CN110205405A - A kind of kit and primer and probe of detection and identification Seneca Valley virus, foot and mouth disease virus O, A and Asial type - Google Patents
A kind of kit and primer and probe of detection and identification Seneca Valley virus, foot and mouth disease virus O, A and Asial type Download PDFInfo
- Publication number
- CN110205405A CN110205405A CN201711420252.XA CN201711420252A CN110205405A CN 110205405 A CN110205405 A CN 110205405A CN 201711420252 A CN201711420252 A CN 201711420252A CN 110205405 A CN110205405 A CN 110205405A
- Authority
- CN
- China
- Prior art keywords
- mouth disease
- foot
- virus
- disease virus
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- 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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
Abstract
The invention discloses the kits and primer and probe of a kind of detection and identification Seneca Valley virus, foot and mouth disease virus O, A and Asial type.For detecting and identifying Seneca Valley virus, O-shaped foot and mouth disease virus, A type foot and mouth disease virus and Asial type foot and mouth disease virus, primer and probe sequence are as follows: Seneca Valley virus detection primer and probe sequence SEQ1, SEQ2 and SEQ3；O-shaped foot and mouth disease virus detection primer and probe sequence SEQ4, SEQ5 and SEQ6；A type foot and mouth disease virus detection primer and probe sequence SEQ7, SEQ8 and SEQ9；Asia1 type foot and mouth disease virus detection primer and probe sequence SEQ10, SEQ11 and SEQ12.Kit of the invention can carry out the detection and identification of Seneca Valley virus and foot and mouth disease virus O, A and Asial type simultaneously, simplify operating procedure, shorten detection time and improve accuracy in detection.
Description
Technical field
The present invention relates to a kind of specific primer that can be used for animal virus detection to and probe, and include specificity
The immue quantitative detection reagent box of primer pair and probe, exactly the present invention be it is a kind of can detect and identify simultaneously Seneca Valley virus,
Foot and mouth disease virus O, A and Asial type one-step method TapMan real-time fluorescence quantitative PCR detection kit.
Background technique
Seneca Valley virus (Seneca Valley virus, SVV) is the RNA virus of the non-segmented negative of single-stranded positive, is
The unique member that Picornaviridae (Picornaviridae) Seneca Valley virus belongs to.Since in Septembers, 2015, American States are more
Vesiculovirus epidemic situation occurs for the swinery in a area, and infection pig walks lamely, rubescent hot around coronary band and hoof wall, and ulcer venereal disease occurs
Become, the nose kiss of pig, coronet shape band portion is bubble sample lesion occur, by laboratory Pathogen identification, is excluded as aftosa, pig blister
The important exotic animals epidemic disease such as disease and vesicular stomatitis is finally diagnosed as Seneca Valley virus infection.Summer in 2015 is at me
There is the first report of the virus in Guangdong Province, state, it is subsequent in April, 2016 Hubei Province also there is the outburst of the virus, mainly face
Bed symptom is that hog snout is kissed, bubble sample lesion occurs in hoof, although Seneca Valley virus will not cause weight huge economic loss, by
The clinical symptoms shown after its infection are difficult to differentiate between with aftosa, so it is badly in need of establishing one kind quickly, sensitive and special
The differential diagnostic method of Sai Nika paddy and aftosa is distinguished, it is most important to the control of the disease.
Seneca Valley virus and foot and mouth disease virus (Foot-and-Mouth Disease are detected currently without identifying simultaneously
Virus, FMDV) O, A and Asial multiple real time fluorescence quantifying detection technique report, use this kit carry out Real
Time RT-PCR reaction can be carried out continuously Seneca Valley virus and the inspection of foot and mouth disease virus O, A and Asial type in same reaction tube
It surveys, easy to operate, sensibility is high, and can effectively prevent pollution.
Summary of the invention
In view of this, the present invention provides it is a kind of special, sensitive, quick and it is convenient and fast can be used for identifying detect Sai Nika paddy
The detection kit of the specific primer pair and probe of virus and foot and mouth disease virus O, A and Asial type.
