CN105925728A - Seneca valley virus real-time fluorescence quantification PCR detection primer and kit - Google Patents
Seneca valley virus real-time fluorescence quantification PCR detection primer and kit Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection, and discloses a Seneca valley virus real-time fluorescence quantification PCR detection primer and a kit. Firstly, the Seneca valley virus real-time fluorescence quantification PCR detection primer and a probe are obtained through design and screening, the sequences of an upstream primer, a downstream primer and the probe are shown as SEQ ID NO: 1-3; the primer and the probe are used to specifically amplify a Seneca valley virus, real-time fluorescence quantification monitors the condition of combination of a double-stranded DNA fluorescent dye with a PCR amplification product in the PCR process in real time, then fluorescent data are acquired, and the SVV (Seneca Valley Virus) is identified according to a CT value; the SVV is identified after the method is used to carry out PCR amplification, the accuracy is high, the specificity and the repeatability are good, the SVV can be identified accurately, rapidly and efficiently, and the popularization and the application in clinical practice benefits are facilitated.
Description
Technical field
The present invention relates to technical field of biological, quantitative more particularly, to a kind of Sai Neijia paddy virus real-time fluorescence
PCR detection primer and test kit.
Background technology
Sai Neijia paddy virus (Seneca Valley virus SVV) is single strand plus RNA virus, is that micro ribonucleic acid is sick
The Typical Representative of poison section Senecavirus Tobamovirus.It is considered as initially the pollutant in PER.C6 cell culture, subsequently
The swinery of America & Canada is separated, vesicle Disease Clinical disease was occurred in the swinery in the U.S. one slaughterhouse in 2007
Shape, the pig of about 80% occurs that cyllopodia, PCR detection Schweineseuche, SVD, herpetic stomatitis and vesicle rash are feminine gender, and
SVV is but positive, thus is considered as constitutional vesicle disease.Pig Sai Neijia paddy virus (SVV) is in Brazil swinery in the recent period
Infection and outburst, it was demonstrated that one of pig Sai Neijia paddy virus constitutional vesicle disease pathogen being likely to be pig.2015
Year China and Brazil occur in that SVV infects swinery and serious clinical onset and dead symptom occurs several times, causes serious warp
Ji loss.Clinical symptoms caused clinically for SVV is very much like with the blister disease of some other pig, in clinical and tissue disease
Being difficult in Neo-Confucianism distinguish, this just brings certain difficulty to making a definite diagnosis of this disease, and its Differential Diagnosis generally need to be by laboratory diagnosis skill
Art, traditional detection method wastes time and energy, and operational approach is loaded down with trivial details, is unfavorable for the timely diagnoses and treatment of disease.
Summary of the invention
The technical problem to be solved is overcome in prior art existing for Sai Neijia paddy Viral diagnosis above-mentioned
Defect, it is provided that the primer of a kind of specific detection Sai Neijia paddy virus real-time fluorescence quantitative PCR.
Second object of the present invention is to provide the test kit containing above-mentioned detection primer.
It is an object of the invention to be achieved by the following technical programs:
A kind of Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and probe, described forward primer, downstream primer and probe
Sequence as shown in SEQ ID NO:1～3.
Preferably, 5 ' end labellings of described probe is fluorescent reporter group；3 ' end labellings are quenching groups.
It is highly preferred that described fluorescent reporter group is FAM, described quenching group is TAMAR.
The present invention also provides for containing described Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and the reagent of probe
Box.
Preferably, the concentration of described forward primer, downstream primer and probe is 10 μMs.
Preferably, described test kit also includes PCR reaction buffer, fluorescent dye, positive criteria product.
It is highly preferred that the preparation method of described positive criteria product is to become from SVV cell virus extracting RNA reverse transcription
CDNA, then through PCR by SVV-3C gene amplification out, obtain the amplified production consistent with purpose clip size.PCR primer reclaims
After, through being connected with PMD19-T carrier, convert, expanding the recombiant plasmid containing purpose fragment, identify through bacterium solution PCR and order-checking,
Confirm the most successfully to construct SVV recombiant plasmid standard substance, named PMD19-T-3C, stand-by as positive criteria product.
Preferably, described test kit carries out the reaction system of quantitative fluorescent PCR and is: Premix Ex Taq(Probe
QPCR) (2 ×) 10 μ L, 10 μMs, 0.4 μ L forward primer, 10 μMs, 0.4 μ L downstream primer, ROX Reference Dye II
(50 ×) 0.2 μ L, 10 μMs, 0.8 μ L probe, DNA profiling 2.0 μ L, ddH2O 6.2μL。
Preferably, described test kit carries out the reaction condition of quantitative fluorescent PCR and is: 95 DEG C 30 seconds, 95 DEG C 5
Second, 60 DEG C 34 seconds, 40 circulations.
