CN105861755A - Specific primer set for fast detecting South African type foot and mouth disease viruses and kit comprising same - Google Patents
Specific primer set for fast detecting South African type foot and mouth disease viruses and kit comprising same Download PDFInfo
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Abstract
The invention discloses a specific primer set for fast detecting South African type foot and mouth disease viruses. The specific primer set is composed of one downstream primer and seven upstream primers, the downstream primer is a single-stranded DNA molecule shown in the sequence 1 in a sequence table, and the upstream primers are single-stranded DNA molecules shown from the sequence 2 to the sequence 8 in the sequence table. The invention further provides a kit comprising the primer set. The specific primer set has the advantages that the specific primer set is used for detecting viral nucleic acid of the SAT FMDVs at the molecular level with the assistance of a series of quality control and negative and positive contrast. The method has the characteristics of being simple, economical, fast, sensitive and specific, is a detection technology capable of detecting three SAT FMDVs at the same time, achieves strategic reserves of diagnosing and monitoring the SAT FMDVs, has important strategic significance in preventing the viruses from being introduced into China and has good application prospects.
Description
Technical field
The present invention relates to foot and mouth disease virus detection technique field, be particularly used for quickly detecting South Africa type foot and mouth disease virus SAT
The specific primer group of FMDV and include the kit of this primer sets.
Background technology
Aftosa (Foot and Mouth Disease, FMD) is a kind of by foot and mouth disease virus (Foot and Mouth Disease
Virus, FMDV) the harm serious acute febrile high degree in contact sexually transmitted disease artiodactylous such as ox, pig, sheep that causes.With
Heating, lip, mucous membrane of mouth, breast and hoof occur that speck or blister venereal disease reason are changed to typical Clinical symptoms.Infect mouth
Fever aphthous not only results in animal production and significantly declines, and has had a strong impact on listing and the outlet of Infected regions livestock products.
Owing to its spread speed is fast, wayward and elimination, serious harm world animal trade, it is classified as on necessary by the World Health Organization
One of report infectious disease.
Foot and mouth disease virus belongs to the Hostis (Aphthovirus) in Picornaviridae (Picornaviridae).
Including A, O, C, Asia 1 (Type Asia 1), SAT 1 (South Africa 1 type), SAT 2 (South Africa 2 type) and SAT 3 (south
Non-3 types) 7 serotypes, without cross-protection between each serotype.The virion diameter 20-25nm of FMDV, for just
Icosahedral structure of virus, sub-circular；Genome is total length 8, the positive chain RNA of 500nt, including an ORFs (open
Reading frame, ORF), 5 '-noncoding region (5 '-Untraslated Region, 5 '-UTR) and 3 '-noncoding region
(3 '-Untraslated Region, 3 '-UTR)；ORF encodes viral polyprotein, depend on self coding protease (L,
2A, 3C) and the host factor of minority, after 3 grades crack, formed 4 kinds of virus structural proteins (VP4, VP2,
VP3 and VP1) and 9 kinds of non-structural proteins (Lab, Lb, 2A, 2B, 2C, 3A, 3B, 3C and 3D)；Four kinds of knots
The each 60 molecular composition virions of structure albumen, the close duck eye being made up of virus surface five former grains of VP1, VP2 and VP3
Being positioned at far-end, VP4 is then fully located at inside capsid.
According to the homology between these 7 serotypes, FMDV is divided into 2 groups, group 1 (Eurasian type) comprise A, O,
C and Asia1 type；Group's 2 (South Africa types) comprise SAT1, SAT2 and SAT3 type.By South Africa type foot and mouth disease virus ((Southern
African Territories Foot and Mouth Disease Virus, SAT FMDV) the South Africa type aftosa that causes initially main office
It is limited to area, sub-Saharan Africa, has obvious geographic limitations, but due to trade and the fast development of personnel transfer,
SAT2 reaches the countries such as Pakistani from Sub-Sahara through north African.Southwest China connects with Pakistan, it is impossible to row
Possibility except the incoming China of SAT FMDV.
