CN105861755B - For quickly detect the specific primer group of South Africa type foot and mouth disease virus and include the primer sets kit - Google Patents
For quickly detect the specific primer group of South Africa type foot and mouth disease virus and include the primer sets kit Download PDFInfo
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Abstract
The invention discloses one group for quickly detecting the specific primer group of South Africa type foot and mouth disease virus, the specific primer group is made of 1 downstream primer and 7 upstream primers；The downstream primer is single strand dna shown in sequence 1 in sequence table, and the upstream primer is single strand dna shown in sequence 2-8 in sequence table；Additionally provide include the primer sets kit.The invention has the benefit that by the present invention in that with specific composite primer and under a series of Quality Controls and negative, positive control auxiliary, it is detected from viral nucleic acid of the molecular level to SAT FMDV, this method has the characteristics that easy, economical, quick, sensitive and special, it is the detection technique that can detect three SAT FMDV simultaneously, realize the strategic reserves for diagnosing and monitoring to SAT FMDV, for preventing the incoming China of those viruses that there is important strategic importance, have a good application prospect simultaneously.
Description
Technical field
The present invention relates to foot and mouth disease virus detection technique fields, and in particular to for quickly detecting South Africa type foot and mouth disease virus
The specific primer group of SAT FMDV and include the primer sets kit.
Background technique
Aftosa (Foot and Mouth Disease, FMD) is one kind by foot and mouth disease virus (Foot and Mouth
Disease Virus, FMDV) caused by endanger the serious acute febrile high degree in contact artiodactylous such as ox, pig, sheep and infect
Disease.With fever, there is speck in lip, mucous membrane of mouth, breast and hoof or blister pathological change is typical Clinical symptoms.Sense
Dye aftosa not only result in animal production sharp fall, and seriously affected Infected regions livestock products listing and
Outlet.Since its spread speed is fast, it is difficult to control and eliminates, seriously endanger world animal trade, be classified as by the World Health Organization
One of infectious disease must be reported.
Foot and mouth disease virus belongs to the Hostis in Picornaviridae (Picornaviridae)
(Aphthovirus).Including A, O, C, Asia 1 (Type Asia 1), SAT 1 (1 type of South Africa), SAT 2 (2 type of South Africa) and SAT 3
(3 type of South Africa) 7 serotypes, without cross-protection between each serotype.The virion diameter 20-25nm of FMDV, is positive two
Decahedron structure, it is approximate circle；Genome is overall length 8, the positive chain RNA of 500nt, including an open reading frame (open
Reading frame, ORF), 5 '-noncoding regions (5 '-Untraslated Region, 5 '-UTR) and 3 '-noncoding regions (3 '-
Untraslated Region, 3 '-UTR)；ORF coding viral polyprotein, the protease encoded dependent on itself (L, 2A,
3C) and a small number of host factors after 3 grades of cracking forms 4 kinds of virus structural proteins (VP4, VP2, VP3 and VP1) and 9
Kind non-structural protein (Lab, Lb, 2A, 2B, 2C, 3A, 3B, 3C and 3D)；Each 60 molecule of four kinds of structural proteins constitutes virion,
VP1 is located at distal end close to the duck eye for being made of virus surface five former grains, VP2 and VP3, and VP4 is then fully located inside capsid.
Be 2 groups by FMDV points according to the homology between this 7 serotypes, group 1 (Eurasian type) include A, O, C and
Asia1 type；2 (South Africa types) of group include SAT1, SAT2 and SAT3 type.By South Africa type foot and mouth disease virus ((Southern African
Territories Foot and Mouth Disease Virus, SAT FMDV) caused by South Africa type aftosa it is initially main
It is confined to sub-Saharan Africa area, there are an apparent geographic limitations, but is due to trade and personnel transfer quick
Development, SAT2 reach the countries such as Pakistan from Sub-Sahara through north African.Southwest China connects with Pakistan, no
It can exclude a possibility that SAT FMDV is passed to China.