Compared with prior art, the method have the advantages that
The present invention obtains a kind of Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and probe, institute by design screening
State the sequence of forward primer, downstream primer and probe as shown in SEQ ID NO:1～3；Utilize this primer and the probe can specificity
Expanding out Sai Neijia paddy virus, real time fluorescent quantitative can be by double-stranded DNA fluorescent dyestuff and PCR during monitoring PCR in real time
The combination situation of amplified production, gathers fluorescence data, differentiates SVV according to CT value；Described method is utilized to carry out after PCR amplification i.e.
Can differentiate SVV, accuracy is high, specificity is good, reproducible, can carry out accurately, fast and efficiently identifying SVV, be conducive to
Popularization and application in clinical practice.
Accompanying drawing explanation
Fig. 1 is the PCR amplification of restructuring plasmid standard PMD19-T-3C.
Fig. 2 is the standard curve of real-time fluorescence quantitative PCR.
Fig. 3 is the susceptiveness test of real-time fluorescence quantitative PCR；Wherein 1 is 1 × 109copies/μL；2 be 1 ×
108copies/μL；3 is 1 × 107copies/μL；4 is 1 × 106copies/μL；5 is 1 × 105copies/μL；6 be 1 ×
104copies/μL；7 is 1 × 103copies/μL；8 is 1 × 102copies/μL；9 is 1 × 101copies/μL；10 be 1 ×
100copies/μL；11 is negative control.
Fig. 4 is the specific test of real-time fluorescence quantitative PCR, and wherein 1 is SVV；2 is FMDV；3 is SVDV；4 is VSV；5
For PRRSV；6 is PCV；7 is PRV；8 is PK-15 cell.
Detailed description of the invention
Further illustrate present disclosure below in conjunction with Figure of description and specific embodiment, but should not be construed as right
The restriction of the present invention.In the case of present invention spirit and essence, the amendment that the inventive method, step, condition are made
Or replace, belong to the scope of the present invention.Unless otherwise noted, experimental technique used in embodiment is people in the art
Conventional method known to Yuan and technology, reagent or material are and are obtained by commercial sources.
The preparation of embodiment 1 standard positive template
One, the extracting of virus total RNA
Extract test kit operation instructions by Invitrogen company's T RIZOL LS Reagent RNA to carry out.At 1.5 mL
Eppendorf pipe adds cleer and peaceful 750 L TRIZOL on the virus liquid of the good cell proliferation gained of 250 L subpackage, fully
Mixing, room temperature places 10 min；Adding the chloroform of 200 L, be aggressively shaken 15sec, room temperature stands 5min, 4 DEG C, 12000 rpm
Centrifugal 15 min；Transfer to supernatant, in 1.5 new mL eppendorf pipes, add 500 L isoamyl alcohol, fully mix, room
Temperature places 10min, and 4 DEG C, 12000 rpm are centrifuged 10 min；Abandoning supernatant, precipitates ice-cold 70% ethanol 1000 L,
Mixing gently, washed once, 4 DEG C, 12000 rpm are centrifuged 10 min；Abandoning supernatant, air-dries；With 20 L DEPC process
Tri-distilled water dissolves RNA, and-80 DEG C preserve or are directly used in reverse transcription.
Two, design of primers
According to SVV-001(DQ641257 in GenBank) 3C regional gene sequence, design PCR primer: forward primer P1:
5 '-ATCCTTTTGCTGCCCATGTG-3 ', downstream primer P2:5 '-AGAACTCTACCCAACGCCTC-3 ', utilize this primer
Amplification 3C gene, reaction system is as follows: PremixTaq 25 μ L, forward primer P1 1 μ L, downstream primer P2 1 μ L, template DNA
1 μ L, finally uses ddH2It is 50 μ L that O supplies reaction system cumulative volume.
Amplification program is: (1) 94 DEG C of denaturation 5min, (2) 94 DEG C of degeneration 1min, and 57 DEG C of annealing 30 s, 72 DEG C extend 30
S, totally 32 circulations；(3) 72 DEG C extend 10min.
Reclaim PCR primer (with reference to the operation instructions of Omega company E.Z.N.A. Gel Extraction Kit),
Carry out agarose gel electrophoresis, be accredited as correct target fragment, for follow-up clone.
