CN101280343A - Primer for quantitative and qualitative determination of blue tongue virus - Google Patents
Primer for quantitative and qualitative determination of blue tongue virus Download PDFInfo
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- CN101280343A CN101280343A CNA2008100940492A CN200810094049A CN101280343A CN 101280343 A CN101280343 A CN 101280343A CN A2008100940492 A CNA2008100940492 A CN A2008100940492A CN 200810094049 A CN200810094049 A CN 200810094049A CN 101280343 A CN101280343 A CN 101280343A
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Abstract
The invention discloses a primer used for blue tongue virus qualitative and quantitative measurement. The upstream primer of the primer is provided with SEQ ID NO. 1 in the sequence table and the downstream primer of the primer is provided with SEQ ID NO. 2 in the sequence table. The primer can be used for qualitative and quantitative analyzing of the blue tongue virus ribonucleic acid of the animals which is infected with the blue tongue virus in clinic and scientific researches, and the primer is provided with significant meaning to the judgment of occurring and recurring of the blue tongue virus, the curing effect evaluation and the dynamic observation of the patients condition; the invention can play an important role in the animal medicine detecting field.
Description
The present invention is May 16 2006 applying date, the dividing an application of the patent application of application number 200610081309.3.
Technical field
The present invention relates to virus be carried out the primer and the probe of qualitative and quantitative analysis with molecular biology method, particularly relate to the primer and the TaqMan probe that blue tongue rims are carried out qualitative and quantitative analysis with real-time fluorescence quantitative PCR (real-time fluorescent quantification PCR, real-time FQPCR) technology.
Background technology
Bluetongue is one of 15 kinds of category-A Animal diseases of OIE (OIE) affirmation, has been subjected to the special concern of whole world the countries concerned.Bluetongue betides South Africa (19 th century later) the earliest, entered for 20th century after, this disease takes place successively all over the world or finds this disease virus.China from 1979 since this disease takes place in Yunnan Shizong County first, successively repeatedly take place again in other area.
(bluetongue virus BTV) is the arboviruses that infects domestic and wild ruminating animal to blue tongue rims.BTV is the line-up of delegates of Reoviridae (Reoviridae) Orbivirus (Orbivirus), has found 25 BTV serotypes (Davies FG, Muugai JN, Pini A.Vet Microbiol 1992 so far; 31:25-32), wherein find 6 serotypes (BTV2,10,11,13,17 and 24), find 1 serotype (BTV10) in China in the U.S..The BTV genome contains the double-stranded RNA (dsRNA) of 10 sections, is long segment by large, medium and small branch: fragment 1-3, and fragment 4-6 is middle long segment, and fragment 7-10 is the short-movie section.The dsRNA of these 10 sections encode respectively 7 structural protein (VP1, VP2, VP3, VP4, VP5, VP6, VP7) and 3 Nonstructural Proteins (NS1, NS2, NS3).Wherein (segment 6, and S6) coding is translated, and the dsRNA fragment of growing up little in the genus is to duplicate relevant a kind of Nonstructural Protein with BTV by fragment 6 for NS1.Wang LF etc. studies show that, NS1 gene (claiming S6 again) is the most conservative (Wang LF, Dior h, Osburn BI.Nucleic Acid Res 1989 in 10 sections genes of BTV serogroups; 17:8002).Jonathan etc. propose BTV NS1 gene and other Orbivirus cross reaction minimum (Jonathan B K, Gary AG, David A A, Karl A A ﹠amp; Kathryan M M, Am J Vet Res, 54 (1993) 2021).
Can cut open to examine to change according to disease symptom, epidemiology and pathology bluetongue is supposed diagnosis, make a definite diagnosis separation and the evaluation or the specific serum test that then must rely on virus.International diagnostic method is to adopt agar immunodiffusion(ID), fluorescence antibody or complement fixation test (CFT) detection specificity antibody.Neutralization test, competitive ELISA, indirect ELISA also can be used for detection specificity antibody.In addition, round pcr can be used for this viral gene test.
