CN102337356B - Swine getah virus reverse transcription-polymerase chain reaction (RT-PCR) detection kit and application thereof - Google Patents
Swine getah virus reverse transcription-polymerase chain reaction (RT-PCR) detection kit and application thereof Download PDFInfo
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- CN102337356B CN102337356B CN 201110321991 CN201110321991A CN102337356B CN 102337356 B CN102337356 B CN 102337356B CN 201110321991 CN201110321991 CN 201110321991 CN 201110321991 A CN201110321991 A CN 201110321991A CN 102337356 B CN102337356 B CN 102337356B
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Abstract
The invention provides a swine getah virus reverse transcription-polymerase chain reaction (RT-PCR) detection kit, which is used for diagnosing and detecting swine getah virus. The kit is developed by designing a specific primer according to a swine getah virus cellulose acetate propionate (CAP) gene sequence, establishing an RT-PCR detection method and optimizing an RT-PCR reaction condition. The invention also provides a method for detecting the swine getah virus by using the kit. The RT-PCR detection kit can quickly and accurately detect the swine getah virus.
Description
Technical field
The present invention relates to the sick viral diagnosis of pig lid tower and detection technique field, in particular, relate to the sick virus RT-PCR detection kit of boar lid tower and detection method.The method is sensitive, special, quick.
Background technology
Pig lid tower disease is by a kind of arthropod borne infection of killing propagation, mainly encroaches on horse and pig.Getah virus (Getah virus, GETV) separates acquisition by United States Army medical research department in nineteen fifty-five at first from Malay culex gelidus (C.gelidus), called after Getah virus, its prototype-strain are MM2021.After this on the ground such as east, South East Asia and Australia of Japan, USSR (Union of Soviet Socialist Republics), from three band kiss culexs and thorn sorrow yellow-fever mosquito and pig blood, also be separated to getah virus.This Tobamovirus Togaviridae (Togaviridae) alphavirus (Alpha virus).Red blood corpuscle to goose has very high compendency, can be at various vertebratess and arthropods proliferation in vivo.It is reported that there are the part cross reaction in the African chikungunya fever virus (Chikungunya virus) in this virus and the alphavirus and Australian Luo Si river (Ross River) virus.Getah virus contains the single-stranded RNA genome, and complete virion has 3 peptide species, and is responsive to chloroform and deoxycholate.There is Vero in permissive cell system, BHK-21, C6/36, MA-104, Hmlu, RK-13 etc.Since the self-discovery getah virus, people pay close attention to pathogenic to animal of this virus always.It is pathogenic to prove first in Japan that getah virus had pig in 1985, thinks one of cause of death of Japanese newborn piglet.The experimental infection pregnant sow proves, but this virus vertical infection and cause stillbirth; After female mouse inoculation to conceived 8d, litter size obviously reduces, and institute galactopoiesis mouse is all dead after female mouse inoculation of conceived 12 d.Detecting at present the sick main method of pig lid tower has pathogen separation, blood clotting and blood clotting inhibition method, and other molecular biology method for quick have no report both at home and abroad.The time that pathogen separation needs is long, and need to carry out in three grades of laboratories of Biosafety, and the needs that the specificity that blood clotting and blood clotting suppress and susceptibility can not satisfy inspection and quarantine.The pig that the present invention adopts the RT-PCR method to detect in the sample is covered the viral nucleic acid of tower, and is highly sensitive, only needs several hours both can obtain the result fast, is particularly suitable for inspection and quarantining for import/export inspection routine needs.The present invention is exactly test kit and the detection method that can detect fast, with sensitivity the sick virus of pig lid tower of developing on the basis of conventional RT-PCR method.
Summary of the invention
The technical problem that (one) will solve
Purpose of the present invention aims to provide the RT-PCR detection kit of boar lid tower virus, is used for the sick Detecting of pig lid tower and Molecule Epidemiology Investigation.