Three, the clone of genes of interest
(1) reclaim product and connect pMD19-T carrier
With reference to pMD 19-T Vector test kit description, coupled reaction system is: PCR primer 2 μ L, pMD19-T of purification
vector 0.5μL、Solution I 2.5μL；The cumulative volume of final coupled reaction system is 5 μ L.Above-mentioned reaction system is mixed
Even and slightly make centrifugal after, 4 DEG C connect overnight.
(2) connect product to convert
The connection product 5 μ L of (1) is added in the competent escherichia coli cell of 50 μ L lightly, ice bath 30min；42 DEG C of heat are stopped
Then gram 90s is quickly transferred in ice bath cool down 2min；Being added in 200 μ L LB fluid mediums, 37 DEG C, 160rpm shakes training
Support 45min, make escherichia coli recover, tolerant gene expression；The competent cell even spread Amp that will recover above+/ LB solid
Culture medium plate, 37 DEG C of overnight incubation.
(3) PCR of recon identifies and screening
Three single bacterium colonies of picking from the flat board of (2) incubated overnight, are inoculated in 1mL respectively and contain 100 μ g/mL Amp+LB liquid
In body culture medium, 37 DEG C, more than 6h (OD600 value 0.3～0.4) is cultivated in 220rpm shaking；Take bacterium solution and carry out PCR qualification, product
Detect with 1% agarose gel electrophoresis；Take 20 μ L and identify that positive bacterium solution is inoculated into 2mL Amp with the ratio of 1:100+'s
In LB fluid medium, 37 DEG C, 220rpm shakes overnight incubation.
(4) extraction of recombiant plasmid and qualification
With reference to the description of Omega company E.Z.N.A. Plasmid Mini Kit I, incubated overnight bacterium solution is carried out plasmid
Extracting, by 1% agarose gel electrophoresis detection plasmid extraction effect.
Being detected with 1% agarose gel electrophoresis by recombiant plasmid extract product, qualification result is shown in Fig. 1, the weight of the identified positive
Group pMD19-T plasmid send Beijing AudioCodes biotechnology Co., Ltd to carry out nucleotide sequencing.
The design of embodiment 2 primer and TaqMan probe and screening
Designing special primer and TaqMan probe according to the 3C conservative fragments in SVV gene order, by Hua Da, company synthesizes, primer
Being shown in Table 1 with probe sequence, the fluorescent reporter group of 5 ' end labellings of probe is FAM, and the quenching group of 3 ' end labellings is TAMRA.
By the primer in table 1 and probe, SVV is carried out PCR, found that: other 3 pairs of primers occur non-specific expansion
Increasing or primer dimer, do not meet requirement of experiment, the most only primer 1 and primer 2 can be as specific amplification detection SVV's
Primer.
Embodiment 3 quantitative fluorescent PCR reaction condition optimization and standard curve making
To primer concentration, concentration and probe concentration and annealing temperature condition optimization, Real-Time PCR 20 μ L reaction system is
Premix Ex Taq(Probe qPCR) (2 ×) 10 μ L, each 0.4 μ L in each specificity upstream and downstream primer (10 μMs), ROX
Reference Dye II(50 ×) 0.2 μ L, fluorescent probe (10 μMs) 0.8 μ L, DNA profiling 2.0 μ L, ddH2O 6.2 μ
L.Optimum reaction condition is: 95 DEG C of 30 s, 95 DEG C of 5 s, 60 DEG C of 34 s, 40 circulations.
The OD of bioassay standard restructuring positive plasmid260nmAnd OD280nmValue and ratio thereof, calculate plasmid concentration, and be converted into and copy
Shellfish number, becomes 7 gradients (10 with 10 times of serial dilutions of distilled water8～103Copies/ μ L).As template, set up 20 μ L reactions
System, according to the PCR reaction system after optimizing, expands on ABI 7500 type quantitative real time PCR Instrument, and gained standard curve is shown in figure
2.As a result, the linear relationship between amplification curve and Ct value is tight, correlation coefficient (R2) up to 0.999.
The susceptiveness test of embodiment 4 real-time fluorescence quantitative PCR
10 times of serial dilutions of SVV positive criteria product carry out real-time fluorescence quantitative PCR (l × 10 as template0～l ×
109Copies/ μ L, totally 10 gradients, to determine their detection lower limit.As a result, with PMD19-T-3C standard substance as template, glimmering
The detection lower limit of Fluorescent Quantitative PCR is 1 × 102Copies/ μ L(Fig. 3).