Aftosa is the threat of cloven-hoofed economic animal it is well known that economic loss caused by its morbidity is still each so far
First of class infectious disease.In order to determine that whether in a certain area presence, the presence tool of FMDV is made a definite diagnosis by pathogeny detection by FMDV
It is significant.General detection method includes virus purification, virus neutralization experiment and the molecular biology for expanding viral nucleic acid
Etc. diagnostic techniques.Although virus purification is considered as the goldstandard for detecting FMDV, costly since time-consuming, there are two
The hidden danger of secondary scattered poison, general base are difficult to carry out this work, and there is also similar drawbacks for same virus neutralization experiment.Amplification disease
The molecular biology method of malicious nucleic acid has become the main method of diagnosis cause of disease now.South Africa type aftosa was not sudden and violent in China
It sent out, diagnostic techniques and weak, thus, it is necessary to set up quick, easy, easy to operate, inexpensive, especially suitable future
In the viral nucleic acid detection method of base's veterinary laboratories application.Classical reverse transcriptional PCR (Reverse
Transcription Polymerase Chain Reaction, RT-PCR) meet above-mentioned requirements just.Due to SAT FMDV,
Especially SAT2, topological type is numerous, and the protein-coding region the VP1 gene 1D degree of variation for type diagnosis is again very big, even if building
Classical RT-PCR method is found there is also great difficulty or with 1 Eurasia type FMDV of group there are cross reactivity, it is specific compared with
Low or exactly presence is failed to pinpoint a disease in diagnosis, and sensibility is again relatively low.It is examined as Reid once designed SAT-1D209F/FMDV-2B208R primer pair
Survey 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.), as a result can only be directed to SAT1 type
Amplify the purpose product of 720bp with SAT2, SAT3 amplified production is using gel electrophoresis detected at all less than can only pass through
The method of PCR-ELISA can just detect；Callens et al. shared downstream primer (P1) and compound upstream primer once also to detect
SAT1 (P126, P151-153), SAT2 (P168-170) and 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 it is easy to cross reaction occur between 3 serotypes.Although foreign scholar has done phase
It should study, but since there are drawbacks described above, so, state, inside and outside corresponding SAT FMDV diagnostic kit not yet are asked at present
Generation.So, it is established that the RT-PCR detection method of SAT FMDV simultaneously develops corresponding diagnostic kit to prevention and control SAT FMD biography
Enter China with important strategic importance.
Summary of the invention
The purpose of the present invention is to above-mentioned defects in the prior art, provide for quickly detecting SAT FMDV's
Specific primer group and include the primer sets kit.
To achieve the goals above, technical solution provided by the invention are as follows: one group for quickly detecting the spy of SAT FMDV
Specific primer group, the specific primer group are made of 1 downstream primer and 7 upstream primers；The downstream primer is sequence
Single strand dna shown in sequence 1 in table, the upstream primer are single strand dna shown in sequence 2-8 in sequence table, this is compound
Type primer sets are named as SAT-D8.
8 special primers are made 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。
A second object of the present invention is to provide above-mentioned specific primer group in preparation for quickly detecting SAT FMDV
Kit in application.
It include above-mentioned spy third object of the present invention is to provide for quickly detecting the kit of SAT FMDV
Specific primer group.
The present invention is directed to using the gene order of synthesis as template, it is established that SAT FMDV conventional RT-PCR diagnostic method draws
Object is designed as establishing the key of RT-PCR method, since the protein-coding region VP1 gene 1D is the foundation for carrying out FMDV parting, and because
It is big (70%-85%) for the 1D genetic mutation degree of SAT FMDV, so, composite primer is the optimal selection of this experiment.By dividing
It analyses compared with, has chosen 1D gene 3 ' and hold the target region designed as upstream primer, downstream primer is in the preferable area 2B of conservative.
It is detected by experiment, finishing screen is selected a set of composite primer being made of 7 upstream primers and 1 downstream primer, is named as
8 primer of SAT-D.