(2) technical scheme
The cap gene of pig lid tower virus is one section conservative special sequence, by the comparison that the pig of GenBank issue is covered the sick viral cap gene sequence of tower, designed, designed a pair of special primer, developed the RT-PCR detection kit and a kind of method of using pig lid tower virus in this test kit rapid detection sample is provided, to realize the quickly and accurately detection to the sick virus of pig lid tower.
The RT-PCR detection kit of wherein said pig lid tower virus comprises:
(1) sample RNA extracting solution
The RNA extracting solution is TRIZOL solution in the sample.
(2) RT-PCR reaction solution
Each 1～10mM of final concentration 4 in dNTPs, final concentration is primer CAP-1 and the CAP-2 of 0.1-0.5 μ M, final concentration is the Mg of 1.5～5.0mM
2+
(3) positive control
The positive plasmid pTCAP of the sick cap gene of pig lid tower.
(4) AMV ThermoScript II, RNase inhibitor enzyme and Taq enzyme add respectively 5U, 40U, 5U by each RT-PCR reaction tubes.
In addition, can also contain also in the test kit of the present invention that chloroform, Virahol, DEPC process without RNase enzyme distilled water, agarose, ethidium bromide and tetrabromophenol sulfonphthalein point sample damping fluid.
The optimizing process of the optimization reaction conditions of test kit of the present invention:
(1) primer concentration optimization
Primer concentration is chosen in 0.1～0.5 μ M carries out RT-PCR amplification test, the result shows that the primer of these several concentration can both amplify the purpose band, and when every primer amount with 20pmol/ μ L, i.e. best results during 0.2 μ M.
(2) MgCL
2
Concentration optimization
Mg is set
2+Concentration range is 2.0～8.0mM, and the result shows the Mg in this concentration range
2+Can both amplify the purpose band, and work as Mg
2+When concentration was 5.0mM, expanding effect was best, and not only without non-specific, and amplified band is also very limpid in sight.
(3) optimization of dNTPs concentration
4 kinds of isocyatic dNTPs take final concentration as 0.5～5 mM carry out the RT-PCR amplification, and the dNTPs in this concentration range all can amplify the purpose band as a result, and are the best when total dNTPs concentration is 1 mM.
RT-PCR test kit of the present invention detects the method for pig lid tower virus, and step is as follows:
1, the pre-treatment of sample: get the sample of detection, the physiological saline that adds 600 μ L sterilization grinds, and-20 ℃～20 ℃ freeze thawing 2 times are got supernatant as for subsequent use.Fully washing sample obtains containing virulent suspension;
2, sample rna extracts: get the pretreated sample of 200 μ l, add 800 μ l RNA extracting solution TRIZOL, and the vibration mixing, room temperature is placed 5min, adds 200 μ l chloroforms, violent jolting 15s, after room temperature is placed 2-3min, the centrifugal 15min(4 of 12,000rpm ℃); Suct layer water in another new Ep pipe, add the 0.5ml Virahol, abundant mixing, behind-20 ℃ of placement 15-30min, the centrifugal 10min(4 of 12,000rpm ℃); Supernatant discarded adds 75% ethanol 1ml, jolting mixing, fully washing precipitation, the centrifugal 5min(4 of 7,500rpm ℃); Supernatant discarded, natural drying at room temperature without the globule, are dissolved in 10 μ l DEPC and process in the water to the tube wall, and-70 ℃ save backup.
3, RT-PCR amplification
(1) get the RT-PCR reaction solution according to amplification number n (n=sample number+2), AMV ThermoScript II, RNase inhibitor enzyme and Taq enzyme mixing are in a centrifuge tube, and by every pipe packing, the lid upper tube cap is for subsequent use.
(2) first negative controls is added in the tubulature in minute, the RNA that gets each sample adds in the corresponding reaction tubes, takes out at last positive control and adds in another reaction tubes, and is centrifugal behind each reaction tubes mark, takes out and puts on the PCR instrument.