The present invention solves above-mentioned technical problem by following technological means:
The present invention is a kind of for that can identify detection Seneca Valley virus and foot and mouth disease virus O, A and Asial type simultaneously
Primer pair and probe gene order are respectively as follows:
Seneca Valley virus detection primer and probe:
SEQ1:(SVV-F) 5 '-AGAATTTGGAAGCCATGCTCT-3 '
SEQ2:(SVV-R) 5 '-GAGCCAACATAGARACAGATTGC-3 '
SEQ3:(SVV-Probe) 5 '-TTCAAACCAGGAACACTACTCGAGA-3 '
(5′FAM,3′BHQ1)
O-shaped FMDV detection primer and probe:
SEQ4:(O/FMDV-F) 5 '-GAAGCAGCCTTGGACAACACC-3 '
SEQ5:(O/FMDV-R) 5 '-GTGTGGTGCCGTGTAGGGCA-3 '
SEQ6:(O/FMDV-Probe) 5 '-CACCAACCCAACGGCGTAYCAYAAGGC-3 '
(5′ROX,3′BHQ1)
A type FMDV detection primer and probe:
SEQ7:(A/FMDV-F) 5 '-ATTCGGGCCACGGAGATCCRC-3 '
SEQ8:(A/FMDV-R) 5 '-GTCTTGCGACGACACCTCC-3 '
SEQ9:(A/FMDV-Probe) 5 '-TTGTGCGCATGAAGCGCGCCGAGCT-3 '
(5′HEX,3′BHQ1)
Asia1 type FMDV detection primer and probe:
SEQ10:(Asia1/FMDV-F) 5 '-TGCCCACTTCCTTCAACTACGG-3 '
SEQ11:(Asia1/FMDV-R) 5 '-CAAGGGCCTGGGGCAGTATGT-3 '
SEQ12:(Asia1/FMDV-Probe) 5 '-CCATCACGGAGCTGTTGATCCGCATG-3 '
(5′CY5,3′BHQ1)
Wherein, Y C/T, R A/G.
The present invention is a kind of for that can detect the real-time of Seneca Valley virus and foot and mouth disease virus O, A and Asial type simultaneously
PCR kit for fluorescence quantitative, in addition to comprising above-mentioned primer and probe sequence further include:
2 × One Step RT-PCR Buffer III (Mixture containing dNTP, Mg2+), TaKaRa Ex Taq HS (5U/
μ L), PrimeScript RT Enzyme Mix II, the RNA without Seneca Valley virus and foot and mouth disease virus O, A and Asial type
Sample contains Seneca Valley virus and foot and mouth disease virus O, A and Asial type as negative sample, and as positive control
RNA sample.It is fixed in the real-time fluorescence that the concrete application present invention detects Seneca Valley virus and foot and mouth disease virus O, A and Asial type
When measuring PCR kit, PCR amplification condition are as follows: 42 DEG C of 10min, 95 DEG C of 2min, 1 circulation → 95 DEG C of 10sec, 60 DEG C of 45sec,
40 circulations, wherein fluorescence signal collection is placed on 60 DEG C of ends 45sec of each circulation.
Kit of the invention is a kind of kit of one-step method TapMan fluorescence quantitative PCR detection, reagent of the invention
It is using the end 5` in box application with fluorescent material, the end 3` has the TapMan probe progress fluorescence detection that substance is quenched, and works as spy
When needle is complete, the end 5` fluorescent material is gone out the restriction of substance by 3` end quenching, cannot issue fluorescence.And when TapMan probe is decomposed
Afterwards, the fluorescent material at the end 5` will separate out, issue fluorescence.Middle probe and template hybridization are reacted in PCR, extension is polymerase
Activity can decompose the fluorescence probe hybridized with template, and the fluorescent material that dissociates issues fluorescence.By glimmering in detection reaction system
Luminous intensity, so as to achieve the purpose that detect PCR product amplification amount.