Cloven-hoofed economic animal is threatened by aftosa it is known that the economic loss that its morbidity causes is the most still all kinds of infections
First of disease.In order to determine whether FMDV exists in a certain area, had with the existence making a definite diagnosis FMDV by pathogeny detection
Significance.General detection method includes that the molecular biology etc. of virus purification, virus neutralization experiment and amplicon virus nucleic acid is examined
Disconnected technology.Although virus purification is considered as the goldstandard of detection FMDV, but it is due to time-consuming long, costly, there is secondary
Dissipating the hidden danger of poison, general basic unit is difficult to carry out this work, and same virus neutralizes experiment and there is also similar drawback.Amplicon virus
The molecular biology method of nucleic acid has become as the main method of diagnosis cause of disease now.South Africa type aftosa was not broken out in China
Cross, diagnostic techniques and weak, thus, it is necessary to set up operation quick, easy, easy, low cost, be especially suitable for not
Carry out the viral nucleic acid detection method in basic unit's veterinary laboratories application.Classical reverse transcriptional PCR (Reverse
Transcription Polymerase Chain Reaction, RT-PCR) just meet above-mentioned requirements.Due to SAT FMDV, especially
It is SAT2, and topological type is numerous, the biggest for the VP1 protein-coding region gene 1D degree of variation of type diagnosis, even if
Set up classical RT-PCR method and there is also great difficulty, or type FMDV Eurasian with group 1 exists cross reactivity, special
Property is relatively low, or is exactly that existence is failed to pinpoint a disease in diagnosis, and sensitiveness is the most on the low side.Once designed SAT-1D209F/FMDV-2B208R to draw such as Reid
Thing is to detecting SAT FMDV (Reid S.M., Ferris N.P., Hutchings G.H., et al.Primary diagnosis of
foot-and-mouth disease by reverse transcription polymerase chain reaction.Journal of Virological
Methods, 2000,89:167-176.), result can only amplify the purpose product of 720bp, SAT3 for SAT1 type and SAT2
Amplified production utilize gel electrophoresis detected at all less than, just can only be detected by the method for PCR-ELISA；Callens
Et al. the most once share downstream primer (P1) and compound upstream primer to detect SAT1 (P126, P151-153), SAT2 (P168-170)
With SAT3 (P130, P158-161) (Callens M., Clercq K.D.Differentiation of the seven serotypes of
foot-and-mouth disease virus by reverse transcriptase polymerase chain reaction.Journal of
Virological Methods, 1997,67:35-44.), but as easy as rolling off a log between 3 serotypes cross reaction occurs.Although abroad learning
Person has done corresponding research, but owing to there is drawbacks described above, so, current state, inside and outside also there is no corresponding SAT FMDV
Diagnostic kit comes out.So, it is established that the RT-PCR detection method of SAT FMDV also develops corresponding diagnostic reagent
Box has important strategic importance to the incoming China of prevention and control SAT FMD.
Summary of the invention
The purpose of the present invention is aiming at above-mentioned defect of the prior art, it is provided that for quickly detecting the spy of SAT FMDV
Specific primer group and include the kit of this primer sets.
To achieve these goals, the technical scheme that the present invention provides is: one group for quickly detecting the special of SAT FMDV
Property primer sets, described specific primer group is made up of 1 downstream primer and 7 upstream primers；Described downstream primer is sequence table
Single strand dna shown in middle sequence 1, described upstream primer is single strand dna shown in sequence 2-8 in sequence table, and this is multiple
Mould assembly primer sets is named as SAT-D8.
Article 8, special primer is made up of 1 downstream primer and 7 upstream primers:
P296-R:5-ACYTTTACCAGGGYTTGGC-3；
P280-F:5-GGCGTTGAGAAACAACTGTG-3；
P281-F:5-GGCGTTGAGAAACAGCTGTG-3；
P282-F:5-GGTGTCGAAAAACAGTTGTG-3；
P283-F:5-GGTGTCGAAAAACAGCTGTG-3；
P310-F:5-GGCGTCGAAAGACAGACCCT-3；
P329-F:5-GCACCAGCCAAACAACTGTG-3；
P331-F:5-AGTGCTGACAAGCAGATGTG-3。
Second object of the present invention there is provided above-mentioned specific primer group in preparation for quickly detecting the examination of SAT FMDV
Application in agent box.
Third object of the present invention there is provided the kit for quickly detecting SAT FMDV, includes above-mentioned special
Property primer sets.
It is contemplated that with synthesis gene order as template, it is established that SAT FMDV conventional RT-PCR diagnostic method, draw
Thing is designed as setting up the key of RT-PCR method, owing to VP1 protein-coding region gene 1D is by depending on of FMDV parting
According to, again because the 1D genetic mutation degree of SAT FMDV is big (70%-85%), so, composite primer is the optimum of this experiment
Select.By analyzing and comparing, have chosen the 1D gene 3 ' end target region as upstream primer design, downstream primer is conservative
The preferable 2B district of property.Being detected by experiment, finishing screen is selected a set of by answering that 7 upstream primers and 1 downstream primer form
Close primer, named SAT-D 8 primer.