(3) RT-PCR amplification program: 50 ℃ of reverse transcription 30 min; 94 ℃ of 2 min makes AMV ThermoScript II inactivation; 94 ℃ of sex change 1 min, 54 ℃ of renaturation 1 min, 72 ℃ are extended 1 min, so carry out 30 circulations; Last 72 ℃ are extended 10 min, should set up the positive and negative control simultaneously;
4, the RT-PCR amplified production is analyzed
Get the sample after 10-20 μ L increases, adopt in the sepharose of 1.2-1.5%, in the ethidium bromide solution of 0.5-1 μ g/mL, dye after electrophoresis 30-40 minute under the 120-150V, under ultraviolet lamp, observe, if there is the 820bpDNA band in positive hole negative hole without situation under, test is set up.Sample well has the 820bpDNA band, contains the sick virus of pig lid tower in the interpret sample, otherwise does not then have.
(3) beneficial effect
Positively effect of the present invention is: test kit preparation rationally, simple, the RT-PCR reaction condition optimization of preparation, high specificity, susceptibility be high, and the result judges objective and accurate, and the sick viral nucleic acid of pig lid tower is had diagnostic effect.
Cardinal principle of the present invention is: with the RNA extracting solution in the test kit viral RNA in the sample is carried out extracting.The PCR Reagent Tube contains polymerase chain reaction reagent, by adding the sick viral special RNA fragment primer of pig lid tower and the compositions such as AMV ThermoScript II, RNase inhibitor enzyme and Taq enzyme, the RNA that extracts in the sample is carried out specific amplification.Because designed primer is that pig lid tower virus institute is peculiar, therefore, if contain the sick virus of pig lid tower in the sample, so behind its RNA process amplification and electrophoresis and the ethidium bromide staining, UV-light detects just can detect the band of certain molecular weight, if there is not the sick virus of pig lid tower the band of same molecular weight also just can not occur in the sample.
The present invention has following advantage:
The present invention is covered the sick viral special primer of tower the design pig and is also successfully set up on the basis of the viral RT-PCR detection method of pig lid tower, further be developed into the RT-PCR detection kit of Simple fast sensitivity, compare with other the sick virus detection techniques of pig lid tower, the outstanding feature of the inventive method is: 1) detect fast, can learn detected result in 3-4 hour, and other method such as viral isolation technique need about a week at least, and need to finish in three grades of laboratories of Biosafety; Be particularly suitable for inspection and quarantining for import/export inspection routine needs; 2) the present invention has guaranteed the accuracy and the specificity that detect according to the peculiar one section conserved sequence design special primer of pig lid tower virus and the comparison of increasing.
Description of drawings
Fig. 1. RT-PCR test kit specific test, wherein 1, Pestivirus suis; 2, encephalitis b virus; 3, west Nile fever vaccine virus; 4, the sick viral pTCAP of pig lid tower; 5, negative control; 6, Marker DL2000;
Fig. 2. the sensitivity test of RT-PCR test kit, wherein 1, negative control; 2,3,4,5,6,7 are respectively 0.1pg, 0.2pg.0.4pg, 0.6pg, 0.8pg, 1.0 pg; 8, Marker DL2000;
Fig. 3. the stability test of RT-PCR test kit; 1, Marker DL2000; 2, negative control; 3 ~ 8 preserve respectively 30,60,90,120,150,180 days;
Fig. 4. the test of RT-PCR test kit preservation condition; Wherein 1～4 preserved respectively 1 month, 3 months, 6 months, 9 months; 5, positive control; 6, negative control; 7, Marker DL2000;
Fig. 5. RT-PCR test kit test sample; Wherein 1, standard molecular weight DL-2000; 2, negative control; 3, the sick virus-positive plasmid 3 ~ 9 of pig lid tower, test sample
Embodiment
The following example is intended to further describe for example the present invention, rather than limit by any way the present invention, under the prerequisite that does not deviate from the spirit and principles in the present invention, any change or change that those of ordinary skills that the present invention is done realize easily all will fall within the claim scope that awaits the reply of the present invention.