Since China does not have SAT FMD in history, in order to ensure biological safety and meet experiment needs, in comparative analysis
On the basis of SAT FMDV nucleic acid sequence homology, the representative strain of 8 SAT1 types, 10 SAT2 types and 3 SAT3 types is picked out,
The 1D2A2B gene of those strains is synthesized, and is connect with PUC57 cloning vector, corresponding recombinant plasmid is constructed.It is heavy with those
Group plasmid establishes FMDV SAT-D8RT-PCR detection method using SAT-D8 as primer for template on DNA level first.With
Above-mentioned 21 plasmids are template, can specifically amplify the purpose band of 257bp.Further prepared by in-vitro transcription method
The 1D2A2B gene RNA transcript of 21 strains can also amplify special purpose band as template out.The party
Method and A, O, C and AsiaI type FMDV and bovine ephemeral diarrhea virus (BVDV), sheep of virus (ORFV), goat capripoxvirus
It GTPV, sheep pox virus SPPV, swine vesicular disease virus (SVDV), blue tongue virus (BTV), american type high-pathogenicity porcine reproductive and exhales
Inhale syndrome virus (NA-HP-PRRSV), the non-highly pathogenic PRRSV (NA-Non-HP- of american type
PRRSV), classic swine fever virus (CSFV), porcine pseudorabies virus (PRV), pig parvoviral (PPV) and pig circular ring virus (PCV)
Deng cross reaction does not occur.Using plasmid as template, the sensitivity of FMDV SAT-D8RT-PCR method can reach 102Copy number/micro-
It rises, using rna transcription sheet as template, sensitivity 103-104Copy number/microlitre.
To come from the SAT1/2/3 inactivation antigen and 125 in aftosa OIE reference laboratory (laboratory Britain Pirbright)
Part is originated from the heart, liver, spleen, lung, kidney, lingual surface tissue and the serum of China's cattle,pig and sheep as measuring samples, after extracting its RNA, carries out
SAT-D8RT-PCR experiment.The result shows that amplifying estimated SAT FMDV positive band using SAT1/2/3RNA as template.Sun
Property band further sequence analysis shows be corresponding SAT1/2/3 type.Other 125 parts of fields sample detection result is all south
Nand-type virus is negative.On the basis of establishing FMDV SAT-D8 conventional RT-PCR detection method, corresponding kit is assembled.
The invention has the benefit that specific primer group and packet provided by the present invention for quickly detecting SAT FMDV
Kit containing the primer sets, by using specific composite primer and in a series of Quality Controls and negative, positive control auxiliary
It helps down, the viral nucleic acid including SAT FMDV is detected from molecular level, this method has easy, economical, quick, sensitive
With special feature, the conventional RT-PCR detection technique that can detect tri- serotypes of SAT1/2/3 simultaneously is realized, it can be complete
The strategic reserves of diagnosis and the monitoring of pairs of SAT FMDV, for preventing its incoming China that there is important strategic importance, simultaneously
It has a good application prospect.
Detailed description of the invention
Fig. 1 is that the SAT FMDV of FMDV SAT-D8RT-PCR method detection synthesis is used to represent strain nucleic acid (to contain
The recombinant plasmid of 1D2A2B genetic fragment be expand template) agarose gel electrophoretogram.Occur at 257bp as the result is shown
With estimated molecular weight sizable specific band, 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 of the 1D2A2B gene of the FMDV SAT1/2/3 of the promoter containing T7
Map.For the carrier sequence design primer being mutually connected with target gene both ends, T7 promoter is added in downstream primer end, with
Obtain the objective gene sequence of the promoter containing T7.
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 control is (with ddH2O is template).
Fig. 3 is the electrophorogram of FMDV SAT2 (JF749864) transcription product after purification.
Wherein, M1:DNA DL2000；M2:RNA 1000maker；1: being with SAT2 (JF749864) T7 glue recovery product
The transcription product of template；2: being directly the transcription product of template with SAT2 (JF749864) T7PCR product；3: not plus transcriptase
SAT2 (JF749864) transcription product；SAT2 (JF749864) transcription product of 4:RNase processing.
Fig. 4 is the electrophorogram of FMDV SAT1/2/3 transcription product after purification.
Wherein, 1:SAT1 (AY593846) transcription product；2:SAT2 (JF749862) transcription product；3:SAT3
(AY593852) transcription product.
Fig. 5 is to carry out the assay sensitivity testing result of FMDV SAT-D8RT-PCR method using plasmid as template.With again
The recombinant plasmid of 1D2A2B genetic fragment than being serially diluted the FMDV containing SAT is template, carries out FMDV SAT-D8RT-PCR inspection
It surveys, agarose gel electrophoresis results show 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 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
Sensitivity for template is 4 × 102copies/μL。
Fig. 6 is to carry out FMDV SAT-D8RT- using the rna transcription sheet of the 1D2A2B gene of the SAT FMDV of synthesis as template
The assay sensitivity of PCR method detects.