Round pcr is as novel gene test means, have fast, accurately, characteristics such as high specificity, become the inexorable trend of method for detecting virus development.
Round pcr has not only become conventional ways and means in biology field since 1989 begin to use, and has obtained using widely in the diagnosis of heredopathia, the detection of pathogenic agent, the many-sides such as evaluation of medical jurisprudence sample.And this technology constantly is improved.1996, U.S. Applied Biosystems company has released real-time fluorescence quantitative PCR (real-time fluorescent quantification PCR, real-time FQ PCR) technology, this technology is to add fluorescent mark to carry out quantitatively on conventional PCR basis, not only realized the leap of PCR from qualitative to quantitative, and compare with conventional PCR, it has characteristics such as specificity stronger, pollution-free (need not to use bromination second to form sediment), level of automation height.This technology is that dna probe is connected fluorescent reagent, after treating this dna probe and the PCR product combining, by detecting the fluorescence light quantity, and carries out assistant analysis with computer software, quantizes to calculate, and sensitivity is 0.1%.Quantitative PCR not only can be applied in fundamental research, clinical detection, can also play an important role aspect customs and the food sanitation quarantine.
The real-time fluorescence quantitative PCR technology, be meant in the PCR reaction system and add a specificity fluorescent probe (TaqMan probe) in a pair of primer of adding, probe only with the template specific combination, its binding site utilizes the fluorescent signal accumulation whole PCR process of monitoring in real time between two primers.Taqman fluorescent probe at present commonly used is an oligonucleotide, its 5 ' end be marked with the report fluorophor (Reporter, R), as FAM, VIC etc., 3 ' end be marked with the cancellation fluorophor (Quencher, Q), as TAMRA etc.When probe was complete, the reporter group fluorescent signal emitted was absorbed by quenching group, so detect less than fluorescence; In the pcr amplification process, 5 ' → 3 ' 5 prime excision enzyme activity of Taq enzyme is cut degraded with the probe enzyme, the report fluorophor is separated with the cancellation fluorophor, thereby the fluorescence monitoring system can receive fluorescent signal.In the pcr amplification process, every through a PCR circulation, fluorescent signal is also the same with the purpose fragment, the process that has a sync index to increase, detect first order fluorescence intensity after each loop ends, entire reaction just can obtain an amplification curve after finishing, amplification curve by the concentration known standard model can obtain a typical curve, can carry out quantitative analysis to unknown template according to the amplification curve of this typical curve and unknown template.
But the present real-time fluorescence quantitative PCR detection technique that still is not specifically designed to the blue tongue rims gene is not because have suitable primer and specificity fluorescent probe.
Summary of the invention
The purpose of this invention is to provide a pair of primer that blue tongue rims (BTV) are carried out qualitative and quantitative analysis of being used for.
Primer provided by the present invention, its upstream primer have the SEQ ID № in the sequence table: 1, and downstream primer has the SEQ ID № in the sequence table: 2.
With upstream primer called after P (NS1-1), the SEQ ID № in the sequence table: 1 by 21 based compositions, and this sequence is positioned at BTV NS1 gene (claiming the S6 gene again) from 5 ' end 10-30 base; With downstream primer called after P (NS1-2), SEQ ID №: 2 by 21 based compositions, and this sequence is the reverse complementary sequence of BTV NS1 gene from 5 ' end 110-130 bit base.Described NS1 gene refers to contain the full gene of 5 ' end non-coding region nucleotide sequence.
Also belong to protection scope of the present invention by above-mentioned primer deutero-primer sequence.Described derived sequence is meant the № at SEQID: 1 and/or SEQ ID №: the primer sequence that obtains through replacement, disappearance or the interpolation of one to ten base on 2 the basis.
Above-mentioned primer also can be used in the conventional PCR detection technique of this virus in both being applicable to that the real-time fluorescence quantitative PCR of each serotype of blue tongue rims detects, its detection to as if the NS1 gene of BTV.If is template with testing sample through the cDNA that reverse transcription forms, under the guiding of above-mentioned primer, carry out pcr amplification, the band of 121bp size is arranged in the amplified production, then contain BTV in the sample.