The preparation of the sick viral cap gene positive colony of pig lid tower
The sick viral cap gene sequence of pig lid tower of delivering according to GenBank, precious biological by Dalian Takara() company is synthetic and be cloned in the pMD-T carrier, and is synthetic
The positive plasmid pTCAP of the sick cap gene of pig lid tower, turn out to be the sick viral cap gene of pig lid tower by order-checking.Concrete gene order:
atcggatccatgaattacattccaactcaaaccttttacggacgccgttggcgaccacgcccggcgtaccgtccatggcg
ggtgccgatgcagccggccccacccatggtgattcctgagctgcaaactccgatcgtccaggcccaacagatgcagcagc
taatcagtgcagtttctgccctgacgaccaagcaaaatggcaaagcaccgaagaagccgaagaaaaagccgcaaaaagcg
aaggctaagaaaaacgaacagcaaaagaagaacgagaacaagaaaccaccacctaagcagaagaatccggctaagaagaa
gaaaccaggaaaaagggaacgcatgtgcatgaagatagagaatgattgcatcttcgaggtcaagcttgacggtaaggtca
cgggatacgcctgcctagtcggggataaagtgatgaagccggcacacgtcaaaggtgtgatcgacaaccccgacctagcg
aagcttacctacaagaaatcgagcaagtatgacctggagtgcgcacagataccagtgcacatgaagtcagatgcttcaaa
gtacacccatgaaaaaccggaagggcactacaattggcatcacggtgcagtgcagtacagcggtggcaggttcacaatcc
cgacaggcgcaggtaaaccaggagacagcggccggccgatcttcgacaacaaaggacgcgtggtggccattgtcctggga
ggggccaacgaaggagccaggactgccctatccgtcgtgacctggaccaaagacatggtcacacggtacaccccagaagg
aacagaagaatgggaattcgag
The composition of embodiment 1 test kit
Test kit contains sample RNA extracting solution 20ml, PCR reaction tubes (5 pipe) (25 μ L/ reaction * 10), and dATP wherein, dTTP, dGTP and dTCP final concentration are 1mM, the final concentration of primer CAP-1 and CAP-2 is 0.2 μ M, Mg
2+Final concentration is 5.0mM.Other reagent: chloroform, Virahol 10mL, agarose 10g, ethidium bromide (10 μ g/ μ L) 100 μ L and 6% tetrabromophenol sulfonphthalein point sample damping fluid, 100 μ L, AMV ThermoScript II, RNase inhibitor enzyme and Taq enzyme be 10 μ L respectively; The positive plasmid 20 μ L of 1 sick viral cap gene of pig lid tower.
The composition of embodiment 2 test kits
Test kit contains sample RNA extracting solution 20ml, PCR reaction tubes (5 pipe) (25 μ L/ reaction * 10), and dATP wherein, dTTP, dGTP and dTCP final concentration are 1mM, the final concentration of primer CAP-1 and CAP-2 is 0.1 μ M, Mg
2+Final concentration is 5.0mM.Other reagent: chloroform, Virahol 10mL, agarose 10g, ethidium bromide (10 μ g/ μ L) 100 μ L and 6% tetrabromophenol sulfonphthalein point sample damping fluid, 100 μ L, AMV ThermoScript II, RNase inhibitor enzyme and Taq enzyme be 10 μ L respectively; The positive plasmid 20 μ L of 1 sick viral cap gene of pig lid tower.
The composition of embodiment 3 test kits
Test kit contains sample RNA extracting solution 20ml, PCR reaction tubes (5 pipe) (25 μ L/ reaction * 10), and dATP wherein, dTTP, dGTP and dTCP final concentration are 1mM, the final concentration of primer CAP-1 and CAP-2 is 0.5 μ M, Mg
2+Final concentration is 5.0mM.Other reagent: chloroform, Virahol 10mL, agarose 10g, ethidium bromide (10 μ g/ μ L) 100 μ L and 6% tetrabromophenol sulfonphthalein point sample damping fluid, 100 μ L, AMV ThermoScript II, RNase inhibitor enzyme and Taq enzyme be 10 μ L respectively; The positive plasmid 20 μ L of 1 sick viral cap gene of pig lid tower.
