CN106676201A - Primer pair for detecting salmon alpha viruses, SYBR Green I fluorescent quantitative PCR (Polymerase Chain Reaction) kit and application thereof - Google Patents
Primer pair for detecting salmon alpha viruses, SYBR Green I fluorescent quantitative PCR (Polymerase Chain Reaction) kit and application thereof Download PDFInfo
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Abstract
The invention discloses a primer pair for detecting salmon alpha viruses, an SYBR Green I fluorescent quantitative PCR (Polymerase Chain Reaction) kit and application thereof. The invention provides the real-time fluorescent quantitative PCR primer pair for detecting the salmon alpha viruses for the first time; and the sequence is shown as SEQ ID NO.2 and SEQ ID NO.3. The invention further provides the SYBR Green I fluorescent quantitative PCR kit containing the primer pair and a method for detecting the salmon alpha viruses. A research shows that the primer or the kit, disclosed by the invention, is used for detecting the salmon alpha viruses, has the advantages of simplicity, rapidness, accuracy, high sensitivity and good specificity and the like, and can be used for detecting six gene types of the salmon alpha viruses. The invention provides an effective technical means for detecting the salmon alpha viruses.
Description
Technical field
The present invention relates to be used for the primer and test kit of Viral diagnosis, more particularly to for detecting salmon Alphaviruses six
The fluorescence quantification PCR primer of the specificity of genotype, the invention further relates to using the primer and the fluorescent dyes of SYBR Green I
Carry out the method and test kit of salmon Alphaviruses detection.The invention belongs to virus detection techniques field.
Background technology
Salmon Alphaviruses (salmonid alphavirus, SAV) are 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 lethargy, fatality 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 sleeping sickness
SAV is separated in trout body, at present the disease is widely current in England, Scotland, Norway, France, Poland, Italy and Spain
Deng European countries, according to statistics 90% is increased up to year by year from nineteen ninety-five to 2007 annual morbidities, every year thus epidemic disease causes huge
Economic loss.Salmon Alphaviruses infection in 2013 is put in 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 in recent years China introduces to salmon fishes import and salmon fishes ovum,
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 the SAV (SAV 1, SAV 2, SAV 3, SAV 4, SAV5, SAV 6) of six genotype.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,
Size is 65nm.Single strand plus RNA virus, gene group leader 11-12kb, and containing two open reading frame (ORF).Genome
Second ORF coding 26S subbases because mRNA, it is final that to produce 5 structural protein, i.e. capsid protein, E3, E2,6K and E1 common
Constitute the membrane glycoprotein of virus.E2 is connected in immaturity with E3, constitutes the precursor of E2, and E3's functions as E2's
Signal peptide.When E2 is 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 at present the virus, 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 Alphaviruses 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 Alphaviruses are predominantly detected
Technology, is widely used in detection and the Molecule Epidemiology Investigation of clinical sample.
The content of the invention
It is an object of the invention to provide a kind of primer of detection salmon Alphaviruses, and the SYBR containing the primer
The PCR kit for fluorescence quantitative of Green I.
Further object is that providing the fluorescence quantifying PCR method of detection salmon Alphaviruses.
For achieving the above object, following technological means be present invention employs：
The present invention compares to known salmon Alphavirus genomes sequence, filters out six genotype of salmon Alphaviruses and guards
One section of conserved sequence (9471-9668bp) in region sequence, i.e. E2 protein gene, its nucleotide sequence such as SEQ ID NO.1 institutes
Show.For sequence inventor Jing repeated screenings and checking, a pair are obtained for detecting six differences of salmon Alphaviruses
The fluorescence quantification PCR primer of genotype, amplified fragments size is 197bp, and primer sequence is as follows：
Forward primer：5’-GCAACATTGCCGATCTGAGGTGTGG-3’(SEQ ID NO.2)
Downstream primer：5’-CGTAACATGCAACGACAGCCAGTGC-3’(SEQ ID NO.3)
Further, present invention also offers a kind of quantitative fluorescent PCRs of SYBR Green I for detecting salmon Alphaviruses
Test kit, it contains primer pair of the present invention and SYBR Green I fluorescent quantitation PCR amplification buffers.