Seneca Valley virus and foot and mouth disease virus O, A and Asial type one-step method are carried out simultaneously using kit of the present invention
TapMan fluorescence quantitative PCR detection, the advantage is that, be drawn according to Seneca Valley virus 3D gene two specificity of conserved regions design
Object and probe, respectively according to the VP1 gene conserved region of foot and mouth disease virus O, A and Asial type epidemic strain, design specific primer and
Probe carries out fluorescent quantitative PCR to tissue sample, is realized respectively to Sai Nika on the basis of respective viral RNA extracts
The quantitative detection of paddy virus and foot and mouth disease virus O, A and Asial type.Real Time RT- is carried out using this kit in PCR
PCR reaction can be carried out continuously in same reaction tube, easy to operate, and can effectively prevent pollution.
It is to Seneca Valley virus and foot and mouth disease virus O, A and Asial when being detected using kit of the invention
Type RNA directly carries out quantitative pcr amplification after extracting, while reaction system and reaction condition is optimized in the present invention, and can
The precision of detection is improved, one-step method TapMan fluorescent quantitation can directly carry out standard curve after the completion of amplification and quantitatively originate
Viral copy number, while its is simple and quick, is highly suitable for the application of field sample epidemiological survey.
Detailed description of the invention
The invention will be further described with reference to the accompanying drawings and examples.
Fig. 1: the gene of the 3D section containing Seneca Valley virus and foot and mouth disease virus O, A and Asial type VP1 gene order recombinate matter
Granzyme cuts identification electrophoretogram,
Wherein: M:5000bp DNA Marker
1) recombinant plasmid pMD-SVV-3D double digestion is identified
2) recombinant plasmid pMD-FMDV-OVP1 double digestion is identified.
3) recombinant plasmid pMD-FMDV-AVP1 double digestion is identified.
4) recombinant plasmid pMD-FMDV-AsialVP1 double digestion is identified.
Fig. 2: Seneca Valley virus one-step method TapMan fluorescence quantitative PCR detection standard curve, in which: ordinate represents Ct
Value.The copy number of abscissa representative sample.
Fig. 3: the O-shaped one-step method TapMan fluorescence quantitative PCR detection standard curve of foot and mouth disease virus, in which: ordinate represents
Ct value.The copy number of abscissa representative sample.
Fig. 4: foot and mouth disease virus A type one-step method TapMan fluorescence quantitative PCR detection standard curve, in which: ordinate represents
Ct value.The copy number of abscissa representative sample.
Fig. 5: foot and mouth disease virus Asial type one-step method TapMan fluorescence quantitative PCR detection standard curve, in which: ordinate
Represent Ct value.The copy number of abscissa representative sample.
Fig. 6: being the amplification curve of detection method specificity.
Specific embodiment
Below with reference to attached drawing, the present invention is described in detail, below in conjunction with specific example and Detailed description of the invention to the present invention
It is described further
A kind of detection kit that can detect and identify Seneca Valley virus, foot and mouth disease virus O, A and Asial type simultaneously,
It is used to detect and identify Seneca Valley virus, O-shaped foot and mouth disease virus, A type foot and mouth disease virus and Asial type foot and mouth disease virus.
Its primer and probe sequence are as follows:
Seneca Valley virus detection primer and probe sequence SEQ1, SEQ2 and SEQ3；
O-shaped foot and mouth disease virus detection primer and probe sequence SEQ4, SEQ5 and SEQ6；
A type foot and mouth disease virus detection primer and probe sequence SEQ7, SEQ8 and SEQ9；
Asia1 type foot and mouth disease virus detection primer and probe sequence SEQ10, SEQ11 and SEQ12.
For detecting the end the 5' fluorescent marker FAM of the probe of Seneca Valley virus, the end 3' quenched label BHQ1；
The end the 5' fluorescent marker ROX, the end 3' quenched label BHQ1 for being used to detect O-shaped foot and mouth disease virus；
The end the 5' fluorescent marker HEX for being used to detect A type foot and mouth disease virus, 3' end quenching go out
Mark BHQ1；
The end the 5' fluorescent marker CY5, the end 3' quenched label BHQ1 for being used to detect Asial type foot and mouth disease virus.