Following examples, the test kit of employing be among the embodiment 1-3 any.
The specific test of embodiment 4 test kits
Each the 1 μ L of RNA that gets 3 check samples such as Pestivirus suis, encephalitis b virus, west Nile fever vaccine virus is the specificity RT-PCR amplification that template is carried out test kit, establishes simultaneously blank, yin and yang attribute contrast.RT-PCR amplification condition: 50 ℃ of reverse transcription 30 min; 94 ℃ of 2 min makes AMV ThermoScript II inactivation; 94 ℃ of sex change 1 min, 54 ℃ of renaturation 1 min, 72 ℃ are extended 1 min, so carry out 30 circulations; Last 72 ℃ are extended 10 min, and amplified production carries out electrophoretic analysis with 1.0% agarose, to determine its specificity.
Electrophoresis result shows that the DNA cloning of the sick Virus Sample of pig lid tower goes out the specific fragment of 820bp, and the DNA of sample all occurs without this amplified band in contrast, sees Fig. 1.
The sensitivity test of embodiment 5 test kits
The gradient that pTCAP after quantitative is carried out respectively 10 times is diluted, and detects with the RT-PCR test kit, and amplification condition is the same, establishes simultaneously blank.Amplified production carries out electrophoretic analysis with 1.0% agarose, to determine its susceptibility.Electrophoresis result shows, detects the DNA concentration that lower limit is low to moderate 0.2pg, all can amplify clear and legible band, sees Fig. 2.
The stability test of embodiment 6 test kits
Except AMV ThermoScript II, RNase inhibitor enzyme and Taq enzyme, the RT-PCR reaction solution, outside the positive template-20 ℃ storage, sample RNA extracting solution and all the other reagent thereof are all preserved under 4 ℃ of conditions.When being 30,60,90,120,150,180 days, period of storage takes out, with the stability of known RT-PCR positive plasmid detection kit.Amplification condition is the same, establishes simultaneously blank.Amplified production carries out electrophoretic analysis with 1.0% agarose, to determine its stability.The result shows at 30,60,90,120,150,180 days day parts and takes out respectively test kit, carries out the RT-PCR amplification with the sick virus-positive plasmid of pig lid tower, all can amplify bright band, and specific band nothing but, and blank is all negative, sees Fig. 3.
The preservation condition test of embodiment 7 test kits
To detect known positive plasmid in room temperature, 4 ℃ and-20 ℃ of test kits (AMV ThermoScript II, RNase inhibitor enzyme and Taq enzyme are-20 ℃ of preservations all the time) of preserving 1 month, 3 months, 6 months, 9 months.Amplification condition is the same, establishes simultaneously blank.Amplified production carries out electrophoretic analysis with 1.0% agarose, to determine its preservation condition.The result shows that this test kit (except the Taq enzyme) can preserve 6 months at least under room temperature, 4 ℃ and-20 ℃, all can amplify bright band, and specific band nothing but, and blank is all negative.AMV ThermoScript II, RNase inhibitor enzyme and Taq enzyme in the suggestion test kit, the RT-PCR reaction solution, positive template-20 ℃ storage, sample RNA extracting solution and all the other reagent thereof are all preserved under 4 ℃ of conditions, and the effect that detects like this can be better, sees Fig. 4.
Gathered certain pig farm ight soil swab sample, and totally 121 parts of pig product samples, numbering, it is to be checked then to put into 4 ℃ of refrigerators.