In test kit of the present invention, it is preferred that the SYBR GreenI fluorescent quantitative PCRs buffer bag
Include dNTPs, Mg2+, SYBR Green I, archaeal dna polymerase and RNase inhibitor.
In test kit of the present invention, it is preferred that the SYBR Green I fluorescent quantitation PCR amplification buffers are
THUNDERBIRD qPCR Mix。
In test kit of the present invention, it is preferred that also include positive control and negative control in described test kit,
Described positive control is the recombiant plasmid standard substance containing salmon Alphaviruses raq gene, and described negative control is deionization
Water.
Further, the invention allows for a kind of using the described PCR kit for fluorescence quantitative of SYBR Green I
The method of detection salmon Alphaviruses, comprises the following steps：With sample total serum IgE as template, reverse transcription is cDNA, using described
The PCR kit for fluorescence quantitative of SYBR Gree n I carries out real-time fluorescence quantitative PCR, while negative control and positive control are set up,
According to amplification curve result of determination.
Wherein, it is preferred that 20 μ l real-time fluorescence quantitative PCR work systems are：THUNDERBIRDqPCR Mix
10 μ l, 0.3 μM of forward primer and each 1 μ l of downstream primer, cDNA templates 3 μ l, ddH2O 5μl。
Wherein, it is preferred that the response procedures of real-time fluorescence quantitative PCR are：95 DEG C of denaturations 30s, 1 circulation；95 DEG C of changes
Property 3s, 60 DEG C annealing 30s, 40 circulation.
Wherein, it is preferred that in the detection two kinds of controls are for during effectively amplification, 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, the invention allows for described primer pair and the described quantitative fluorescent PCRs of SYBR Green I
Application of the test kit in the reagent for preparing detection salmon Alphaviruses, it is preferred that described salmon Alphaviruses include following six
Genotype：SAV 1, SAV 2, SAV 3, SAV 4, SAV5 and SAV 6.
To sum up, the invention provides the specific primer for detecting six genotype of salmon Alphaviruses, establishes detection
The fluorescence quantifying PCR methods of SYBR Green I of salmon Alphaviruses, with very high detection sensitivity, can detect 15 and copy
The salmon Alphaviruses cDNA of shellfish；And the method high specificity, its is reproducible, can improve as the means of detection clinical samples
The positive rate of six genotype detection of salmon Alphaviruses, simple to operate, detection process is only needed 90 minutes, greatly shortens detection week
Phase, testing result is directly observed using quantitative real time PCR Instrument, not there is a problem of that ethidium bromide pollutes.The inspection that the present invention is provided
Test agent box, its reaction system contains above-mentioned primer, feminine gender and positive control, the popularization and application of the test kit will for preventing and treating and
Monitoring salmon onychonosus viral disease provides technical support.
Description of the drawings
Fig. 1 is the fluorescent quantitative PCR kinetic curves of salmon Alphaviruses SYBR Green I；
Fig. 2 is the quantitative fluorescent PCR standard curves of salmon Alphaviruses SYBR Green I；
Fig. 3 is the fluorescent quantitative PCR product solubility curves of salmon Alphaviruses SYBR Green I；
Fig. 4 is that regular-PCR method detects that the present invention sets up the susceptiveness nucleic acid electrophoresis figure of primer；
Fig. 5 is the susceptiveness nucleic acid electrophoresis for detecting salmon Alphaviruses primer that regular-PCR method detects OIE approvals
Figure；
Fig. 6 expands kinetic curve for the specific test of the quantitative fluorescent PCRs of salmon Alphaviruses SYBR Green I；
Fig. 7 is the specific test amplified production solubility curve of the quantitative fluorescent PCRs of salmon Alphaviruses SYBR Green I.