The present invention also provides the TaqMan probe that is used for blue tongue rims are carried out qualitative and quantitative analysis.
TaqMan probe provided by the present invention can have the dna sequence dna of SEQ ID NO:3 in the sequence table or SEQ ID NO:4; Described probe is fluorescently-labeled for process, and its 5 ' end is marked with the report fluorophor, and 3 ' end is marked with the cancellation fluorophor.
Described report fluorophor can be FAM, VIC etc., and the fluorescent quenching group can be TAMRA, MGB etc.
Be extended when preventing pcr amplification, 3 ' end of described probe will be handled through phosphorylation.
The TaqMan probe called after TaqManProbe1 (NS1) that will have SEQ ID NO:3 in the sequence table, the SEQ ID № in the sequence table: 3 by 25 based compositions, and this sequence is the reverse complementary sequence of BTV NS1 gene from 5 ' end 70-94 bit base;
The TaqMan probe called after TaqManProbe2 (NS1) that will have SEQ ID NO:4 in the sequence table, the SEQ ID № in the sequence table: 4 by 23 based compositions, and this sequence is that BTV NS1 gene is from 5 ' end 72-94 bit base.
The derived sequence of above-mentioned TaqMan probe sequence also belongs to protection scope of the present invention.Described derived sequence is meant the № at SEQ ID: 3 or SEQ ID №: on 4 the basis, add, reduce the sequence that one or more bases obtain again at the 5 ' end and/or the 3 ' end of sequence.
The 3rd purpose of the present invention provides a kind of real-time fluorescence quantitative PCR test kit that detects blue tongue rims.
The real-time fluorescence quantitative PCR test kit of detection blue tongue rims provided by the present invention can comprise the primer and the TaqMan probe that are used for blue tongue rims are carried out qualitative and quantitative analysis.
The invention provides the primer and the TaqMan probe that are used for blue tongue rims are carried out qualitative and quantitative analysis, by extracting total RNA of testing sample, and obtain cDNA by reverse transcription, in conjunction with the real-time fluorescence quantitative PCR detection technique, can reach the purpose of blue tongue rims RNA in the accurate quantitative sample to be measured again.Primer provided by the present invention and probe can be used in clinical and the scientific research blue tongue rims RNA in the animal that infects bluetongue being carried out qualitative and quantitative analysis, to judging generation, the recurrence of bluetongue, dynamic observing of the treatment effectiveness evaluation and the state of an illness is significant.The present invention will play a significant role at the animal medicine detection range.
Below in conjunction with specific embodiment the present invention is described in further detail.
Description of drawings
Fig. 1 is the agarose gel electrophoresis detected result of BTV-3,5,8,10,11,21,22, (A) sample P CR amplified production
Fig. 2 is the agarose gel electrophoresis detected result of bacterium colony PCR product
The agarose gel electrophoresis detected result of the positive cloned plasmids pGEM-T120 of Fig. 3
Fig. 4 A is the real-time fluorescence quantitative PCR amplification curve of different dilution pGEM-T120 standard substance
Fig. 4 B is the real-time fluorescence quantitative PCR amplification curve of 6 kinds of BTV serotype samples
Fig. 5 is the real-time fluorescence quantitative PCR typical curve of detection BTV and the site of sample on typical curve of 6 kinds of BTV serotypes
Fig. 6 is the real-time fluorescence quantitative PCR amplification curve of 2 kinds of other BTV serotype samples
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.The primer is synthetic by three rich companies, and it is synthetic that used TaqMan probe is given birth to the worker by Shanghai, and the work of all sequences mensuration is finished by magnificent major company.