It include one-step method fluorescence quantitative RT-PCR primer and probe in the kit.
Be provided in the kit: 2 × One Step RT-PCR Buffer III, TaKaRa Ex Taq HS,
PrimeScript RT Enzyme Mix II, without Seneca Valley virus, O-shaped foot and mouth disease virus, A type foot and mouth disease virus and
The RNA sample of Asial type foot and mouth disease virus contains Seneca Valley virus, O-shaped foot and mouth disease virus, A type respectively as negative control
The RNA sample of foot and mouth disease virus and Asial type foot and mouth disease virus is as positive control.
The kit RT-PCR condition are as follows: 42 DEG C of -10min, 95 DEG C of -2min, 1 circulation be 95 DEG C of -10sec, 60 DEG C -
45sec, 40 circulations, wherein fluorescence signal collection is placed on 60 DEG C of ends -45sec of each circulation.
Embodiment 1
1. the design and synthesis of Seneca Valley virus and foot and mouth disease virus O, A and Asial type fluorescence quantification PCR primer:
Utilize known Seneca Valley virus 3D gene order and foot and mouth disease virus O, A and Asial type VP1 gene order ratio
To rear, Primer Express3.0 software Design primers and probe are utilized according to conservative region, precious bioengineering (Dalian) is sent to have
The synthesis of limit company.Upstream and downstream primer and probe are respectively as follows:
Seneca Valley virus detection primer and probe:
SEQ1:(SVV-F) 5 '-AGAATTTGGAAGCCATGCTCT-3 '
SEQ2:(SVV-R) 5 '-GAGCCAACATAGARACAGATTGC-3 '
SEQ3:(SVV-Probe) 5 '-TTCAAACCAGGAACACTACTCGAGA-3 ' (5 ' FAM, 3 ' BHQ1)
O-shaped FMDV detection primer and probe:
SEQ4:(O/FMDV-F) 5 '-GAAGCAGCCTTGGACAACACC-3 '
SEQ5:(O/FMDV-R) 5 '-GTGTGGTGCCGTGTAGGGCA-3 '
SEQ6:(O/FMDV-Probe) 5 '-CACCAACCCAACGGCGTAYCAYAAGGC-3 '
(5′ROX,3′BHQ1)
A type FMDV detection primer and probe:
SEQ7:(A/FMDV-F) 5 '-TCGGGCCACGGAGATCCRC-3 '
SEQ8:(A/FMDV-R) 5 '-GTCTTGCGACGACACCTCC-3 '
SEQ9:(A/FMDV-Probe) 5 '-TTGTGCGCATGAAGCGCGCCGAGCTC-3 '
(5′HEX,3′BHQ1)
Asia1 type FMDV detection primer and probe:
SEQ10:(Asia1/FMDV-F) 5 '-TGCCCACTTCCTTCAACTACGG-3 ' SEQ11:(Asia1/FMDV-R)
5 '-CAAGGGCCTGGGGCAGTATGT-3 ' SEQ12:(Asia1/FMDV-Probe) 5 '-
CCATCACGGAGCTGTTGATCCGCATG-3’
(5′CY5,3′BHQ1)
Wherein, Y C/T, R A/G.
2. the preparation of standard items
(1) amplification of Seneca Valley virus 3D gene and VP 1 Gene of Foot-and-Mouth Disease virus and clone identification
With reference to the sequence that Seneca Valley virus and foot and mouth disease virus O, A and Asial type log on NCBI, divide by comparing
After analysis, the upstream and downstream primer that can expand Seneca Valley virus the 3D section gene and VP 1 Gene of Foot-and-Mouth Disease virus sequence is designed, is sent
Precious bioengineering (Dalian) Co., Ltd synthesis.The upstream and downstream primer of amplification Seneca Valley virus 3D gene is respectively as follows: SEQ13:
(SVV-3DF) 5 '-TGTCGATGTGCCCTCCCTGGC-3 ' SEQ14:(SVV-3DR) 5 '-TCCCAGAATCGCCGGCGGCACC-
3'.The amplification general upstream and downstream primer of VP 1 Gene of Foot-and-Mouth Disease virus is respectively as follows: SEQ15:(FMDV-VP1F) 5 '-
GCGCTGGCAAAGACTTTGA-3 ' SEQ16:(FMDV-VP1R) 5 '-GACATGTCCTCCTGCATCTGGTTGA-3 '.
