CN102337356A - 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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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 sick viral diagnosis of pig lid tower and detection technique field, in particular, relate to sick virus RT-PCR detection kit of boar lid tower and detection method.This 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 GETV) separates acquisition by United States Army medical research department in nineteen fifty-five to getah virus 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, the FSU, also be separated to getah virus with stinging sorrow yellow-fever mosquito and the pig blood from three band kiss culexs.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 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 finding getah virus, people pay close attention to this virus pathogenic to animal 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 female mouse inoculation back institute galactopoiesis mouse of conceived 12 d is all dead.Detecting the sick main method of pig lid tower at present has pathogen separation, blood clotting and blood clotting inhibition method, and other molecular biology method for quick do not see that report is arranged both at home and abroad.The time that pathogen separation needs is long, and need carry out in three grades of laboratories of Biosafety, and the needs that specificity that blood clotting and blood clotting suppress and susceptibility can not satisfy inspection and quarantine.The present invention adopts the RT-PCR method to detect the viral nucleic acid of pig lid tower in the sample, 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 the test kit and the detection method of on the basis of conventional RT-PCR method, developing that can detect the sick virus of pig lid tower fast, with sensitivity.
Summary of the invention
The technical problem that (one) will solve
The object of the invention aims to provide the RT-PCR detection kit of boar lid tower virus, is used for the detection and the Molecule Epidemiology Investigation of pig lid tower virus.
(2) technical scheme
The cap gene of pig lid tower virus is one section conservative special sequence; Through the comparison that the pig of GenBank issue is covered the sick viral cap gene sequence of tower; Designed a pair of special primer voluntarily; 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 detection quickly and accurately 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 a 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 5U, 40U, 5U respectively by each RT-PCR reaction tubes.
No RNase enzyme distilled water, agarose, ethidium bromide and the tetrabromophenol sulfonphthalein point sample damping fluid that can also also contain in addition, chloroform, Virahol, DEPC processing in the test kit of the present invention.
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 is optimized
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, did not only have non-specificly, and amplified band is also very limpid in sight.
(3) optimization of dNTPs concentration
With final concentration is that 4 kinds of isocyatic dNTPs of 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 when total dNTPs concentration is 1 mM, is the best.
RT-PCR test kit of the present invention detects the method for pig lid tower virus, and step is following:
1, The pretreatment: get the sample of detection, the saline water that adds 600 μ L sterilization grinds, and-20 ℃～20 ℃ freeze thawing 2 times are got supernatant as subsequent use.The thorough washing sample obtains containing the suspension of virus;
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, shaken 15s, after room temperature is placed 2-3min, 12, the centrifugal 15min of 000rpm (4 ℃); Suct layer water in another new Ep pipe, add the 0.5ml Virahol, abundant mixing, behind-20 ℃ of placement 15-30min, 12, the centrifugal 10min of 000rpm (4 ℃); Supernatant discarded adds 75% ethanol 1ml, jolting mixing, thorough washing deposition, 7, the centrifugal 5min of 500rpm (4 ℃); Supernatant discarded does not have the globule on natural drying at room temperature to the tube wall, is dissolved in the 10 μ l DEPC treating water, and-70 ℃ of preservations are subsequent use.
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 subsequent use.
(2) earlier negative controls is added in the branch tubulature, the RNA that gets each sample adds in the corresponding reaction tubes, takes out positive control at last and adds in another reaction tubes, and is centrifugal behind each reaction tubes mark, takes out and puts on the PCR appearance.
(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 uv lamp, observe; Under the situation that negative hole does not have if there is the 820bpDNA band in positive hole, 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: the RNA extracting solution with in the test kit carries out extracting to viral RNA in the sample.PCR reagent pipe contains polymerase chain reaction reagent, through adding sick viral special RNA fragment primer of pig lid tower and compositions such as AMV ThermoScript II, RNase inhibitor enzyme and Taq enzyme, the RNA that is extracted in the sample is carried out specific amplification.Because institute's designed primer is that pig lid tower virus institute is peculiar; Therefore; If contain the sick virus of pig lid tower in the sample; Behind its RNA process amplification and electrophoresis and the ethidium bromide staining, UV-light detects just can detect the band of certain molecular weight so, 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 sensitive; 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 stripping technique need about a week at least, and need accomplish 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 specificity test, wherein 1, CSFV; 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, pig lid tower disease virus-positive plasmid 3 ~ 9, test sample
Embodiment
The following example is intended to further describe for example the present invention; Rather than limit the present invention by any way; Under the prerequisite that does not deviate from spirit of the present invention and principle, any change or change that those of ordinary skills that the present invention did are realized 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 delivering according to GenBank of pig lid tower, company is synthetic and be cloned in the pMD-T carrier by Dalian Takara (precious biological), 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 through 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 specificity test of embodiment 4 test kits
Each the 1 μ L of RNA that gets 3 check samples such as CSFV, encephalitis b virus, west Nile fever vaccine virus is the specificity RT-PCR amplification that template is carried out test kit, establishes blank, yin and yang attribute contrast simultaneously.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 confirm its specificity.