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 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.Turned with RNA
Record is originally that the sensitivity of template is 4 × 103copies/μL。
Fig. 7 is to carry out FMDV SAT-D8RT-PCR to analyze non-SAT FMDV virus itself mirror used by specific detection
Fixed RT-PCR or PCR amplification 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 plants)；18:PCV2 (Qingyang3 plants)；19:PCV2 (Sichuan plants)；20:PPV
(AV30 plants)；21:PPV (AV31 plants)；22:CSFV；23:NA-HP-PRRSV (QH-08 plants)；24:NA-Non-HP-PRRSV
(Heli plants)；25:NA-HP-PRRSV (GW plants).
Fig. 8 is the analysis specific detection result of FMDV SAT-D8RT-PCR method.For analyzing the disease of specific detection
Poison include FMDV (A, O, C, Asia Ι) and BVDV, ORFV, SPPV/GTPV, SVDV, BTV, PRRSV, CSFV, PRV, PPV,
PCV).It is amplification template with other non-SAT FMDV viral nucleic acids, carries out the Ago-Gel of FMDV SAT-D8RT-PCR detection
Electrophorogram.The method is special as the result is shown, with other viral no cross reactions.
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 to carry out using the RNA of the FMDV SAT1/2/3 inactivation antigen from the laboratory Britain Pirbright as template
FMDV SAT-D8RT-PCR amplified production agarose gel electrophoretogram.From aftosa OIE reference laboratory (Britain
The laboratory Pirbright) introduce SAT1, SAT2 and SAT3 inactivation antigen in extract RNA, as template, using SAT-D8
Its progress conventional RT-PCR detection of primer pair.The results show that being capable of detecting when three kinds of South Africa types using FMDV SAT-D8RT-PCR
Foot and mouth disease virus amplifies target fragment of corresponding size.
M:DL2000 Relative molecular weight markers；1,2:SAT1 recombinant plasmid；3,4:SAT1 inactivation antigen；5,6: negative right
According to；7:SAT2 inactivation antigen；8:SAT3 inactivation antigen；9:SAT2 recombinant plasmid；10:SAT3 recombinant plasmid Figure 10 uses FMDV
The agarose gel electrophoresis of 125 portion pigs of the SAT-D8RT-PCR method detection from China, ox or the field of sheep tissue sample
Map.The results show that 125 parts of field tissue samples are that South Africa type foot and mouth disease virus is negative.
Wherein, M:DL2000 relative molecular mass；A, b, c swimming lane are tri- recombinant plasmids of SAT1/2/3 (as positive right
According to)；3: remaining 125 swimming lane is 125 portions of oxen, sheep, the heart of pig, liver, spleen, lung, kidney, lingual surface tissue or serum detection knot respectively
Fruit.
Specific embodiment
Embodiment 1:
South Africa type aftosa conventional RT-PCR detection method, that is, FMDV SAT-D8RT-PCR foundation:
1, SAT FMDV detects the synthesis of target segment 1D2A2B gene:
The complete genome sequence that SAT1/2/3-FMDV is downloaded from GenBank Database, utilizes bioinformatics software
Clustal W Method method in DNAStar7.0 compares the similitude of the complete genome sequence of analysis SAT-FMDV, final to select
The sequence of similitude lower (< 85%) is taken to represent SAT1 (8), SAT2 (10), SAT3 (3).Table 1 is shown as SAT FMDV
Represent 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 reacts:
This kit designs and synthesizes 8 and draws referring to the sequence of the 1D2A2B gene of the Genbank SAT FMDV logged in
Object.Primer sets sequence is as follows:
Primer sets are made 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。
Using the SAT FMDV 1D2A2B gene of synthesis as template, PCR amplification is carried out, 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: 20 μ L are added to.
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, and 68 DEG C
10min。
The reaction product of PCR detects and result judgement: 2.0% agarose gel electrophoresis of product after reaction, 10V/cm,
Ethidium bromide staining after 30 minutes, whether observation has purpose band to occur 257bp under the ultraviolet lamp of 260nm wavelength.
PCR amplification is carried out to 21 SAT-FMDV-1D2A2B sequences using SAT-D8 primer sets, amplified production segment is big
Small to be consistent (257bp) with expected size, negative control is (with pUC57 carrier and ddH2O is template) occur without band.Such as Fig. 1 institute
Show.The result shows that the SAT-FMDV that the SAT-D8 primer sets screened can successfully identify synthesis represents strain gene.