Appropriate virus liquid is taken, the RNA of virus is extracted according to the method that Trizol extracts RNA, then uses TaKaRa company
One step RT-PCR kit Prime Script One Step RT-PCR Kit is operated as follows:
RT-PCR amplification, which is carried out, as template using RNA extract prepares 25 μ l systems,
It mixes, in the automatic amplification instrument of low-speed centrifugal postposition PCR, expands according to the procedure below: 50 DEG C of 30min, 94 DEG C of 2min
One circulation → 94 DEG C of 30sec, 57 DEG C of 30sec, 72 DEG C of 45sec 35 circulations.PCR product is pure with 1% agarose gel electrophoresis
Change recycling, connect overnight using T4 ligase with 16 DEG C of pMD-20-T Vector, converts DH5 α competent cell, choose monoclonal
Spot is in LB (Amp+) in expand culture, with plasmid extraction kit purify plasmid pMD-FMDV-VP1 and pMD-SVV-3D, pass through
SpeI/EcoRI digestion identification, determines positive plasmid, sees attached drawing 1.
(2) it is transcribed in vitro and obtains standard items RNA
Using the plasmid of above-mentioned linearisation as template, purpose RNA is obtained using external reverse transcription reagent box.Reaction system are as follows:
10 × T7RNA polymerase buffer, 24 μ L, RNase inhibitor of μ L, DTT2 μ L, DNTP Mixture, 0.5 μ
1 μ L, DEPC H of L, Template DNA0.5 μ g, T7RNA polymerase220 μ L of O to, reaction condition: after 37 DEG C of 1h,
The RNA that appropriate DEPC water dissolution is finally obtained according to the extraction and purification step of RNA, freezes after measuring concentration in -80 DEG C.It utilizes
Formula :=[(number × 10 ng (copies/ μ L)-9)×(6.02×1023)] ÷ (base number × 340) converses copying for purpose RNA
Shellfish number.
3. the foundation of standard curve
According to the copy number obtained after quantitative, standard sample is made to the examination criteria product of equal 6 gradient dilutions, for reduction
Error improves the repeatability of experiment, and each dilution sample is designed with three repeating holes, determines Ct value (Ct i.e. cycle
Threshold refers to the recurring number experienced when the fluorescence signal in reaction tube reaches the threshold value of setting), so that it is determined that amplification
The best standard curve of effect.
Amplification system is 25 μ L, wherein including 2 × One Step RT-PCR Buffer III (Mixture containing dNTP, Mg2
+), TaKaRa Ex Taq HS (5U/ μ L), PrimeScript RT Enzyme Mix II, RNase Free dH2O, upstream draw
Object, downstream primer, probe and the 2 μ L of standard items diluted.PCR amplification uses Agilent Techologies-
Mx3005pPCR amplification instrument, program are as follows: 42 DEG C of 10min, 95 DEG C of 2min, 1 circulation → 95 DEG C of 10sec, 60 DEG C of 45sec, 40
Circulation, wherein fluorescence signal collection is placed on 60 DEG C of ends 45sec of each circulation.The analysis of PCR fluorescence signal is prompt using peace
Human relations company MxPro software, Buddhist nun blocks paddy virus and the standard of the obtained quantitative fluorescent PCR of foot and mouth disease virus O, A and Asial type is bent
Line is shown in attached drawing 2,3,4 and 5 respectively.