The specific test of embodiment 5 real-time fluorescence quantitative PCR
With cDNA or DNA of SVV, FMDV, SVDV, VSV, PRRSV, PCV, PRV, PEDV and normal PK-15 cell as template, should
The reaction system and the reaction condition that optimize by the embodiment 3 set up carry out real-time fluorescence quantitative PCR amplification, found that should
PCR amplification only SVV is positive (Fig. 4).
The replica test of embodiment 6 real-time fluorescence quantitative PCR
The positive criteria quality grain of SVV is carried out 109、107、105Copies/ μ L totally 3 gradients are template, carry out organizing interior and group
Between Repeatability checking, each gradient will be repeated 5 times, and does 3 secondary responses altogether.The method being set up SVV carries out PMD19-
T-3C standard substance plasmid Repeatability checking.Shown in result table 2: the coefficient of variation (CV) in group and between group is respectively less than 0.96%.
The composition of embodiment 7 test kit
According to following composition be formulated for detect pig Sai Neijia paddy virus test kit: 10 μMs of primers 1,10 μMs of primer 2s, 10 μ
M probe, ROX Reference Dye II(50 ×), Premix Ex Taq(Probe qPCR) (2 ×).
A kind of reaction system of this test kit can be: Premix Ex Taq(Probe qPCR) (2 ×) 10 μ L, 10 μ
M, 0.4 μ L primer 1,10 μMs, 0.4 μ L primer 2, ROX Reference Dye II(50 ×) 0.2 μ L, 10 μMs, 0.8 μ L visits
Pin, DNA profiling 2.0 μ L, ddH2O 6.2 μ L, reaction cumulative volume is 20 μ L.
The reaction condition utilizing this test kit to carry out real-time fluorescence quantitative PCR is: 95 DEG C 30 seconds, 95 DEG C 5 seconds, 60 DEG C
34 seconds, 40 circulations.
SEQUENCE LISTING
<110>Agricultural University Of South China
<120>a kind of Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and test kit
<130>
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>primer 1
<400> 1
gagcttcaat ctcctaga 18
<210> 2
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<212> DNA
<213>primer 2
<400> 2
gtgtcatcat tctcgttag 19
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<212> DNA
<213>probe 1
<400> 3
cagacattcg agccaagcaa caa 23
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<213>primer 3
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ccctaacgag aatgatga 18
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<212> DNA
<213>primer 4
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cactgccaag attatcaag 19
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<212> DNA
<213>probe 2
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ccgcagctag agcaagacct 20
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<212> DNA
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ggctctgtag aactagag 18
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tagggctctg tagaacta 18
Claims (9)
1. Yi Zhong Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and probe, it is characterised in that described forward primer,
The sequence of downstream primer and probe is as shown in SEQ ID NO:1～3.
Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and probe the most according to claim 1, it is characterised in that
5 ' end labellings of described probe are fluorescent reporter group；3 ' end labellings are quenching groups.
Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and probe the most according to claim 2, it is characterised in that
Described fluorescent reporter group is FAM, and described quenching group is TAMAR.
4. contain Sai Neijia paddy virus real-time fluorescence quantitative PCR detection primer and the examination of probe described in any one of claims 1 to 3
Agent box.
Test kit the most according to claim 4, it is characterised in that the concentration of described forward primer, downstream primer and probe
It it is 10 μMs.
Test kit the most according to claim 5, it is characterised in that described test kit also includes PCR reaction buffer, fluorescence
Dyestuff, positive criteria product.
Test kit the most according to claim 6, it is characterised in that described positive criteria product are PMD19-T-3C.