SAT FMD is not had in history, in order to ensure biological safety and meet experiment needs, in comparative analysis due to China
On the basis of SAT FMDV nucleic acid sequence homology, pick out 8 SAT1 types, 10 SAT2 types and 3 SAT3 types
Represent strain, synthesize the 1D2A2B gene of those strains, and be connected with PUC57 cloning vector, construct corresponding restructuring
Plasmid.With those recombinant plasmids as template, with SAT-D8 as primer, on DNA level, first establish FMDV
SAT-D8RT-PCR detection method.With above-mentioned 21 plasmids as template, can specifically amplify the purpose bar of 257bp
Band.The 1D2A2B gene RNA transcript of 21 strains is prepared further, as template, also by in-vitro transcription method
Special purpose band can be amplified.The method and A, O, C and AsiaI type FMDV and bovine ephemeral diarrhoeal diseases
Poison (BVDV), sheep of virus (ORFV), goat capripoxvirus GTPV, sheep pox virus SPPV, swine vesicular disease virus
(SVDV), blue tongue virus (BTV), american type highly pathogenic PRRSV
(NA-HP-PRRSV), the non-highly pathogenic PRRSV of american type (NA-Non-HP-PRRSV),
Classic CSFV (CSFV), PRV (PRV), pig parvoviral (PPV) and pig circular ring virus (PCV)
Deng cross reaction does not occur.With plasmid as template, the sensitivity of FMDV SAT-D8RT-PCR method can reach 102Copy
Number/microlitre, with rna transcription originally as template, sensitivity is 103-104Copy number/microlitre.
With from the SAT1/2/3 inactivation antigen of aftosa OIE reference laboratory (Britain Pirbright laboratory) and 125 parts
It is derived from the heart of China's cattle,pig and sheep, liver, spleen, lung, kidney, lingual surface tissue and serum as measuring samples, after extracting its RNA,
Carry out SAT-D8RT-PCR experiment.Result shows, with SAT1/2/3RNA as template, amplifies anticipated SAT
FMDV positive band.The further sequence analysis of positive band is shown to be corresponding SAT1/2/3 type.Other 125 parts of fields
It is negative that sample detection result is all South Africa type virus.Establishing FMDV SAT-D8 conventional RT-PCR detection method basis
On, assemble corresponding kit.
The invention have the benefit that the specific primer group for quickly detection SAT FMDV that the present invention provides and comprise
There is the kit of this primer sets, by using specific composite primer and in a series of Quality Controls and negative, the auxiliary of positive control
Under, from molecular level, the viral nucleic acid including SAT FMDV is detected, the method have simplicity, economy, quickly,
Sensitive and special feature, it is achieved that can simultaneously detect the conventional RT-PCR detection technique of tri-serotypes of SAT1/2/3,
The diagnosis to SAT FMDV and the strategic reserves of monitoring can be completed, for preventing its incoming China to have important strategy meaning
Justice, has a good application prospect simultaneously.
Accompanying drawing explanation
Fig. 1 is that the SAT FMDV using the detection synthesis of FMDV SAT-D8RT-PCR method represents strain nucleic acid (to contain
The recombinant plasmid of 1D2A2B genetic fragment be amplification template) agarose gel electrophoretogram.Result shows to go out at 257bp
Having showed specific band sizable with estimated molecular weight, negative control is (with ddH2O is template) occur without band.
Wherein, M:DL2000 molecular weight standard (2000,1000,750,500,250,100bp)；P:PUC57 plasmid；1:SAT1
(AY593839)；2:SAT1(AY593840)；3:SAT1(AY593841)；4:SAT1(AY593846)；
5:SAT(AY593838)；6:SAT1(HM067706)；7:SAT1 (JF749860)；8:SAT1(AY593842)；
9:SAT2 (JF749864)；10:SAT2 (AF540910)；11:SAT2(AY593848)；12:SAT2 (JF749861)；
13:SAT2 (JF749862)；14:SAT2 (A593847)；15:SAT2 (HM067705)；16:SAT2 (AJ251473)；
17:SAT2 (JHM067704)；18:SAT2 (KC440884)；19:SAT3 (AY593850)；20:SAT3 (AY593852)；
21:SAT3 (AY593853)；W: negative control.
Fig. 2 is the pcr amplification product agarose gel electrophoresis figure of the 1D2A2B gene of the FMDV SAT1/2/3 containing T7 promoter
Spectrum.For the carrier sequence design primer being connected mutually with genes of interest two ends, add T7 promoter at downstream primer end, with
Obtain the genes of interest sequence containing T7 promoter.