4. the detection of specificity
With the common epidemic disease cause of disease RNA of following pig: Porcine epidemic diarrhea virus, transmissible gastroenteritis of swine, pig indigo plant youngster virus, pig
Pseudorabies virus and swine foot-and-mouth disease virus, detect the specificity of built cube method, and obtained fluorescent quantitative PCR result is shown in attached drawing
6.What this method can only be specific as the result is shown amplifies Seneca Valley virus and foot and mouth disease virus O, A and Asial type RNA positive
Control, specificity are good.
5. the detection of sensibility
Seneca Valley virus and foot and mouth disease virus O, A and Asial type standard sample are respectively prepared 1 × 107-1×100
(copies/ μ L) 8 dilutions, detect the sensibility of built cube method, and to ensure the accuracy detected, high pressure sterilization water is arranged
For background values control, the sample for playing peak that≤32 recycle is determined as the positive, this fluorescence quantitative detecting method can be detected accurately
Cause of disease to more than 10 copies, and regular-PCR intelligent measurement is to 1 × 104It is more than a copy, it can be seen that the present invention detection and
Identify Seneca Valley virus, the detection kit sensibility of quantitative fluorescent PCR of foot and mouth disease virus O, A and Asial type is common
The 10 of PCR3Times, sensibility is good.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with
Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the scope of the claims of invention.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>a kind of detection and identification Seneca Valley virus, the kit of foot and mouth disease virus O, A and Asial type and primer and spy
Needle
<160> 0
<170> SIPOSequenceListing 1.0
Claims (6)
1. the kit of a kind of detection and identification Seneca Valley virus, foot and mouth disease virus O, A and Asial type, it is characterised in that: use
In detection and identify Seneca Valley virus, O-shaped foot and mouth disease virus, A type foot and mouth disease virus and Asial type foot and mouth disease virus.
2. a kind of detection according to claim 1 and identification Seneca Valley virus, foot and mouth disease virus O, A and Asial type
The primer and probe of kit, which is characterized in that its primer and probe sequence are as follows:
Seneca Valley virus detection primer and probe sequence SEQ1, SEQ2 and SEQ3；
O-shaped foot and mouth disease virus detection primer and probe sequence SEQ4, SEQ5 and SEQ6；
A type foot and mouth disease virus detection primer and probe sequence SEQ7, SEQ8 and SEQ9；
Asia1 type foot and mouth disease virus detection primer and probe sequence SEQ10, SEQ11 and SEQ12.
3. a kind of detection according to claim 2 and identification Seneca Valley virus, foot and mouth disease virus O, A and Asial type
The primer and probe of kit, it is characterised in that: for detecting the end the 5' fluorescent marker FAM of the probe of Seneca Valley virus, 3'
Hold quenched label BHQ1；
The end the 5' fluorescent marker ROX, the end 3' quenched label BHQ1 for being used to detect O-shaped foot and mouth disease virus；
The end the 5' fluorescent marker HEX, the end 3' quenched label BHQ1 for being used to detect A type foot and mouth disease virus；
The end the 5' fluorescent marker CY5, the end 3' quenched label BHQ1 for being used to detect Asial type foot and mouth disease virus.
4. a kind of detection according to claim 1 and identification Seneca Valley virus, foot and mouth disease virus O, A and Asial type
Kit, it is characterised in that: include one-step method fluorescence quantitative RT-PCR primer and probe in the kit.
5. a kind of detection according to claim 4 and identification Seneca Valley virus, foot and mouth disease virus O, A and Asial type
Kit, which is characterized in that be provided in the kit: 2 × One Step RT-PCR Buffer III, TaKaRa Ex
Taq HS, PrimeScript RT Enzyme Mix II are free of Seneca Valley virus, O-shaped foot and mouth disease virus, A type hoof-and-mouth disease
The RNA sample of poison and Asial type foot and mouth disease virus contains Seneca Valley virus, O-shaped hoof-and-mouth disease respectively as negative control
Poison, A type foot and mouth disease virus and the RNA sample of Asial type foot and mouth disease virus are as positive control.
