CN101280343B - 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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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 molecular biology method virus be carried out primer and the probe of qualitative and quantitative analysis, 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 subject 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 occurs successively all over the world or finds this disease virus.China from 1979 since this disease occurs in Yunnan Shizong County first, successively repeatedly occur in other area again.
Blue tongue rims (bluetongue virus, BTV) are the arbovirusess that infects domestic and wild ruminating animal.BTV is the line-up of delegates of Reoviridae (Reoviridae) Orbivirus (Orbivirus), has found so far 25 BTV serotypes (Davies FG, Muugai JN, Pini A.Vet Microbiol 1992; 31:25-32), wherein find 6 serotypes (BTV2,10,11,13,17 and 24) in the U.S., find 1 serotype (BTV10) in China.The BTV genome contains the double-stranded RNA (dsRNA) of 10 sections, and by large, medium and small minute: fragment 1-3 was long segment, 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 copy relevant a kind of Nonstructural Protein with BTV by fragment 6 for NS1.Wang LF etc. studies show that, NS1 gene (claiming again S6) 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 suppose diagnosis to bluetongue according to disease symptom, epidemiology and pathological anatomical change, make a definite diagnosis separation and 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, the 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, and has been widely used in the diagnosis of heredopathia, the detection of pathogenic agent, the many-sides such as evaluation of medical jurisprudence sample since 1989 begin to use.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 specificity stronger, pollution-free (need not to use bromination second to form sediment), level of automation high.This technology is that dna probe is connected fluorescent reagent, after the PCR product is combined, by detecting the fluorescence light quantity, and carries out assistant analysis with computer software until this dna probe, 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.
Real-Time Fluorescent Quantitative PCR Technique, add a specificity fluorescent probe (TaqMan probe) when referring in the PCR reaction system, add pair of primers, probe only with the template specific combination, its binding site utilizes the whole PCR process of fluorescent signal accumulation Real-Time Monitoring between two primers.At present Taqman fluorescent probe commonly used is oligonucleotide, and its 5 ' end is marked with report fluorophor (Reporter, R), and such as FAM, VIC etc., 3 ' end is marked with cancellation fluorophor (Quencher, Q), such as TAMRA etc.When probe was complete, the fluorescent signal of reporter group emission was absorbed by quenching group, so can't detect 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, whole 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 for blue tongue rims (BTV) are carried out qualitative and quantitative analysis.
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 again the S6 gene) 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 that BTV NS1 gene is from the reverse complementary sequence of 5 ' end 110-130 bit base.Described NS1 gene refers to contain the full gene of 5 ' end non-coding region nucleotide sequence.
The primer sequence of being derived by above-mentioned primer also belongs to protection scope of the present invention.Described derived sequence refers to 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.
During the real-time fluorescence quantitative PCR that above-mentioned primer both had been applicable to each serotype of blue tongue rims detects, also can be used in the conventional PCR detection technique of this virus, its detection to as if the NS1 gene of BTV.If the cDNA that forms through reverse transcription take testing sample carries out pcr amplification as template under the guiding of above-mentioned primer, 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 carries out qualitative and quantitative analysis for to blue tongue rims.
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 pass through phosphatizing treatment.
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 that BTV NS1 gene is from the reverse complementary sequence of 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 refers to the № at SEQ ID: 3 or SEQ ID №: on 4 the basis, add, reduce the sequence that one or more bases obtain at 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 for the primer and the TaqMan probe that blue tongue rims are carried out qualitative and quantitative analysis.
The invention provides for the primer and the TaqMan probe that 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 analysis 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, the dynamic observation for the treatment of effectiveness evaluation and the state of an illness is significant.The present invention will play a significant role at the animal medicine detection field.
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 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, choose the conservative region sequence of NS1 gene 5 ' end 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, then according to comparison result design primer and TaqMan probe, sequence is as follows:
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 again the S6 gene) 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 that BTV NS1 gene is from the reverse complementary sequence of 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 that BTV NS1 gene is from the reverse complementary sequence of 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 phosphatizing treatment.
1, the processing of BTV sample
Select the BTV of 8 serotypes (being respectively BTV3, BTV5, BTV8, BTV10, BTV11, BTV21, BTV22, BTV (A) type) in BTV, blood plasma and the tissue through cell cultures as BTV sample to be measured.
With following method to processing through the BTV of cell cultures sample: inoculate first BTV to BHK-21 cell, at 37 ℃ CO
2Being cultured to cytopathy in the incubator reaches more than 80%, then under aseptic condition, sick cell is moved in the sterilization centrifuge tube, the centrifugal 30min of 8000rpm, under aseptic condition supernatant being moved into places under the room temperature temporarily in another centrifuge tube, add supernatant with behind the cell precipitation multigelation three times, the piping and druming mixing, 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 process.
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 take step 2 respectively is as template, 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 rapidly 5min on the frozen water, 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) at ice chest again, mixing, 42 ℃ of reaction 1h.
4, conventional PCR detects
Respectively take the cDNA of the synthetic different B TV sample of step 3 reverse transcription as template, 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; Then 94 ℃ of 45s, 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 expected results, shown that the conventional PCR that primer of the present invention can be used for blue tongue rims detects.
1, the cultivation of BTV and processing
Select BTV through cell cultures as BTV sample to be measured, and use with embodiment 2 in identical method to processing 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 take step 2 is as 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, then use Wizard SVGel and PCR Clean-up System (Promega) and reference reagent box specification sheets that this purpose segment is reclaimed and purifying.
(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 that legal system is standby, rotate gently mixing, place 30min on the frozen water, then 42 ℃ of heat shock 2min, put rapidly again 5min on the frozen water, 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, at 37 ℃ of lower 12-24h that cultivate.
(4) bacterium colony PCR detects
Picking 3 hickies selecting flat board to grow in the LB resistance 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, then uses high purity plasmid extraction kit (Fa Tejie) to extract plasmid, with this plasmid called after pGEM-T120.Get 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 recombinant 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
Be 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-T1201 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; Then 94 ℃ of 30s, 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, amplification curve is seen Fig. 4 A, 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).
The 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 real-time FQ PCR detection is carried out in the testing sample (BTV5, BTV8, BTV10, BTV21, BTV22, BTV (A)) of 6 BTV serotypes, outside the removing template (cDNA that is formed 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 being carried out real-time FQ PCR detects, 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
Claims (2)
1. be used for blue tongue rims are carried out the primer of qualitative detection, 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. detect the real-time fluorescence quantitative PCR test kit of blue tongue rims, comprise the primer for blue tongue rims being carried out qualitative detection claimed in claim 1.
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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 |
| CN108342510B (en) * | 2018-03-23 | 2021-08-03 | 重庆出入境检验检疫局检验检疫技术中心 | Multiple RT-PCR kit for BTV-11 type, 17 type, 20 type, 23 type and 24 type genotype typing identification and detection method thereof |
| 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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