Embodiment 1, be used for blue tongue rims (BTV) are carried out the design of the primer and the TaqMan probe of qualitative and quantitative analysis
According to the NS1 gene order of 8 serotypes B TV among the GenBank, utilize DNAStar software that the NS1 gene of these 8 serotypes B TV is carried out sequence alignment, the conservative region sequence of choosing NS1 gene 5 ' end is as detecting sequence.Owing to there are 6 serotypes B TV NS1 gene orders (GenBankAccession No.AY462225, M97762, NC 006025, X15891, X56735, Y00422) to comprise 5 ' long non-coding area sequence in these 8 serotypes B TV NS1 genes, utilize DNAStar software that these 6 serotypes/strain BTV NS1 gene order is compared, according to comparison result design primer and TaqMan probe, sequence is as follows then:
Upstream primer P (NS1-1): 5 '-GTTCTCTAGTTGGCAACCACC-3 ' (SEQ ID NO:1 in the sequence table), Tm is 57 ℃;
Downstream primer P (NS1-2): 5 '-GTGACTGCAAGTCCATTGAGG-3 ' (SEQ ID NO:2 in the sequence table), Tm is 57 ℃.
TaqManProbe1 (NS1): 5 ' FAM-AGTTCTCGTGGCATTTGCGTAATCC-TAMRA3 ' (SEQ IDNO:3 in the sequence table), Tm is 67 ℃.
TaqManProbe2 (NS1): 5 ' FAM-ATTACGCAAATGCCACGAGAACT-TAMRA3 ' (SEQ IDNO:4 in the sequence table), Tm is 62 ℃.
With upstream primer called after P (NS1-1), the SEQ ID № in the sequence table: 1 by 21 based compositions, and this sequence is positioned at BTV NS1 gene (claiming the S6 gene again) from 5 ' end 10-30 base; With downstream primer called after P (NS1-2), SEQ ID №: 2 by 21 based compositions, and this sequence is the reverse complementary sequence of BTV NS1 gene from 5 ' end 110-130 base.Described NS1 gene refers to contain the full gene of 5 ' end non-coding region nucleotide sequence.
The TaqMan probe called after TaqManProbe1 (NS1) that will have SEQ ID NO:3 in the sequence table, the SEQ ID № in the sequence table: 3 by 25 based compositions, and this sequence is the reverse complementary sequence of BTV NS1 gene from 5 ' end 70-94 bit base.
The TaqMan probe called after TaqManProbe2 (NS1) that will have SEQ ID NO:4 in the sequence table, the SEQ ID № in the sequence table: 4 by 23 based compositions, and this sequence is that BTV NS1 gene is from 5 ' end 72-94 bit base.
With 5 ' end mark report fluorophor FAM of above-mentioned TaqMan probe, 3 ' holds mark cancellation fluorophor TAMRA, and 3 ' end of probe is carried out the phosphorylation processing.
1, the processing of BTV sample
The BTV that selects 8 serotypes (being respectively BTV3, BTV5, BTV8, BTV10, BTV11, BTV21, BTV22, BTV (A) type) in BTV, blood plasma and the tissue through cell cultures for use is as BTV sample to be measured.
With following method to handling through the BTV of cell cultures sample: first inoculation BTV to BHK-21 cell, being cultured to cytopathy in 37 ℃ CO2 incubator reaches more than 80%, under aseptic condition, sick cell is moved in the sterilization centrifuge tube then, the centrifugal 30min of 8000rpm, under aseptic condition, supernatant moved into and place temporarily in another centrifuge tube under the room temperature, add supernatant, the piping and druming mixing behind the cell precipitation multigelation three times, the centrifugal 30min of 8000rpm gets the extraction that supernatant is used for BTV RNA.
BTV sample in blood plasma and the tissue need not to handle.
2, the extraction of BTV RNA
Above-mentioned BTV sample is respectively got 300 μ l, extracts RNA with Body-fluid viral DNA/Viral RNA Mini-prep test kit (V-Gene) and reference reagent box specification sheets.
3, reverse transcription
The RNA of the different B TV sample that extracts with step 2 is a template respectively, its cDNA is synthesized in reverse transcription, reaction system and reaction conditions are: get BTV RNA 9 μ l, add downstream Auele Specific Primer P (NS1-2) 2 μ l (50pg/ μ l), mixing, 97 ℃ of water-bath 5min, place 5min on the frozen water rapidly, on ice chest, add M-MLV damping fluid 4 μ l, dNTPs (2.5mM each) 4 μ l (TaKaRa), M-MLV RT 1 μ l (TaKaRa), RNasin 0.5 μ l (TaKaRa) again, mixing, 42 ℃ of reaction 1h.