6. a kind of detection according to claim 5 and identification Seneca Valley virus, foot and mouth disease virus O, A and Asial type
Kit, which is characterized in that the kit RT-PCR condition are as follows: 42 DEG C of -10min, 95 DEG C of -2min, 1 circulation for 95 DEG C -
10sec, 60 DEG C of -45sec, 40 circulations, wherein fluorescence signal collection is placed on 60 DEG C of ends -45sec of each circulation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711420252.XA CN110205405B (en) | 2017-12-25 | 2017-12-25 | Kit, primer and probe for detecting and identifying O, A and Asial types of Seneca Valley virus, foot-and-mouth disease virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711420252.XA CN110205405B (en) | 2017-12-25 | 2017-12-25 | Kit, primer and probe for detecting and identifying O, A and Asial types of Seneca Valley virus, foot-and-mouth disease virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110205405A true CN110205405A (en) | 2019-09-06 |
| CN110205405B CN110205405B (en) | 2022-09-23 |
Family
ID=67778635
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711420252.XA Active CN110205405B (en) | 2017-12-25 | 2017-12-25 | Kit, primer and probe for detecting and identifying O, A and Asial types of Seneca Valley virus, foot-and-mouth disease virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110205405B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110747293A (en) * | 2019-12-05 | 2020-02-04 | 安阳工学院 | Triple fluorescence RT-PCR detection kit for identifying swine vesicular disease virus, foot and mouth disease virus and seneca valley virus |
| CN110964856A (en) * | 2019-12-23 | 2020-04-07 | 广西壮族自治区动物疫病预防控制中心 | Multiplex fluorescence quantitative RT-PCR kit for simultaneously detecting SVA and FMDV and detection method |
| CN114164299A (en) * | 2021-05-26 | 2022-03-11 | 郑州中道生物技术有限公司 | Foot-and-mouth disease (OAA 1) triple fluorescence RT-PCR detection kit |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695628A (en) * | 2016-03-07 | 2016-06-22 | 华南农业大学 | HRM detecting primers and method for distinguishing foot-mouth disease virus and Seneca Valley virus |
| CN107184969A (en) * | 2017-04-18 | 2017-09-22 | 中农威特生物科技股份有限公司 | A kind of A types Sai Nika paddy viral inactivation vaccines and its preparation method and application |
| WO2017181070A1 (en) * | 2016-04-15 | 2017-10-19 | Kansas State University Research Foundation | Vaccine against seneca valley virus |
| CN107326100A (en) * | 2017-07-17 | 2017-11-07 | 河南省动物疫病预防控制中心 | Aftosa and the double real-time fluorescence quantitative PCR detection kit of Sai Nika paddy virus |
-
2017
- 2017-12-25 CN CN201711420252.XA patent/CN110205405B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695628A (en) * | 2016-03-07 | 2016-06-22 | 华南农业大学 | HRM detecting primers and method for distinguishing foot-mouth disease virus and Seneca Valley virus |
| WO2017181070A1 (en) * | 2016-04-15 | 2017-10-19 | Kansas State University Research Foundation | Vaccine against seneca valley virus |
| CN107184969A (en) * | 2017-04-18 | 2017-09-22 | 中农威特生物科技股份有限公司 | A kind of A types Sai Nika paddy viral inactivation vaccines and its preparation method and application |
| CN107326100A (en) * | 2017-07-17 | 2017-11-07 | 河南省动物疫病预防控制中心 | Aftosa and the double real-time fluorescence quantitative PCR detection kit of Sai Nika paddy virus |
Non-Patent Citations (2)
| Title |
|---|
| VERONICA L.FOWLER等: "Development of a novel real-time RT-PCR assay to detect Seneca Valley virus-1 associated with emerging cases of vesicular disease in pigs", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| 樊晓旭等: "塞尼卡谷病毒TaqMan荧光定量PCR检测方法的建立", 《中国预防兽医学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110747293A (en) * | 2019-12-05 | 2020-02-04 | 安阳工学院 | Triple fluorescence RT-PCR detection kit for identifying swine vesicular disease virus, foot and mouth disease virus and seneca valley virus |
| CN110747293B (en) * | 2019-12-05 | 2022-12-23 | 安阳工学院 | Triple fluorescence RT-PCR detection kit for identifying swine vesicular disease virus, foot and mouth disease virus and seneca valley virus |