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 made to the inventive method, step or condition or replacement belong to the present invention
Scope.
If not specializing, the conventional meanses that technological means used are well known to those skilled in the art in embodiment.
The design of the six Serotype-dependent primers of salmon Alphaviruses of embodiment 1
According to salmon Alphaviruses (SAV) the raq gene sequence delivered in Genebank, compare 6 genotype differences of SAV
The raq gene sequence of strain, filters out one section of conserved sequence (9471-9668bp).During design primer, note avoiding the prominent of sequence
Height, Jing repeated screenings and checking, obtain a pair quantitative fluorescent PCRs for detecting six different genotypes of salmon Alphaviruses and draw
Thing, amplified fragments size is 197bp, and its nucleotide sequence is as shown in SEQ ID NO.1：
Forward primer：5’-GCAACATTGCCGATCTGAGGTGTGG-3’(SEQ ID NO.2)
Downstream primer：5’-CGTAACATGCAACGACAGCCAGTGC-3’(SEQ ID NO.3)
The foundation of the fluorescent quantitative PCR detection methods of SYBR Green I of the detection salmon Alphaviruses of embodiment 2
1st, the preparation of salmon Alphaviruses raq gene recombiant plasmid standard substance：
The total serum IgE of salmon Alphaviruses SAV 4640 is extracted according to Total RNAs extraction description, with reference to TOYOBO Reverse Transcriptions
Box description carries out reverse transcription, and reverse transcription gained cDNA is carried out with the specific primer of amplification salmon Alphaviruses E2 full-length genes
Standard PCR amplification, 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)
The agarose gel electrophoresiies of PCR primer Jing 1.0% detect that amplification E2 total lengths are 1375bp, are returned with glue reclaim test kit
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 substance, 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, recombiant plasmid is carried out into 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 recombiant plasmid of copies/ μ l, as standard substance template.
Sample all sets up 1 positive control sample and negative control sample.Positive control sample is above-mentioned recombiant plasmid；It is cloudy
Property control sample be deionized water.
2nd, the fluorescence quantitative PCR detections of SYBR Green I
Concentration is taken respectively for 1.5 × 1010、1.5×109、1.5×108、1.5×107、1.5×106The weight of copies/ μ l
Group plasmid pET32 a-SAV E2 carry out the amplification of the quantitative fluorescent PCRs of SYBR Green I for template, obtain expanding kinetics song
Line and standard curve, as shown in Figures 1 and 2, the correlation coefficient of standard curve is 0.996, and slope is -3.733, it can thus be seen that
Linear relationship is good.
The μ l PCR reaction systems of salmon Alphaviruses fluorescent quantitation 20 are shown in Table 1：
The salmon Alphaviruses quantitative fluorescent PCR reaction system of table 1
3rd, real-time fluorescence quantitative PCR reaction condition
5 DEG C of denaturations 30s, 1 circulation；95 DEG C of degeneration 3s, 60 DEG C of annealing 30s, 40 circulations.
Simultaneously no template control and positive control are set up, according to amplification curve result of determination.
After reaction terminates, the solubility curve of quantitative fluorescent PCR is confirmed, as shown in figure 3, only a single set of peak, explanation is drawn
Thing high specificity, amplification efficiency highest, the solution temperature of standard substance is all at 86.5 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 control effect can be played to testing sample；If testing sample amplification curve Ct values are less than or wait
In 40, then illustrate that sample is that SAV nucleic acid is positive；If testing sample is without amplification curve, or amplification curve Ct values are more than 40, then sample
It is negative for SAV nucleic acid.
The evaluating characteristics of the salmon Alphaviruses fluorescent quantitative PCR detection method of embodiment 3
1st, sensitivity evaluation, the i.e. lowest detectable limit of real-time PCR.