4, conventional PCR detects
CDNA with step 3 reverse transcription synthetic different B TV sample is a template respectively, under the guiding of primer P of the present invention (NS1-1) and P (NS1-2), carry out pcr amplification, 25 μ l PCR reaction systems are: 10 * PCR damping fluid, 2.5 μ l, dNTPs (2.5mM each) 2 μ l, P (NS1-1) 0.5 μ l (50pg), P (NS1-2) 0.5 μ l (50pg), cDNA 5 μ l, Taq enzyme 1 μ l (TaKaRa), ddH
2O 13.5 μ l.The PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 45s then, 45 ℃ of 45s, 72 ℃ of 1min, totally 35 circulations; Last 72 ℃ of 7min, 4 ℃ of termination reactions.After reaction finishes, get each 6 μ l of pcr amplification product and carry out 2.5% agarose gel electrophoresis detection (70V electrophoresis 30min), detected result is (swimming lane M:Marker DL2000 as shown in Figure 1, swimming lane A:BTV3, swimming lane B:BTV5, swimming lane C:BTV8, swimming lane D:BTV10, swimming lane E:BTV11, swimming lane F:BTV21, swimming lane G:BTV22, swimming lane H:BTV (A)), the result is through the BTV sample (BTV-3 of 8 serotypes of cell cultures, 5,8,10,11,21,22, (A)) all obtained the purpose band of 121bp through pcr amplification, conformed to, shown that the conventional PCR that primer of the present invention can be used for blue tongue rims detects with expected results.
1, the cultivation of BTV and processing
Select for use BTV through cell cultures as BTV sample to be measured, and use with embodiment 2 in identical method to handling through the BTV of cell cultures sample.
2, the extraction of BTV RNA
Get the BTV sample 300 μ l that step 1 obtains, use with embodiment 2 in identical method extract RNA.
3, reverse transcription
The RNA of the BTV sample that extracts with step 2 is a template, and its cDNA is synthesized in reverse transcription, and is identical among reaction system and reaction conditions and the embodiment 2.
4, the preparation of real-time FQ PCR standard substance
(1) pcr amplification product of BTV-5 reclaims
Get the pcr amplification product 50 μ l of the test sample BTV5 of embodiment 2 acquisitions, it is carried out 2.5% agarose gel electrophoresis (70V electrophoresis 30min), under ultraviolet lamp, cut the purpose band of 121bp, use Wizard SVGel and PCR Clean-up System (Promega) and reference reagent box specification sheets that this purpose segment is reclaimed and purifying then.
(2) connect
The purpose segment of the 121bp that step (1) is reclaimed is connected with pGEM-T Easy Vector, and linked system and condition are: recovery product 8 μ l, 2 * T
4Dna ligase damping fluid 10 μ l, pGEM-T Easy Vector 1 μ l (Promega), T
4Dna ligase 1 μ l (Promega), mixing behind 16 ℃ of reaction 2h, is placed 12-24h for 4 ℃.
(3) transform
Get the connection product 20 μ l of step (2), add 100 μ l CaCl
2The DH5 α competent cell of method preparation, rotate mixing gently, place 30min on the frozen water, 42 ℃ of heat shock 2min then, put 5min on the frozen water more rapidly, add 800 μ l LB liquid nutrient mediums, 37 ℃ of shaking tables are cultivated 1h, the centrifugal 5min of 6000rpm abandons about 800 μ l supernatants, suspends with the piping and druming of residue supernatant and precipitates, and bacteria suspension is coated the LB resistance select flat board (to contain penbritin (Amp) 50mg/L, 50mg/mL X-gal 16 μ l, 200mg/mL IPTG 4 μ l) on, cultivate 12-24h down at 37 ℃.