Wherein, M:DL2000 Relative molecular weight markers；1:SAT1(AY593839)；2:SAT1(AY593840)；3:SAT1
(AY593841)；4:SAT1(AY593846)；5:SAT1(AY593838)；6:SAT1(HM067706)；
7:SAT1 (JF749860)；8:SAT1(AY593842)；9:SAT2 (JF749864)；10:SAT2 (AF540910)；
11:SAT2(AY593848)；12:SAT2 (JF749861)；13:SAT2 (JF749862)；14:SAT2 (A593847)；
15:SAT2 (HM067705)；16:SAT2 (AJ251473)；17:SAT2 (JHM067704)；18:SAT2 (KC440884)；
19:SAT3 (AY593850)；20:SAT3 (AY593852)；21:SAT3 (AY593853)；W: negative right
According to (with ddH2O is template).
Fig. 3 is FMDV SAT2 (JF749864) transcription product electrophoresis pattern after purification.
Wherein, M1:DNA DL2000；M2:RNA 1000maker；1: reclaiming product with SAT2 (JF749864) T7 glue is
The transcription product of template；2: the direct transcription product as template with SAT2 (JF749864) T7PCR product；3: do not add and transcribe
SAT2 (JF749864) transcription product of enzyme；SAT2 (JF749864) transcription product that 4:RNase processes.
Fig. 4 is FMDV SAT1/2/3 transcription product electrophoresis pattern after purification.
Wherein, 1:SAT1 (AY593846) transcription product；2:SAT2 (JF749862) transcription product；3:SAT3 (AY593852)
Transcription product.
Fig. 5 is with plasmid as template, carries out the assay sensitivity testing result of FMDV SAT-D8RT-PCR method.With multiple proportions system
The recombinant plasmid of the row dilution 1D2A2B genetic fragment containing SAT FMDV is template, carries out FMDV SAT-D8RT-PCR
Detection, agarose gel electrophoresis result shows that the sensitivity of the method is 4 × 102copies/μL。
Wherein, (A) SAT1 type represents strain (AY593846) sensitivity technique result；(B) SAT2 type represents strain (JF749862)
Sensitivity technique result；(C) SAT3 type represents strain (AY593852) sensitivity technique result；1-9 swimming lane is with plasmid
SAT1/2/3 is template, and copy number is followed successively by 4 × 108-4×100copies/μL；10 swimming lanes are negative controls.With plasmid as template
Sensitivity be 4 × 102copies/μL。
Fig. 6 be with synthesis SAT FMDV 1D2A2B gene rna transcription this as template, carry out FMDV SAT-D8
The assay sensitivity detection of RT-PCR method.
Wherein, (A) SAT1 type represents strain (AY593846) sensitivity technique result；(B) SAT2 type represents strain (JF749862)
Sensitivity technique result；(C) SAT3 type represents strain (AY593852) sensitivity technique result；1-9 swimming lane is with plasmid
SAT1/2/3 is template, and copy number is followed successively by 4 × 109-4×101copies/μL；10 swimming lanes are negative controls.With rna transcription
This is 4 × 10 for the sensitivity of template3copies/μL。
The non-SAT FMDV virus that Fig. 7 is used by carrying out FMDV SAT-D8RT-PCR analysis specific detection is identified self
RT-PCR or PCR expands agarose gel electrophoretogram.
Wherein, M:DL2000 molecular weight standard；1:FMDV/A/AF72/MF4；2:FMDV/A/HB/WH/09/MF4；3:
FMDV/A/FJ/YX/2014/MF5；4:FMDV/O/GD/BYQ/2010/S/33/MF6；5:FMDV/O/NX/99/MF4；
6:FMDV/O/HN/XH/BF13-15/MF2；7:FMDV/O/GS/LT/101209/MF2；8:FMDV/
Asia1/JS/WX/05/MF4；9:FMDV/Asia1/58/MF3；10:FMDV/Asia1/XJ-KLMY/MF4；11:
ORFV；12:BVDV；13:BTV；14:GTPV；15:SPPV；16:SVDV；17:PRV (AV25 strain)；
18:PCV2 (Qingyang3 strain)；19:PCV2 (Sichuan strain)；20:PPV (AV30 strain)；21:PPV
(AV31 strain)；22:CSFV；23:NA-HP-PRRSV (QH-08 strain)；24:NA-Non-HP-PRRSV (Heli
Strain)；25:NA-HP-PRRSV (GW strain).
Fig. 8 is the analysis specific detection result of FMDV SAT-D8RT-PCR method.For analyzing the virus bag of specific detection
Include FMDV (A, O, C, Asia Ι) and BVDV, ORFV, SPPV/GTPV, SVDV, BTV, PRRSV,
CSFV、PRV、PPV、PCV).With other non-SAT FMDV viral nucleic acid for amplification template, carry out FMDV SAT-D8
The agarose gel electrophoretogram of RT-PCR detection.Result shows that the method is special, with other virus no cross reaction.