Positive reference DNA original liquid concentration is 1.5 × 1010copies/μl.First by its 10 times of gradient dilutions to 1.5 ×
101Copies/ μ l, then respectively with each dilution solution as template, the fluorescent quantitation set up using the embodiment of the present invention 2
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.
In order to the fluorescence quantification PCR primers of SYBR Green I and the OIE accreditations of foundation relatively more of the present invention are for detecting salmon
The susceptiveness of Alphaviruses conventional RT-PCR primer (E2F/E2R), same template is detected with regular-PCR to evaluate the minimum inspection of primer
Survey limit.OIE ratifies the common PCR primers sequence for detecting SAV：
E2F:5’-CCGTTGCGGCCACACTGGATG-3’(SEQ ID NO.6)
E2R:5’-CCTCATAGGTGATCGACGGCAG-3’(SEQ ID NO.7)
The primer specificity amplification salmon Alphaviruses raq gene, amplification purpose fragment size is 516bp.Therefore this is selected
The standard substance plasmid pET32 a-SAV E2 of bright foundation carry out 10 times of gradient dilutions with the deionized water of sterilizing, dilute plasmid concentration
For 1.5 × 1010Copies/ μ l to 1.5 × 101Copies/ μ l, then with each dilution solution as template, respectively with this
Identification salmon Alphaviruses primer (E2F/E2R) of the fluorescence quantification PCR primers of SYBR Green I of bright foundation and OIE accreditations is carried out
Regular-PCR, deionized water detects same template to evaluate the lowest detectable limit of primer as negative control.
The common PCR reaction system of detection salmon Alphaviruses is shown in Table 2：
Table 2 detects salmon Alphaviruses 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 altogether 30 circulations；72℃10min.
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 altogether 30 circulations；72℃10min.
The nucleic acid gel electrophoresis of PCR primer Jing 2.5% that will be obtained, as shown in Figure 4 and Figure 5.Fig. 4 shows what the present invention set up
The minimum plasmid concentrations of primer designed by the fluorescent quantitations of SYBR Green I are 1.5 × 106Copies/ μ l, amplified fragments
Size is 197bp；The minimum plasmids detection concentration of E2F/E2R primers is 1.5 × 107Copies/ μ l, amplified fragments size is
516bp (Fig. 5).As can be seen here the primer sensitivity of the quantitative fluorescent PCRs of SYBR Green I that the present invention sets up is that OIE assert
10 times of common PCR primers used by identification salmon Alphaviruses；The minimum detectable amount of fluorescence quantifying PCR method that the present invention sets up
For 1.5 × 101Copies/ μ l, its susceptiveness is significantly larger than regular-PCR method detection salmon Alphaviruses, illustrates the invention detection
Method sensitivity is high.
2nd, Evaluation on specificity
The fluorescence quantifying PCR method set up using the embodiment of the present invention 2, to salmon Alphaviruses (SAV), infectious hematopoietic device
Official's IPNV (IHNV), infectivity pancreatic necrosis virus (IPNV), SVCV (SVCV), avian infectious method
Family name's capsule virus (IBDV), bovine rota (BRV), transmissible gastro-enteritis viruss (TGEV) nucleic acid detected as template,
Every kind of sample do three it is parallel.As a result Fig. 6 and Fig. 7 is seen.Fig. 6 shows that only SAV samples (arrow indication) have amplification curve, other
Viral cDNA illustrates the inventive method to IHNV without amplification curve, IPNV, SVCV, IBDV, BRV and TGEV no cross reaction,
Positive is presented to salmon Alphaviruses testing result.Fig. 7 shows that solubility curve expands tri- parallel samples of SAV and has list at 86 DEG C or so
One specificity set peak, and the not visible solubility curve of other virus sample, illustrate that the inventive method has excellent specificity.
3rd, reproducibility.