Test kit the most according to claim 6, it is characterised in that described test kit carries out the reactant of quantitative fluorescent PCR
System is: Premix Ex Taq(Probe qPCR) (2 ×) 10 μ L, 10 μMs, 0.4 μ L forward primer, 10 μMs, 0.4 μ L downstream is drawn
Thing, ROX Reference Dye II(50 ×) 0.2 μ L, 10 μMs, 0.8 μ L probe, DNA profiling 2.0 μ L, ddH2O 6.2μL。
Test kit the most according to claim 6, it is characterised in that described test kit carries out the reaction bar of quantitative fluorescent PCR
Part is: 95 DEG C 30 seconds, 95 DEG C 5 seconds, 60 DEG C 34 seconds, 40 circulations.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107034313A (en) * | 2017-05-10 | 2017-08-11 | 广东温氏食品集团股份有限公司 | The RT PCR detection primers and RT PCR detection methods of a pair of Sai Nika paddy virus |
| CN107937617A (en) * | 2017-12-28 | 2018-04-20 | 广州维佰生物科技有限公司 | Detect the RT LAMP primer compositions thing and its kit and method of Sai Neijia paddy viruses |
| CN107937618A (en) * | 2017-12-29 | 2018-04-20 | 中国检验检疫科学研究院 | The droplet numeral RT PCR detection primers of A types Senecan virus and probe and its application |
| CN107955840A (en) * | 2017-12-13 | 2018-04-24 | 华南农业大学 | For detecting double PCR primer, detection method and the kit of swine foot-and-mouth disease virus and Sai Neijia paddy virus |
| CN108384893A (en) * | 2018-05-03 | 2018-08-10 | 中国农业科学院兰州兽医研究所 | Real-time fluorescence quantitative RT-PCR kit for detecting foot and mouth disease virus and Sai Nika paddy viruses and its application |
| CN109097495A (en) * | 2018-08-16 | 2018-12-28 | 金宇保灵生物药品有限公司 | Senecan virus and foot and mouth disease virus dual real-time fluorescence quantitative PCR detection kit |
| CN109234464A (en) * | 2018-11-23 | 2019-01-18 | 山东新希望六和集团有限公司 | For detecting primer and probe, the PCR kit for fluorescence quantitative and methods and applications of Seneca Valley virus |
| CN110093320A (en) * | 2019-03-27 | 2019-08-06 | 华南农业大学 | GD-SVA-2018 plants of pig Senecan virus and its application |
| CN110229933A (en) * | 2019-06-24 | 2019-09-13 | 派生特（福州）生物科技有限公司 | A kind of primer sets, kit and application for the viral RT-Nested PCR detection of pig card inside competition |
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| CN110527746A (en) * | 2019-07-30 | 2019-12-03 | 华南农业大学 | For detecting double PCR primer, detection method and the kit of pig Senecan paddy virus Yu 3 type of annulus virus |
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| CN107034313A (en) * | 2017-05-10 | 2017-08-11 | 广东温氏食品集团股份有限公司 | The RT PCR detection primers and RT PCR detection methods of a pair of Sai Nika paddy virus |
| CN107955840A (en) * | 2017-12-13 | 2018-04-24 | 华南农业大学 | For detecting double PCR primer, detection method and the kit of swine foot-and-mouth disease virus and Sai Neijia paddy virus |
| CN107937617A (en) * | 2017-12-28 | 2018-04-20 | 广州维佰生物科技有限公司 | Detect the RT LAMP primer compositions thing and its kit and method of Sai Neijia paddy viruses |
| CN107937618A (en) * | 2017-12-29 | 2018-04-20 | 中国检验检疫科学研究院 | The droplet numeral RT PCR detection primers of A types Senecan virus and probe and its application |
| CN110257554A (en) * | 2018-03-12 | 2019-09-20 | 金宇保灵生物药品有限公司 | The real-time fluorescence quantitative PCR detection kit and its primer special and TaqMan probe of Seneca Valley virus |
| CN108384893A (en) * | 2018-05-03 | 2018-08-10 | 中国农业科学院兰州兽医研究所 | Real-time fluorescence quantitative RT-PCR kit for detecting foot and mouth disease virus and Sai Nika paddy viruses and its application |
| CN109097495A (en) * | 2018-08-16 | 2018-12-28 | 金宇保灵生物药品有限公司 | Senecan virus and foot and mouth disease virus dual real-time fluorescence quantitative PCR detection kit |
| WO2020034317A1 (en) * | 2018-08-16 | 2020-02-20 | 金宇保灵生物药品有限公司 | Dual real-time fluorescent quantitative pcr detection reagent and reagent kit for seneca virus a and foot-and-mouth disease virus |
| CN109234464A (en) * | 2018-11-23 | 2019-01-18 | 山东新希望六和集团有限公司 | For detecting primer and probe, the PCR kit for fluorescence quantitative and methods and applications of Seneca Valley virus |
| CN110093320A (en) * | 2019-03-27 | 2019-08-06 | 华南农业大学 | GD-SVA-2018 plants of pig Senecan virus and its application |
| CN110229933A (en) * | 2019-06-24 | 2019-09-13 | 派生特（福州）生物科技有限公司 | A kind of primer sets, kit and application for the viral RT-Nested PCR detection of pig card inside competition |
| CN110229933B (en) * | 2019-06-24 | 2022-06-07 | 派生特（福州）生物科技有限公司 | Primer group and kit for nested RT-PCR (reverse transcription-polymerase chain reaction) detection of porcine Saikovia virus and application of primer group and kit |
| CN110527746A (en) * | 2019-07-30 | 2019-12-03 | 华南农业大学 | For detecting double PCR primer, detection method and the kit of pig Senecan paddy virus Yu 3 type of annulus virus |
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