Wherein, M:DL2000；1,12,28,35,36,37:SAT-FMDV；2:FMDV/A/AF72/MF4；3:
FMDV/A/HB/WH/09/MF4；4:FMDV/A/FJ/YX/2014/MF5；5:
FMDV/O/GD/BYQ/2010/S/33/MF6；6:FMDV/O/NX/99/MF4；7:FMDV
/O/HN/XH/BF13-15/MF2；8:FMDV/O/GS/LT/101209/MF2；9:FMDV/Asia1/JS/WX/05/MF4；
10:FMDV/Asia1/58/MF3；11:FMDV/Asia1/XJ-KLMY/MF4；13:ORFV；14:BVDV；15:
BTV；16:GTPV；17:SPPV；18:SVDV；19:PRV (AV25)；20:PCV2 (Qingyang3)；
21:PCV2 (Sichuan)；22:PPV (AV30)；23:PPV (AV31)；24:CSFV；25:PRRSV
(QH08)；26:PRRSV (Heli)；27:PRRSV (GW)；29:FMDV/C (DQ409188)；30:
FMDV/C(AY593809)；31:FMDV/C (AY593810)；32:FMDV/C (AJ007347)；33:
pUC57；34:DEPC-treated water.
Fig. 9 is with the RNA of the FMDV SAT1/2/3 inactivation antigen from Britain Pirbright laboratory as template, carries out FMDV
SAT-D8RT-PCR amplified production agarose gel electrophoretogram.From aftosa OIE reference laboratory (Britain Pirbright
Laboratory) SAT1, SAT2 and SAT3 inactivation antigen of introducing extracts RNA, as template, use SAT-D8
Primer carries out conventional RT-PCR detection to it.Result shows, uses FMDV SAT-D8RT-PCR to be capable of detecting when three kinds
South Africa type foot and mouth disease virus, amplifies correspondingly sized purpose fragment.
M:DL2000 Relative molecular weight markers；1,2:SAT1 recombinant plasmid；3,4:SAT1 inactivation antigen；5,6: negative
Comparison；7:SAT2 inactivation antigen；8:SAT3 inactivation antigen；9:SAT2 recombinant plasmid；10:SAT3 recombinant plasmid
Figure 10 uses the detection of FMDV SAT-D8RT-PCR method from the field tissue sample of 125 portions of pigs, ox or the sheep of China
Agarose gel electrophoretogram.Result shows, 125 parts of field tissue samples are South Africa type foot and mouth disease virus feminine gender.
Wherein, M:DL2000 relative molecular mass；A, b, c swimming lane is tri-recombinant plasmids (as positive control) of SAT1/2/3；
3: remaining 125 swimming lane is 125 portions of oxen, sheep, the heart of pig, liver, spleen, lung, kidney, lingual surface tissue or Virus monitory respectively
Result.
Detailed description of the invention
Embodiment 1:
The foundation of South Africa type aftosa conventional RT-PCR detection method i.e. FMDV SAT-D8RT-PCR:
1, the synthesis of SAT FMDV detection target fragment 1D2A2B gene:
From GenBank Database, download the complete genome sequence of SAT1/2/3-FMDV, utilize bioinformatics software
The similitude of the complete genome sequence of SAT-FMDV is analyzed in Clustal W Method method comparison in DNAStar7.0, finally
The sequence choosing similitude relatively low (< 85%) represents SAT1 (8), SAT2 (10), SAT3 (3).Table 1
It is shown as SAT FMDV and represents strain sequence accession number.