The assay sensitivity and analysis specificity experiments of embodiment 2FMDV SAT-D8RT-PCR method:
1, the assay sensitivity experiment of FMDV SAT RT-PCR:
1.1 using SAT-FMDV-1D2A2B segment recombinant plasmid dna as template, detection FMDV SAT-D8RT-PCR method spirit
Sensitivity:
Using the SAT1/2/3-FMDV-1D2A2B-pUC57 recombinant plasmid of known measurement concentration as template, copy number is calculated,
With 4 × 108Copies/ μ L is starting template, and 10 times of doubling dilutions are carried out to it, measures disease according to above-mentioned PCR testing process
Malicious minimum detectability.The result shows that measuring viral minimum detectability result is 4 × 102Copies/ μ L, as shown in Fig. 2.
SAT-FMDV-1D2A2B-PUC57 belongs to double-stranded DNA, and copy number calculation 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；
Average molecular weight (MW g/mol): dsDNA=(base number) × (660 dalton/base)；SsDNA=(base
Number) × (330 dalton/base)
1.2 using the external rna transcription sheet of SAT-FMDV-1D2A2B segment as template, detects the side SAT-D8RT-PCR FMDV
Method sensitivity:
1.2.1SAT-FMDV-1D2A2B this preparation of the rna transcription of DNA fragmentation:
Recombinant plasmid can not be directly transcribed in vitro without promoter.So first against connection target fragment 1D2A2B
Then the pUC57 carrier sequence design primer at both ends adds T7 promoter in prime end, carries out by template of recombinant plasmid
PCR amplification obtains the 1D2A2B aim sequence DNA of the promoter containing T7, can be directly as template after the recycling of pcr amplification product glue
It is transcribed in vitro.The aim sequence clip size of the promoter containing T7 gone out by PCR amplification is consistent with expection, as a result such as Fig. 3
Shown, the product for respectively selecting 2 samples at random from SAT1, SAT2, SAT3 respectively is sent to the sequencing of Jin Weizhi company, sequencing result
It is compared correctly with target fragment, can be used as in-vitro transcription template.With one of positive recombinant plasmid SAT2-FMDV-
According to mMESSAGE for 1D2A2B-pUC57 (JF749864)Kit (P/N:AM1344,25 times/box)
Kit specification is transcribed in vitro, then according to RNeasy MinElute Cleanup Kit (Cat.No.74204,50
Secondary/box) operating process of kit purifies transcription product, the results showed that not plus the sample of transcriptase and at RNase
The sample of reason occurs without purpose band, and normal reaction sample has the appearance of purpose band at the place 1300nt or so, such as Fig. 4 institute
Show, is consistent with expection, shows successfully to prepare transcript folder RNA.Since RNA is unstable, be not suitable for long-term storage, so SAT1/2/3
It respectively selects a positive recombinant plasmid and carries out in-vitro transcription and prepare transcript folder RNA, as a result as shown in figure 5, being subsequently used for SAT-D8
The template of primer sensitivity technique.
1.2.2 using the external rna transcription sheet of SAT-FMDV-1D2A2B segment as template, FMDV SAT-D8RT-PCR is detected
Method sensitivity:
Using above by SAT1//2/3-FMDV-1D2A2B-pUC57 aim sequence transcript that preparation is transcribed in vitro
RNA is template, with 4 × 109Copies/ μ L is starting template, and 10 times of doubling dilutions are carried out to it, according to following testing processes
Utilize PrimeScriptTM One Step RT-PCR Kit Ver.2 (Lot:RR055A) kit measurement of TaKaRa company
Viral minimum detectability.The result shows that the minimum detectability of SAT-D8 primer detection SAT FMDV is about 4 using RNA as template
×103Copies/ μ L, as shown in Fig. 6.
Using the SAT FMDV nucleic acid 1D2A2B transcript folder RNA of synthesis as template, RT-PCR amplification, RT-PCR reactant are carried out
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, and totally 37
Circulation, 72 DEG C of 10min.
Transcript is single stranded RNA, and copy number calculation 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.
Average molecular weight (MW g/mol): ssRNA=(base number) × (340 dalton/base).