(4) bacterium colony PCR detects
3 hickies growing on the LB resistance is selected flat board of picking carry out colony PCR amplification at random, used identical of PCR reaction system and reaction conditions and the conventional PCR among the embodiment 2.After reaction finishes, get pcr amplification product 6 μ l, it is carried out 2.5% agarose gel electrophoresis detect (70V electrophoresis 30min), detected result is (swimming lane M:Marker DL2000 as shown in Figure 2, swimming lane A: bacterium colony 1, swimming lane B: bacterium colony 2, swimming lane C: bacterium colony 3), the purpose band of 121bp has appearred in result wherein 1 clone (bacterium colony 1) through pcr amplification.
(5) plasmid of extraction positive colony
Picking step (4) is through the positive monoclonal of preliminary evaluation, it is inoculated in the LB liquid nutrient medium that contains 50mg/L Amp, shaking table is cultivated 14h under 37 ℃, 300rpm, uses high purity plasmid extraction kit (Fa Tejie) to extract plasmid then, with this plasmid called after pGEM-T120.Get the plasmid pGEM-T120 5 μ l of extraction, it is carried out 2.5% agarose gel electrophoresis detect (70V electrophoresis 30min), detected result is (swimming lane M:Marker DL2000 as shown in Figure 3, swimming lane A and swimming lane B:pGEM-T120), the stripe size of plasmid pGEM-T120 is about 3kb, conforms to expected results.
(6) order-checking
The reorganization bacterium called after DH5 α (pGEM-T120) of pGEM-T120 will be carried, getting DH5 α (pGEM-T120) bacterium liquid checks order, the entrained amplification segment of pGEM-T120 has SEQ ID № in the sequence table as a result: 5 nucleotide sequence, the amplification segment and the sequence to be amplified in the BTV NS1 gene (BTV NS1 gene is from 5 ' end 10-130 bit base) that show the 121bp that pGEM-T120 is entrained are in full accord, and the plasmid sequence of structure is correct.
(7) concentration determination of pGEM-T120
Get pGEM-T120 stoste 20 μ l, be diluted to 100 μ l ddH
2Among the O, measure OD with ultraviolet spectrophotometer
260Value, the concentration that the result records pGEM-T120 stoste is 31.7 μ g/mL, 1 μ l pGEM-T120 stoste contains 9 * 10
9Individual copy.
5, with TaqManProbe1 (NS1) the BTV sample being carried out real-time FQ PCR detects
(1) drawing standard curve
Is 10 with pGEM-T120 stoste by 10 * serial dilution
4, 10
5, 10
6, 10
7Individual copy carries out real-time FQPCR and detects, 25 μ l reaction systems are: 10 * PCR damping fluid, 2.5 μ l, dNTPs (2.5mM each) 2 μ l, P (NS1-1) 1 μ l (50pg), P (NS1-2) 1 μ l (50pg), TaqManProbe1 (NS1) 0.5 μ l (25pg), pGEM-T120 1 μ l, Taq enzyme 1 μ l (TaKaRa), MgCl
2(25mM) 2.5 μ l (Promega), ddH
2O 13.5 μ l.Reaction conditions is: 94 ℃ of 3min of elder generation; 94 ℃ of 30s then, 45 ℃ of 45s, 72 ℃ of 20s, totally 40 circulations.Each dilution pGEM-T120 all can produce fluorescent signal as a result, the Ct value is respectively 29.63,24.83,21.06,18.08, and amplification curve is seen Fig. 4 A, and quantitative data sees Table 1, the slope of standard curve that is obtained by amplification curve is-3.933, and typical curve is seen Fig. 5 (Y=-3.933X+45.257).