Table 1
| SAT1-FMDV | SAT2-FMDV | SAT3-FMDV | C-FMDV |
| AY593839 | JF749864 | AY593850 | DQ409188 |
| AY593840 | AF540910 | AY593852 | AY593809 |
| AY593841 | AY593848 | AY593853 | AY593810 |
| AY593846 | JF749861 | AJ007347 | |
| AY593838 | JF749862 | ||
| HM067706 | AY593847 | ||
| JF749860 | HM067705 | ||
| AY593842 | AJ251473 | ||
| HM067704 |
2, FMDV SAT-D8RT-PCR reaction:
The sequence of the 1D2A2B gene of the SAT FMDV that this kit logs in reference to Genbank, designs and synthesizes 8 and draws
Thing.Primer sets sequence is as follows:
Primer sets is made up of 1 downstream primer and 7 upstream primers:
P296-R:5-ACYTTTACCAGGGYTTGGC-3；
P280-F:5-GGCGTTGAGAAACAACTGTG-3；
P281-F:5-GGCGTTGAGAAACAGCTGTG-3；
P282-F:5-GGTGTCGAAAAACAGTTGTG-3；
P283-F:5-GGTGTCGAAAAACAGCTGTG-3；
P310-F:5-GGCGTCGAAAGACAGACCCT-3；
P329-F:5-GCACCAGCCAAACAACTGTG-3；
P331-F:5-AGTGCTGACAAGCAGATGTG-3。
With synthesis SAT FMDV 1D2A2B gene as template, carry out PCR amplification, PCR reaction system is as follows:
PrimeSTAR GLX (PrimeSTAR) enzyme (1.25U/ μ L): 0.4 μ L；
5x PrimeSTAR Buffer(Mg2+Plus): 4 μ L；
DNTP Mixture (each 2.5mM): 1.6 μ L；
F-primer: each 5pmol；
R-primer:10pmol；
DNA template:1 μ L；
DEPC water: add to 20 μ L.
PCR amplification condition is as follows: 95 DEG C of 5min, 94 DEG C of 30s, 60 DEG C of 30s, 68 DEG C of 30s, totally 36 circulations, 68 DEG C
10min。
The product detection of PCR and result judge: reacted product 2.0% agarose gel electrophoresis, 10V/cm, 30
Whether ethidium bromide staining after minute, observe under the uviol lamp of 260nm wavelength and have purpose band to occur at 257bp.
Utilize SAT-D8 primer sets that 21 SAT-FMDV-1D2A2B bar sequences are carried out PCR amplification, amplified production fragment
Size is consistent (257bp) with expection size, and negative control is (with pUC57 carrier and ddH2O is template) occur without band.
As shown in Figure 1.Result shows that screened SAT-D8 primer sets can successfully identify the SAT-FMDV representative of synthesis
Strain gene.
The assay sensitivity of embodiment 2 FMDV SAT-D8RT-PCR method and analysis specificity experiments:
1, the assay sensitivity experiment of FMDV SAT RT-PCR:
1.1 with SAT-FMDV-1D2A2B fragment recombinant plasmid dna as template, detects FMDV SAT-D8RT-PCR method
Sensitivity:
With the SAT1/2/3-FMDV-1D2A2B-pUC57 recombinant plasmid of known mensuration concentration as template, calculate copy number, with
4×108Copies/ μ L is starting template, and it is carried out 10 times of doubling dilutions, measures virus according to above-mentioned PCR testing process
Minimum detectability.Result shows that measuring virus minimum detectability result is 4 × 102Copies/ μ L, as shown in Figure 2.
SAT-FMDV-1D2A2B-PUC57 belongs to double-stranded DNA, and copy number computing formula is as follows:
(6.02×1023Copy number/mole) × (concentration g/ml)/(MW g/mol) × 10-3=copy number/μ l
Or (6.02 × 1023Copy number/mole) × (concentration ng/ μ l) x10-6/ (MW g/mol) × 10-3=copy number/μ l；
Mean molecule quantity (MW g/mol): dsDNA=(base number) × (660 dalton/base)；SsDNA=(base number) × (330
Dalton/base)
1.2 with the external rna transcription of SAT-FMDV-1D2A2B fragment this as template, detect FMDV SAT-D8RT-PCR
Method sensitivity:
The rna transcription of DNA fragmentation 1.2.1SAT-FMDV-1D2A2B is originally prepared:
Recombinant plasmid is without promoter, it is impossible to directly carry out in-vitro transcription.So first against connecting purpose fragment 1D2A2B two ends
PUC57 carrier sequence design primer, then at prime end plus T7 promoter, carry out PCR with recombinant plasmid for template
Amplification obtains the 1D2A2B aim sequence DNA containing T7 promoter, and pcr amplification product glue can be directly as template after reclaiming
Carry out in-vitro transcription.The aim sequence clip size containing T7 promoter amplified by PCR is consistent with expection, result such as figure
Shown in 3, the product respectively selecting 2 samples respectively from SAT1, SAT2, SAT3 at random is sent to the order-checking of Jin Weizhi company, surveys
Sequence result is correct with purpose fragment comparison, can be as in-vitro transcription template.With one of them positive recombinant plasmid
According to mMESSAGE as a example by SAT2-FMDV-1D2A2B-pUC57 (JF749864)Kit(P/N:
AM1344,25 times/box) kit specification carries out in-vitro transcription, then according to RNeasy MinElute Cleanup Kit
Transcription product is purified by the operating process of (Cat.No.74204,50 times/box) kit, and result shows not add transcriptase
Sample and the sample processed with RNase all occur without purpose band, and normal reaction sample has purpose bar at about 1300nt place
Band occurs, as shown in Figure 4, is consistent with expection, shows successfully to prepare transcript folder RNA.Owing to RNA is unstable, be not suitable for
Long-term storage, so SAT1/2/3 respectively selects a positive recombinant plasmid and carries out in-vitro transcription and prepare transcript folder RNA, result is such as
Shown in Fig. 5, it is subsequently used for the template of SAT-D8 primer sensitivity technique.