1, the pre-treatment of sample: get the sample of detection, the physiological saline that adds 600 μ L sterilization grinds, and-20 ℃～20 ℃ freeze thawing 2 times are got supernatant as for subsequent use.Fully washing sample obtains containing virulent suspension;
2, sample rna extracts: get the pretreated sample of 200 μ l, add 800 μ l RNA extracting solution TRIZOL, and the vibration mixing, room temperature is placed 5min, adds 200 μ l chloroforms, violent jolting 15s, after room temperature is placed 2-3min, the centrifugal 15min(4 of 12,000rpm ℃); Suct layer water in another new Ep pipe, add the 0.5ml Virahol, abundant mixing, behind-20 ℃ of placement 15-30min, the centrifugal 10min(4 of 12,000rpm ℃); Supernatant discarded adds 75% ethanol 1ml, jolting mixing, fully washing precipitation, the centrifugal 5min(4 of 7,500rpm ℃); Supernatant discarded, natural drying at room temperature without the globule, are dissolved in 10 μ l DEPC and process in the water to the tube wall, and-70 ℃ save backup.
3, RT-PCR amplification
(1) get the RT-PCR reaction solution according to amplification number n (n=sample number+2), AMV ThermoScript II, RNase inhibitor enzyme and Taq enzyme mixing are in a centrifuge tube, and by every pipe packing, the lid upper tube cap is for subsequent use.
(2) first negative controls is added in the tubulature in minute, the RNA that gets each sample adds in the corresponding reaction tubes, takes out at last positive control and adds in another reaction tubes, and is centrifugal behind each reaction tubes mark, takes out and puts on the PCR instrument.
(3) RT-PCR amplification program: 50 ℃ of reverse transcription 30 min; 94 ℃ of 2 min makes AMV ThermoScript II inactivation; 94 ℃ of sex change 1 min, 54 ℃ of renaturation 1 min, 72 ℃ are extended 1 min, so carry out 30 circulations; Last 72 ℃ are extended 10 min, should set up the positive and negative control simultaneously;
4, the RT-PCR amplified production is analyzed
Get the sample after 10-20 μ L increases, adopt in the sepharose of 1.2-1.5%, in the ethidium bromide solution of 0.5-1 μ g/mL, dye after electrophoresis 30-40 minute under the 120-150V, under ultraviolet lamp, observe, the results are shown in Figure 5.
As seen from Figure 5: there is the 820bpDNA band in positive hole, negative hole without, sample well has no the 820bpDNA band, does not have the sick virus of pig lid tower in the interpret sample.
Detected result: do not detect the sick virus of pig lid tower in institute's sample thief.
SEQUENCE LISTING
＜110〉Jiangsu Bureau of Emigration ﹠ Ingression Examination ﹠. Quarantine, PRC
＜120〉the sick virus RT-PCR detection kit of pig lid tower and application thereof
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 822
<212> DNA
＜213〉artificial sequence
<400> 1
atcggatcca tgaattacat tccaactcaa accttttacg gacgccgttg gcgaccacgc 60
ccggcgtacc gtccatggcg ggtgccgatg cagccggccc cacccatggt gattcctgag 120
ctgcaaactc cgatcgtcca ggcccaacag atgcagcagc taatcagtgc agtttctgcc 180
ctgacgacca agcaaaatgg caaagcaccg aagaagccga agaaaaagcc gcaaaaagcg 240
aaggctaaga aaaacgaaca gcaaaagaag aacgagaaca agaaaccacc acctaagcag 300
aagaatccgg ctaagaagaa gaaaccagga aaaagggaac gcatgtgcat gaagatagag 360
aatgattgca tcttcgaggt caagcttgac ggtaaggtca cgggatacgc ctgcctagtc 420
ggggataaag tgatgaagcc ggcacacgtc aaaggtgtga tcgacaaccc cgacctagcg 480
aagcttacct acaagaaatc gagcaagtat gacctggagt gcgcacagat accagtgcac 540
atgaagtcag atgcttcaaa gtacacccat gaaaaaccgg aagggcacta caattggcat 600
cacggtgcag tgcagtacag cggtggcagg ttcacaatcc cgacaggcgc aggtaaacca 660
ggagacagcg gccggccgat cttcgacaac aaaggacgcg tggtggccat tgtcctggga 720
ggggccaacg aaggagccag gactgcccta tccgtcgtga cctggaccaa agacatggtc 780
acacggtaca ccccagaagg aacagaagaa tgggaattcg ag 822
<210> 2
<211> 30
<212> DNA
＜213〉artificial sequence
<400> 2
cgcctcgaga tgaattacat tccaactcaa 30
<210> 3
<211> 30
<212> DNA
＜213〉artificial sequence
<400> 3
ctcctgcagc cattcttctg ttccttctgg 30
Claims (1)
1. the sick virus RT-PCR detection kit of boar lid tower is characterized in that it contains:
(1), RNA extracting solution in the sample;
(2), RT-PCR single stage method reaction solution, it contains the Auele Specific Primer of each 0.1-0.5 μ M of final concentration:
CAP-1: 5'- cgcctcgagatgaattacattccaactcaa-3'
CAP-2: 5'- ctcctgcagccattcttctgttccttctgg-3'
(3), the positive plasmid pTCAP of the sick viral cap gene of pig lid tower;
(4), AMV ThermoScript II, RNase inhibitor enzyme and Taq enzyme.