Using the positive plasmid pET32 a-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, and every weekly check 1 time, continuous detecting 2 weeks calculates the coefficient of variation of Cq values between different batches.As a result variation within batch is shown
1% or so, interassay coefficient of variation illustrates that the method has preferably repeatability (see the table below 3) to coefficient within 1%；
The replica test result of the present invention of table 3
The clinical practice and susceptiveness evaluation of the detection salmon Alphaviruses of embodiment 4
Because China not yet finds at present the infection of salmon Alphaviruses, thus using artificial challenge SAV fry as treating
Examine pathological material of disease to evaluate the fluorescence quantifying PCR methods of SYBR Green I of the detection SAV of present invention foundation.Using SAV1 (V4640)
The rainbow trout parr for infecting health with SAV2 (V4583) cell culture and virus 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 description, with reference to TOYOBO reverse transcription reagent box
Description carries out reverse transcription, obtain cDNA and preserve to -80 DEG C it is standby.In order to be directed to E1 bases with the same detection SAV for delivering
Because the susceptiveness of the fluorescent quantitative PCR detection methods of SYBR Green I for designing is contrasted, by the cDNA sterilizings for obtaining
Deionized water carries out 10 doubling dilutions, detects same template to evaluate the lowest detectable limit of primer with both approaches respectively, often
Group sample do three it is parallel.
The fluorescence quantification PCR primer sequences of SYBR Green I for E1 gene design delivered are：
227-F：5’-GACTGGCCTCCTTACGGGG-3’(SEQ ID NO.8)
227-R：5’-TTACAACCGTGCGGTGCTGT-3’(SEQ ID NO.9)
Pcr amplification reaction system is 20 μ l, and reaction system is as shown in table 4 below：
The salmon Alphaviruses quantitative fluorescent PCR reaction system of table 4
Pcr amplification reaction program is：
(1) 95 DEG C of denaturation 30s；
(2) 95 DEG C of degeneration 3s, 60 DEG C of annealing 30s, this step is a circulation, totally 40 circulations；
As shown in Table 5, the present invention is detectable minimum dilute to the cDNA for infecting SAV1 (V4640) tissue pathological material of disease for result of the test
Degree of releasing is 10-6, its sensitivity is 100 times of the fluorescence quantitative detecting methods of SYBR Green I delivered；To infecting SAV2
(V4583) the detectable minimum dilution factors of the cDNA of tissue pathological material of disease are 10-5, its sensitivity is that the SYBR Green I for having delivered are glimmering
10 times of light quantitative detecting method.As can be seen here, the quantitative fluorescent PCRs of SYBR Green I for SAV that the present invention sets up are examined
Survey method has higher susceptiveness for the detection of salmon Alphaviruses clinical sample.
The two methods of table 5 are compared to the susceptiveness for infecting the clinical sample detection of SAV
Although above with a general description of the specific embodiments the present invention is described in detail,
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, without departing from theon the basis of the spirit of the present invention these modifications or improvements, belong to the scope of protection of present invention.
Sequence table
<110>Northeast Agricultural University
<120>A kind of primer pair of detection salmon Alphaviruses, the PCR kit for fluorescence quantitative of SYBR Green I 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 fluorescence quantification PCR primer pair for detecting salmon Alphaviruses, it is characterised in that described primer pair is by upstream
Primer and downstream primer are constituted, and as shown in SEQ ID NO.2, the sequence of the downstream primer is such as the sequence of the forward primer
Shown in SEQ ID NO.3.
2. a kind of PCR kit for fluorescence quantitative of SYBR Green I for detecting salmon Alphaviruses, it is characterised in that containing having the right
Profit requires the primer pair described in 1 and SYBR Green I fluorescent quantitation PCR amplification buffers.
3. the PCR kit for fluorescence quantitative of SYBR Green I according to claim 2, it is characterised in that described
SYBR Green I fluorescent quantitation PCR amplification buffer includes dNTPs, Mg2+, SYBR Green I, archaeal dna polymerase and RNase suppression
Preparation.