1.2.2 with the external rna transcription of SAT-FMDV-1D2A2B fragment this as template, detect FMDV SAT-D8RT-PCR
Method sensitivity:
Utilize the SAT1//2/3-FMDV-1D2A2B-pUC57 aim sequence transcript folder RNA prepared above by in-vitro transcription
For template, with 4 × 109Copies/ μ L is starting template, and it is carried out 10 times of doubling dilutions, utilizes according to following testing process
PrimeScriptTM One Step RT-PCR Kit Ver.2 (Lot:RR055A) the kit measurement virus of TaKaRa company is minimum
Detection limit.Result shows with RNA as template, and the minimum detectability of SAT-D8 primer detection SAT FMDV is about 4 × 103
Copies/ μ L, as shown in Figure 6.
With synthesis SAT FMDV nucleic acid 1D2A2B transcript folder RNA as template, carry out RT-PCR amplification, RT-PCR
Reaction system is as follows:
RT-PCR reaction system:
RT-PCR amplification condition is as follows: 50 DEG C of 30min, 95 DEG C of 5min, 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s,
Totally 37 circulations, 72 DEG C of 10min.
Transcript is single stranded RNA, and copy number computing formula is as follows:
(6.02×1023Copy number/mole) × (concentration g/ml)/(MW g/mol) × 10-3=copy number/μ l
Or (6.02 × 1023Copy number/mole) × (concentration ng/ μ l) x10-6/ (MW g/mol) × 10-3=copy number/μ l.
Mean molecule quantity (MW g/mol): ssRNA=(base number) × (340 dalton/base).
2, the analysis specificity experiments of FMDV SAT RT-PCR:
Analyze strain FMDV (A, O, Asia I) and BVDV used in specificity experiments, ORFV, SPPV/GTPV,
The extraction of SVDV, BTV, PRRSV, CSFV, PRV, PPV, PCV and self identification experiment.
Use viral RNA/DNA extraction kit that QIAGEN company provides, extract virus according to kit specification
RNA/DNA.Then utilize each virus self Idiotype primer and amplification condition to carry out PCR or RT-PCR to identify, product electricity
Swimming result shows that every kind of virus has been taken amplified production band out of at purpose bar and occurred, all positives of testing result, as shown in Figure 7.
Amplified production carries out order-checking further to be identified, sequencing result Blast result on NCBI proves to be strictly these virus, permissible
Test for subsequent analysis specificity verification.
Respectively with A, Asia I and O type foot and mouth disease virus and the BVDV through confirming, ORFV, SPPV/GTPV, SVDV,
RNA or DNA of BTV, PRRSV, CSFV, PRV, PPV, PCV reacts mould as SAT FMDV RT-PCR
Plate, then according to the RT-PCR system of the SAT FMDV in embodiment 2 and condition are reacted, product agar
Detected through gel electrophoresis, 3 SAT FMDV positive controls have purpose band to occur near 257bp, and the inspection of above-mentioned intersection
Surveying strain to occur without band, result as shown in Figure 8, illustrates SAT-D8 primer and other virus no cross reaction.
The above results show the multiple PCR method of the SAT FMDV that this experiment set up with above-mentioned 12 kinds of viruses without intersecting instead
Should.
3, employing FMDV SAT-D8RT-PCR detection clinical sample:
The preparation of 3.1 clinical samples:
With from the inactivation antigen SAT1/2/3 of aftosa OIE reference laboratory (Britain Pirbright laboratory) and 125 parts
It is derived from the heart of China's cattle,pig and sheep, liver, spleen, lung, kidney, lingual surface tissue and serum as field measuring samples, extracts its RNA
After, then according to the system of embodiment 2 and amplification condition carry out SAT-D8RT-PCR experiment.
3.2RT-PCR testing result:
With above-mentioned SAT1/2/3 inactivation antigen (from Britain Pirbright laboratory) and the nucleic acid RNA of 125 parts of field samples
For template, detecting by the FMDV SAT-D8RT-PCR detection method set up, result shows, goes out with SAT1/2/3
Active antigen RNA is template, amplifies anticipated SAT FMDV positive band.The further sequence analysis of positive band shows,
Those inactivation antigens are corresponding SAT type foot and mouth disease virus.It is negative that other 125 parts of field sample detection results are all SAT.