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| CN 201110321991 CN102337356B (en) | 2011-10-21 | 2011-10-21 | Swine getah virus reverse transcription-polymerase chain reaction (RT-PCR) detection kit and application thereof |
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| CN108950080B (en) * | 2018-08-08 | 2021-11-19 | 广东省疾病预防控制中心 | Primer probe set and kit for jointly detecting Sindbis virus and Getavirus based on dual-fluorescence PCR method |
| CN111363854A (en) * | 2020-04-24 | 2020-07-03 | 云南省畜牧兽医科学院 | Nucleic acid group for detecting Japanese encephalitis virus and Getavirus and detection method |
| CN112662819A (en) * | 2021-01-28 | 2021-04-16 | 沈阳农业大学 | Detection kit and detection method for porcine gata virus NSP1 gene |
| CN113943837A (en) * | 2021-12-03 | 2022-01-18 | 广东方道基因生物科技有限公司 | Porcine gatifloxacin virus loop-mediated isothermal amplification detection primer group and kit |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP1386926A1 (en) * | 2002-07-29 | 2004-02-04 | Bioxtal | Methods for producing labelled recombinant polypeptides and uses thereof |
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| EP1386926A1 (en) * | 2002-07-29 | 2004-02-04 | Bioxtal | Methods for producing labelled recombinant polypeptides and uses thereof |
Non-Patent Citations (8)
| Title |
|---|
| Complete sequence characterization of isolates of Getah virus (genus Alphavirus, family Togaviridae) from China;You-gang Zhai et al.;《Journal of General Virology》;20081231;第89卷;1446-1456 * |
| You-gangZhaietal..CompletesequencecharacterizationofisolatesofGetahvirus(genusAlphavirus family Togaviridae) from China.《Journal of General Virology》.2008 |
| 我国分离的盖塔病毒衣壳蛋白基因和3’非翻译区分子特征研究;翟友刚等;《病毒学报》;20070731;第23卷(第4期);270-275 * |
| 我国新分离盖塔病毒的部分基因组分子特征研究;陈维欣等;《中华微生物学和免疫学杂志》;20100531;第30卷(第5期);400-404 * |
| 海南岛两株甲病毒基因组3’末端核苷酸序列的克隆与分析;赵文忠等;《中华实验和临床病毒学杂志》;20000930;第14卷(第3期);213-217 * |
| 翟友刚等.我国分离的盖塔病毒衣壳蛋白基因和3’非翻译区分子特征研究.《病毒学报》.2007,第23卷(第4期),270-275. |
| 赵文忠等.海南岛两株甲病毒基因组3’末端核苷酸序列的克隆与分析.《中华实验和临床病毒学杂志》.2000,第14卷(第3期),213-217. |
| 陈维欣等.我国新分离盖塔病毒的部分基因组分子特征研究.《中华微生物学和免疫学杂志》.2010,第30卷(第5期),400-404. |
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