2, the analysis specificity experiments of FMDV SAT RT-PCR:
Analyze specificity experiments used in strain FMDV (A, O, Asia I) and BVDV, ORFV, SPPV/GTPV, SVDV,
The extraction of BTV, PRRSV, CSFV, PRV, PPV, PCV and itself identification experiment.
Viral RNA/the DNA extraction kit provided using QIAGEN company extracts virus according to kit specification
RNA/DNA.Then it carries out PCR or RT-PCR using itself each viral Idiotype primer and amplification condition to identify, product electrophoresis knot
Fruit shows that every kind of virus has amplified production band to occur out in purpose band, all positives of testing result, as shown in Figure 7.Expand
Increase production the sequencing identification of object further progress, sequencing result Blast result on NCBI proves to be strictly these viruses, can be used for
The experiment of subsequent analysis specificity verification.
Respectively with by confirming A, Asia I and O-shaped foot and mouth disease virus and BVDV, ORFV, SPPV/GTPV, SVDV,
The RNA or DNA of BTV, PRRSV, CSFV, PRV, PPV, PCV are as SAT FMDV RT-PCR reaction template, then according to implementation
The RT-PCR system and condition of SAT FMDV in example 2 is reacted, and reaction product is detected with agargel electrophoresis, 3 SAT
FMDV positive control has the appearance of purpose band near 257bp, and above-mentioned cross detection strain occurs without band, as a result as attached
Shown in Fig. 8, illustrate SAT-D8 primer and other viral no cross reactions.
The above results show that the multiple PCR method for the SAT FMDV that this experiment is established and above-mentioned 12 kinds of virus are anti-without intersecting
It answers.
3, clinical sample is detected using FMDV SAT-D8RT-PCR:
The preparation of 3.1 clinical samples:
To come from the inactivation antigen SAT1/2/3 and 125 in aftosa OIE reference laboratory (laboratory Britain Pirbright)
The heart, liver, spleen, lung, kidney, lingual surface tissue and serum of the part from China's cattle,pig and sheep are as field measuring samples, after extracting its RNA,
Then SAT-D8RT-PCR experiment is carried out according to the system and amplification condition of embodiment 2.
3.2RT-PCR testing result:
With the nucleic acid of above-mentioned SAT1/2/3 inactivation antigen (coming from the laboratory Britain Pirbright) and 125 parts of field samples
RNA is template, is detected with the FMDV SAT-D8RT-PCR detection method established, the results showed that, it is inactivated with SAT1/2/3
Antigen RNA is template, amplifies estimated SAT FMDV positive band.The further sequence of positive band analysis shows, those
Inactivation antigen is corresponding SAT type foot and mouth disease virus.Other 125 parts of fields sample detection result is all SAT feminine gender.As a result such as table
2, shown in Fig. 9, Figure 10.
Table 2
4 conclusions
It is above-mentioned experiments have shown that the FMDV SAT-D8RT-PCR detection method established have sensibility height, high specificity, fastly
The features such as speed, experimental facilities are simple and operation is easy is suitable for laboratory and clinic to the South Africa i.e. SAT of type foot and mouth disease virus
FMDV carries out quick diagnosis, can be used as a kind of strategic reserves resource of China prevention and control South Africa type aftosa.
Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention
Within protection scope.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>for quickly detect South Africa type foot and mouth disease virus specific primer group and include the primer sets examination
Agent box
<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 for quickly detecting the specific primer group of South Africa type foot and mouth disease virus SAT FMDV, which is characterized in that described
Specific primer group is made of 1 downstream primer and 7 upstream primers；The downstream primer is single shown in sequence 1 in sequence table
Ssdna molecule, the upstream primer are single strand dna shown in sequence 2-8 in sequence table.
2. specific primer group according to claim 1 is in preparation for quickly detecting South Africa type foot and mouth disease virus SAT
Application in the kit of FMDV.
3. the kit for quickly detecting South Africa type foot and mouth disease virus SAT FMDV, which is characterized in that include claim 1
The specific primer group.
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| 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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| 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 |
Non-Patent Citations (2)
| Title |
|---|
| Differentiation of the seven serotypes of foot-and-mouth disease virus by reverse transcriptase polymerase chain reaction;M. Callens等;《Journal of Virological Methods》;19970831;第67卷(第1期);35-44 * |
| 应用寡核苷酸芯片检测猪口蹄疫病毒的初步研究;杨林等;《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》;20061026;127-130 * |
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