Different extent of dilution pGEM-T120 of table 1 and BTV-5,8,10,21,22, (A) fluorescent quantitative PCR
Quantitative data
(2) the real-time FQ PCR of BTV sample detects
At first the testing sample (BTV5, BTV8, BTV10, BTV21, BTV22, BTV (A)) to 6 BTV serotypes carries out real-time FQ PCR detection, outside the removing template (cDNA that forms by the RNA reverse transcription), reaction system and reaction conditions are identical with step (1), amplification curve is seen Fig. 4 B, quantitative data sees Table 1, Fig. 6 is seen in site on typical curve, its Ct value is followed successively by 22.50,35.92,32.98,26.24,28.18,28.07, and amplification initial concentration copy number is followed successively by 5.58 * 10
5, 1.73 * 10
2, 1.03 * 10
3, 6.03 * 10
4, 2.00 * 10
4, 2.12 * 10
4
Then other 2 BTV serotype samples (BTV3, BTV11) are carried out real-time FQ PCR with same procedure and detect, amplification curve is seen Fig. 6, and its Ct value is followed successively by 22.20,19.37, and the initial copy number that increases is followed successively by 7.21 * 10
5, 3.85 * 10
6
In addition, with TaqManProbe2 (NS1) above-mentioned BTV sample is carried out real-time FQ PCR and detect, conclusion is identical, shows that TaqManProbe1 of the present invention (NS1) and TaqManProbe2 (NS1) all can be used for the real-time FQ PCR detection of BTV.
Sequence table
<160>5
<210>1
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
gttctctagt?tggcaaccac?c 21
<210>2
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
gtgactgcaa?gtccattgag?g 21
<210>3
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
agttctcgtg?gcatttgcgt?aatcc 25
<210>4
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
attacgcaaa?tgccacgaga?act 23
<210>5
<211>121
<212>DNA
＜213〉blue tongue rims (bluetongue virus, BTV)
<400>5
gttctctagt?tggcaaccac?caaacatgga?gcgctttttg?agaaaataca?acatcagtgg 60
ggattacgca?aatgccacga?gaactttttt?ggctatttca?cctcaatgga?cttgcagtca 120
c 121
Claims (4)
1, be used for blue tongue rims are carried out the primer of qualitative and quantitative analysis, its upstream primer is by the SEQ ID № in the sequence table: 1 expression, downstream primer are by the SEQ ID № in the sequence table: 2 expressions.
2, the derived sequence of the described primer of claim 1.
3, derived sequence according to claim 2 is characterized in that: described derived sequence is at SEQ ID №: 1 and/or SEQ ID №: the primer sequence that obtains through replacement, disappearance or the interpolation of one to ten base on 2 the basis.
4, detect the real-time fluorescence quantitative PCR test kit of blue tongue rims, comprise the described primer that is used for blue tongue rims are carried out qualitative and quantitative analysis of claim 1.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104561382A (en) * | 2015-01-12 | 2015-04-29 | 云南省畜牧兽医科学院 | Specific primers and probe for fluorescence RT-PCR detection for bluetongue virus-16 |
| CN108342510A (en) * | 2018-03-23 | 2018-07-31 | 重庆出入境检验检疫局检验检疫技术中心 | The multiple RT-PCR kit and its detection method that BTV-11 types, 17 types, 20 types, 23 types, 24 type genotypings differentiate |
| CN108588275A (en) * | 2018-03-23 | 2018-09-28 | 重庆出入境检验检疫局检验检疫技术中心 | The multiple RT-PCR kit and its detection method of BTV-10 types, 20 types, 23 type genotypings |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1556216A (en) * | 2004-01-02 | 2004-12-22 | 武汉大学 | Detecting method of blue tongue virus and reagent box |
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2006
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101986157A (en) * | 2010-10-29 | 2011-03-16 | 中国人民解放军军事医学科学院野战输血研究所 | Novel biological bar code detection method and application thereof |
| CN104561382A (en) * | 2015-01-12 | 2015-04-29 | 云南省畜牧兽医科学院 | Specific primers and probe for fluorescence RT-PCR detection for bluetongue virus-16 |
| CN108342510A (en) * | 2018-03-23 | 2018-07-31 | 重庆出入境检验检疫局检验检疫技术中心 | The multiple RT-PCR kit and its detection method that BTV-11 types, 17 types, 20 types, 23 types, 24 type genotypings differentiate |
| CN108588275A (en) * | 2018-03-23 | 2018-09-28 | 重庆出入境检验检疫局检验检疫技术中心 | The multiple RT-PCR kit and its detection method of BTV-10 types, 20 types, 23 type genotypings |
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