4. the PCR kit for fluorescence quantitative of SYBR Green I according to claim 3, it is characterised in that the SYBR
Green I fluorescent quantitations PCR amplification buffer is THUNDERBIRDqPCR Mix。
5. the PCR kit for fluorescence quantitative of SYBR Green I according to any one of claim 2-4, it is characterised in that institute
Also include positive control and negative control in the test kit stated, described positive control is the weight containing salmon Alphaviruses raq gene
Group plasmid standard, described negative control is deionized water.
6. the PCR kit for fluorescence quantitative of SYBR Green I as described in any one of claim 2-5, it is characterised in that be used for
During detection salmon Alphaviruses, comprise the following steps：With sample total serum IgE as template, reverse transcription is cDNA, using claim 2-5
The PCR kit for fluorescence quantitative of SYBR Green I described in any one carries out real-time fluorescence quantitative PCR, while setting up negative control
And positive control, according to amplification curve result of determination.
7. the PCR kit for fluorescence quantitative of SYBR Green I as claimed in claim 6, it is characterised in that its 20 μ l is glimmering in real time
Fluorescent Quantitative PCR work system is：THUNDERBIRDThe μ l of qPCR Mix 10,0.3 μM of forward primer and downstream are drawn
The each 1 μ l of thing, cDNA templates 3 μ l, ddH2O 5μl。
8. the PCR kit for fluorescence quantitative of SYBR Green I as claimed in claim 7, it is characterised in that real time fluorescent quantitative
The response procedures of PCR are：95 DEG C of denaturations 30s, 1 circulation；95 DEG C of degeneration 3s, 60 DEG C of annealing 30s, 40 circulations.
9. the PCR kit for fluorescence quantitative of SYBR Green I as claimed in claim 7, it is characterised in that two kinds in the detection
Compare as during effectively amplification, sample results criterion is as follows：Sample result is the positive during Ct value≤40.0；Ct values ＞ 40.0
Sample result is feminine gender.
10. the fluorescent quantitations of SYBR Green I described in primer pair described in claim 1 and any one of claim 2-9
Application of the PCR kit in the reagent for preparing detection salmon Alphaviruses, it is preferred that described salmon Alphaviruses include following six
Individual genotype：SAV 1, SAV 2, SAV 3, SAV 4, SAV5 and SAV 6.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710093483.8A CN106676201A (en) | 2017-02-21 | 2017-02-21 | Primer pair for detecting salmon alpha viruses, SYBR Green I fluorescent quantitative PCR (Polymerase Chain Reaction) kit and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110894544A (en) * | 2018-09-13 | 2020-03-20 | 杭州众测生物科技有限公司 | RAA constant temperature fluorescence detection method and reagent for Salmon Alphavirus (SAV) |
Citations (1)
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|---|---|---|---|---|
| WO2015051456A1 (en) * | 2013-10-08 | 2015-04-16 | University Of Prince Edward Island | Multiplex diagnostic assay for detecting salmonid pathogens |
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| WO2015051456A1 (en) * | 2013-10-08 | 2015-04-16 | University Of Prince Edward Island | Multiplex diagnostic assay for detecting salmonid pathogens |
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| D.A.GRAHAM等: "Development and evaluation of a one-step real-time reverse transcription polymerase chain reaction assay for the detection of salmonid alphaviruses in serum and tissues", 《DISEASES OF AQUATIC ORGANISMS》 * |
| KJARTAN HODNELAND等: "Sensitive and specific detection of Salmonid alphavirus using real-time PCR (TaqMan®)", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| 刘敏等: "鲑鱼甲病毒阳性核酸物质制备及RT-PCR检测方法的建立", 《东北农业大学学报》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110894544A (en) * | 2018-09-13 | 2020-03-20 | 杭州众测生物科技有限公司 | RAA constant temperature fluorescence detection method and reagent for Salmon Alphavirus (SAV) |
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