Result is as shown in table 2, Fig. 9, Figure 10.
Table 2
4 conclusions
Above-mentioned experiment proves that the FMDV SAT-D8RT-PCR detection method set up has sensitiveness height, high specificity, soon
Speed, experimental facilities be simple and the feature such as processing ease, is suitable for laboratory and clinical to the South Africa i.e. SAT of type foot and mouth disease virus
FMDV carries out quick diagnosis, can be as a kind of strategic reserves resource of China's prevention and control South Africa type aftosa.
Finally it is noted that the foregoing is only the preferred embodiments of the present invention, it is not limited to the present invention, although
Being described in detail the present invention with reference to previous embodiment, for a person skilled in the art, it still can be to front
State the technical scheme described in each embodiment to modify, or wherein portion of techniques feature is carried out equivalent.All at this
Within bright spirit and principle, any modification, equivalent substitution and improvement etc. made, should be included in protection scope of the present invention
Within.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>for quickly detecting the specific primer group of South Africa type foot and mouth disease virus and including the kit of this primer sets
<210> 1
<211> 19
<212> DNA
<213> P296-R
<400> 1
acytttacca
gggyttggc
19
<210> 1
<211> 20
<212> DNA
<213> P280-F
<400> 2
ggcgttgaga
aacaactgtg
20
<210> 1
<211> 20
<212> DNA
<213> P281-F
<400> 3
ggcgttgaga
aacagctgtg
20
<210> 1
<211> 20
<212> DNA
<213> P282-F
<400> 4
ggtgtcgaaa
aacagttgtg
20
<210> 1
<211> 20
<212> DNA
<213> P283-F
<400> 5
ggtgtcgaaa
aacagctgtg
20
<210> 1
<211> 20
<212> DNA
<213> P310-F
<400> 6
ggcgtcgaaa
gacagaccct
20
<210> 1
<211> 20
<212> DNA
<213> P329-F
<400> 7
gcaccagcca
aacaactgtg
20
<210> 1
<211> 20
<212> DNA
<213> P331-F
<400> 8
agtgctgaca
agcagatgtg
20
Claims (3)
1. one group of specific primer group for quickly detection South Africa type foot and mouth disease virus SAT FMDV, it is characterised in that described specific primer group is made up of 1 downstream primer and 7 upstream primers；Described downstream primer is single strand dna shown in sequence 1 in sequence table, and described upstream primer is single strand dna shown in sequence 2-8 in sequence table.
The specific primer group the most according to claim 1 application in preparation is used for the kit of quickly detection South Africa type foot and mouth disease virus SAT FMDV.
3. for quickly detecting the kit of South Africa type foot and mouth disease virus SAT FMDV, it is characterised in that include the specific primer group described in claim 1.
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| CN113564276A (en) * | 2021-05-19 | 2021-10-29 | 中国农业科学院兰州兽医研究所 | Establishment of foot-and-mouth disease virus visual RT-LAMP detection system and method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724711A (en) * | 2008-10-30 | 2010-06-09 | 中国检验检疫科学研究院 | Real-time fluorescence PCR primer and probe for Asia-Europe type foot-and-mouth disease virus detection |
| WO2011054011A2 (en) * | 2009-11-02 | 2011-05-05 | The Trustees Of The University Of Pennsylvania | Foot and mouth disease virus (fmdv) consensus proteins, coding sequences therefor and vaccines made therefrom |
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2016
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| CN101724711A (en) * | 2008-10-30 | 2010-06-09 | 中国检验检疫科学研究院 | Real-time fluorescence PCR primer and probe for Asia-Europe type foot-and-mouth disease virus detection |
| WO2011054011A2 (en) * | 2009-11-02 | 2011-05-05 | The Trustees Of The University Of Pennsylvania | Foot and mouth disease virus (fmdv) consensus proteins, coding sequences therefor and vaccines made therefrom |
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| Title |
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| M. CALLENS等: "Differentiation of the seven serotypes of foot-and-mouth disease virus by reverse transcriptase polymerase chain reaction", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| 杨林等: "应用寡核苷酸芯片检测猪口蹄疫病毒的初步研究", 《中国畜牧兽医学会家畜传染病学分会第六届理事会第二次会议暨教学专业委员会第六届代表大会论文集》 * |
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| CN113564276A (en) * | 2021-05-19 | 2021-10-29 | 中国农业科学院兰州兽医研究所 | Establishment of foot-and-mouth disease virus visual RT-LAMP detection system and method |
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