| CN110964856A (en) * | 2019-12-23 | 2020-04-07 | 广西壮族自治区动物疫病预防控制中心 | Multiplex fluorescence quantitative RT-PCR kit for simultaneously detecting SVA and FMDV and detection method |
| CN114164299A (en) * | 2021-05-26 | 2022-03-11 | 郑州中道生物技术有限公司 | Foot-and-mouth disease (OAA 1) triple fluorescence RT-PCR detection kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110205405B (en) | 2022-09-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107513584B (en) | A kind of five heavy fluorescence quantitative kits detecting enterovirus | |
| CN106947838B (en) | African swine fever virus non-structural gene real-time fluorescence LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method | |
| CN107299155A (en) | A kind of primer and probe of goose astrovirus real-time fluorescence quantitative PCR detection | |
| CN107326100A (en) | Aftosa and the double real-time fluorescence quantitative PCR detection kit of Sai Nika paddy virus | |
| CN103045755B (en) | A kind of fluorescent quantitative PCR detection method detecting Ebola virus and primer thereof and test kit | |
| CN105695634A (en) | PCR primer for detecting African swine fever virus, kit and application thereof | |
| CN110273027A (en) | Norovirus G I, G II and IV type nucleic acid parting detecting reagent of G and detection method | |
| CN108504778B (en) | Kit that is a kind of while detecting porcine circovirus 2 type and porcine pseudorabies virus and application | |
| CN109097495A (en) | Senecan virus and foot and mouth disease virus dual real-time fluorescence quantitative PCR detection kit | |
| CN107034309A (en) | The real-time fluorescence RPA kits of quick detection PRV, test strips RPA kits and application thereof | |
| CN110205405A (en) | A kind of kit and primer and probe of detection and identification Seneca Valley virus, foot and mouth disease virus O, A and Asial type | |
| CN103602757A (en) | Development and application of multiple fluorescence RT-PCR detection method for foot-and-mouth disease, vesicular stomatitis and swine vesicular disease | |
| CN106435033A (en) | Dual real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting and identifying wild strain and vaccine strain of CSFV (classical swine fever virus) in swine umbilical cord blood and application of dual real-time fluorescence RT-PCR kit | |
| CN105603123A (en) | Real-time fluorescence RPA reagent kit and test strip RPA reagent kit for rapidly detecting porcine parvovirus and application of reagent kits | |
| CN105624329A (en) | Real-time fluorescence nucleic acid isothermal amplification detection kit for human herpesvirus 1 | |
| CN108034761A (en) | A kind of reagent, method and application differentiated for FMDV and SVA | |
| CN107937618A (en) | The droplet numeral RT PCR detection primers of A types Senecan virus and probe and its application | |
| CN101979667A (en) | Fluorescence quantitative PCR kit for synchronously detecting herpes simplex virus I and II | |
| CN103725793A (en) | Multiple fluorescent quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) method for detecting PRRSV (porcine reproductive and respiratory syndrome virus) and application thereof | |
| CN102071263B (en) | Nested fluorescence reverse transcription-polymerase chain reaction (RT-PCR) detection reagent for avian influenza virus (AIV) H5 subtype and detection kit | |
| CN110438260A (en) | A kind of African swine fever virus nucleic acid test strips detection kit | |
| CN103225000B (en) | Bird flu H7N9 virus detection reagents and detection kit | |
| CN107974514A (en) | A kind of reagent, detection method and application for pig A type Senecan viral diagnosis | |
| CN101724712A (en) | Animal insect-borne disease multi-RT-PCR distinguishing and detecting reagent as well as preparation method and application | |
| CN110305986A (en) | SVA, the triple real-time fluorescence quantitative PCR detection primers of one-step method of O-shaped FMDV and A type FMDV and probe |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |