table 3 are compared to the sensitivity for infecting the clinical sample detection of SAV
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Northeast Agricultural University
<120>A kind of Eva Green PCR kit for fluorescence quantitative for detecting salmon Alphavirus and its application
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 197
<212> DNA
<213> SAV
<400> 1
gcaacattgc cgatctgagg tgtggcttgc cctgggtgat actggcagcc gttaggactg 60
gctcctcaca cgtaaacgtc tgggagtcgc tggtgaaagt gacggttgcc aagtcagcgc 120
tcttgcattt cttgtcgaat ttcactgtgg taccaggtgg caccctcact gtgcactggc 180
tgtcgttgca tgttacg 197
<210> 2
<211> 25
<212> DNA
<213>Artificial sequence
<400> 2
gcaacattgc cgatctgagg tgtgg 25
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence
<400> 3
cgtaacatgc aacgacagcc agtgc 25
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
agcttcttgc cgtcaccacc t 21
<210> 5
<211> 23
<212> DNA
<213>Artificial sequence
<400> 5
gcggccgctt ggtccacaag tag 23
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence
<400> 6
ccgttgcggc cacactggat g 21
<210> 7
<211> 22
<212> DNA
<213>Artificial sequence
<400> 7
cctcataggt gatcgacggc ag 22
<210> 8
<211> 19
<212> DNA
<213>Artificial sequence
<400> 8
gactggcctc cttacgggg 19
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence
<400> 9
ttacaaccgt gcggtgctgt 20
Claims (10)
1. a kind of Eva Green PCR kit for fluorescence quantitative for detecting salmon Alphavirus, it is characterised in that containing being used for
The fluorescence quantification PCR primer pair and Eva Green fluorescent quantitative PCR buffer solutions of salmon Alphavirus are detected, wherein described
Primer pair be made up of sense primer and anti-sense primer, the sequence of the sense primer as shown in SEQ ID NO.2, the downstream
The sequence of primer is as shown in SEQ ID NO.3.
2. Eva Green PCR kit for fluorescence quantitative according to claim 1, it is characterised in that the Eva Green
Fluorescent quantitative PCR buffer solution includes dNTPs, Mg2+, Eva Green, archaeal dna polymerase and RNase inhibitor.
3. Eva Green PCR kit for fluorescence quantitative according to claim 2, it is characterised in that the Eva Green
Fluorescent quantitative PCR buffer solution is Golden HS EVA Green qPCR Mix.
4. the Eva Green PCR kit for fluorescence quantitative according to claim any one of 1-3, it is characterised in that described
Also include positive control and negative control in kit, described positive control is the restructuring matter containing salmon Alphavirus raq gene
Grain standard items, described negative control is deionized water.
5. the Eva Green PCR kit for fluorescence quantitative as described in claim any one of 1-4, it is characterised in that for detecting
During salmon Alphavirus, comprise the following steps：
(1) sample to be tested is homogenized, is centrifuged, taking supernatant carries out Total RNAs extraction；
(2) as template, reverse transcription synthesizes total cDNA to the total serum IgE for being obtained with step (1), and total cDNA Sample storages are standby to -80 DEG C
With；
(3) total cDNA, positive reference substance and the negative controls respectively template obtained with step (2), in quantitative real time PCR Instrument
Enterprising performing PCR amplification；
(4) after PCR reactions terminate, the Ct values and initial DNA copy number of sample are recorded.
6. Eva Green PCR kit for fluorescence quantitative as claimed in claim 5, it is characterised in that pcr amplification reaction system
For：μ l, 50 × ROX the Reference Dye0.4 μ l of 5 × Golden HS EVA Green qPCR Mix 4, concentration is 0.3 μM
Sense primer and each 0.5 μ l of anti-sense primer, the μ l of template 1, finally with sterilizing deionized water mend to 20 μ l.
7. Eva Green PCR kit for fluorescence quantitative as claimed in claim 6, it is characterised in that real-time fluorescence quantitative PCR
Response procedures be：(1) 95 DEG C of predegeneration 15min；(2) 95 DEG C of denaturation 10s, 60 DEG C of annealing 30s, totally 40 circulations.
8. Eva Green PCR kit for fluorescence quantitative as claimed in claim 5, it is characterised in that two kinds of controls in the detection
During effectively to expand, sample results criterion is as follows：Sample result is the positive during Ct value≤40.0；The sample of Ct values ＞ 40.0
Result is feminine gender.
9. the Eva Green PCR kit for fluorescence quantitative described in any one of claim 2-8 is preparing detection salmon Alphavirus
Application in reagent.
10. application as claimed in claim 9, it is characterised in that described salmon Alphavirus includes following six genotype：SAV
1st, SAV 2, SAV 3, SAV 4, SAV5 and SAV 6.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107760802A (en) * | 2017-12-01 | 2018-03-06 | 广州利洋水产科技股份有限公司 | Luohu virus-specific RT PCR detection kits and detection method |
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| US20100105035A1 (en) * | 2006-11-22 | 2010-04-29 | Syed Anwar Hashsham | Electroluminescent-based fluorescence detection device |
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| US20100105035A1 (en) * | 2006-11-22 | 2010-04-29 | Syed Anwar Hashsham | Electroluminescent-based fluorescence detection device |
| CN106048092A (en) * | 2016-07-28 | 2016-10-26 | 河南牧业经济学院 | EvaGreen real-time fluorescent quantitative PCR (Polymerase Chain Reaction) primers and detection method for RNA (Ribonucleic Acid) viruses of swine reproductive disorder diseases |
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Application publication date: 20170531 |