Electrophoresis result shows that the DNA cloning of the sick Virus Sample of pig lid tower goes out the particular segment of 820bp, and does not all have this amplified band appearance as the DNA of check sample, sees Fig. 1.
The sensitivity test of embodiment 5 test kits
The gradient that pTCAP after quantitative is carried out 10 times is respectively diluted, and detects with the RT-PCR test kit, and amplification condition is the same, establishes blank simultaneously.Amplified production carries out electrophoretic analysis with 1.0% agarose, to confirm 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 that 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., period of storage takes out when being 30,60,90,120,150,180 days, with the stability of known RT-PCR positive plasmid detection kit.Amplification condition is the same, establishes blank simultaneously.Amplified production carries out electrophoretic analysis with 1.0% agarose, to confirm its stability.The result is illustrated in 30,60,90,120,150,180 days day parts and takes out test kit respectively, 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 blank simultaneously.Amplified production carries out electrophoretic analysis with 1.0% agarose, to confirm its preservation condition.The result shows that this test kit (except that 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 to put into 4 ℃ of refrigerators then.
1, The pretreatment: get the sample of detection, the saline water that adds 600 μ L sterilization grinds, and-20 ℃～20 ℃ freeze thawing 2 times are got supernatant as subsequent use.The thorough washing sample obtains containing the suspension of virus;
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, shaken 15s, after room temperature is placed 2-3min, 12, the centrifugal 15min of 000rpm (4 ℃); Suct layer water in another new Ep pipe, add the 0.5ml Virahol, abundant mixing, behind-20 ℃ of placement 15-30min, 12, the centrifugal 10min of 000rpm (4 ℃); Supernatant discarded adds 75% ethanol 1ml, jolting mixing, thorough washing deposition, 7, the centrifugal 5min of 500rpm (4 ℃); Supernatant discarded does not have the globule on natural drying at room temperature to the tube wall, is dissolved in the 10 μ l DEPC treating water, and-70 ℃ of preservations are subsequent use.
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 subsequent use.
(2) earlier negative controls is added in the branch tubulature, the RNA that gets each sample adds in the corresponding reaction tubes, takes out positive control at last and adds in another reaction tubes, and is centrifugal behind each reaction tubes mark, takes out and puts on the PCR appearance.
(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 uv lamp, observe, the result sees Fig. 5.
Visible from Fig. 5: there is the 820bpDNA band in positive hole, and negative hole does not have, and sample well is not seen 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>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 (8)
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.
2. the sick virus RT-PCR detection kit of boar lid tower according to claim 1 is characterized in that the RNA extracting solution is a TRIZOL solution in the sample wherein.
3. according to sick virus RT-PCR detection kit of the said boar lid of claim 1 tower and application thereof, it is characterized in that described RT-PCR reaction solution, it contains the Auele Specific Primer of each 0.2 μ M of final concentration.
4. the sick virus RT-PCR detection kit of boar lid tower according to claim 1 is characterized in that RT-PCR reaction solution wherein also contains the dATP of each 0.5～5mM of final concentration, dTTP, dGTP and dTCP, the Mg of 2.0～8.0mM
2+
5. the sick virus RT-PCR detection kit of a boar according to claim 4 lid tower is characterized in that wherein dATP in the RT-PCR reaction solution, dTTP, and 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.
6. according to the sick virus RT-PCR detection kit of the said boar lid of claim 1 tower, it is characterized in that, also contain no RNase enzyme distilled water, agarose, ethidium bromide and the tetrabromophenol sulfonphthalein point sample damping fluid of chloroform, Virahol, DEPC processing.
7. according to the sick virus RT-PCR detection kit of the said boar lid of claim 1 tower, it is characterized in that described AMV ThermoScript II is that 5U/ μ L, RNase inhibitor enzyme are that 40U/ μ L and Taq enzyme are 5U/ μ.
8. the application of the sick virus RT-PCR detection kit of boar lid tower is characterized in that, may further comprise the steps:
(1) The pretreatment;
(2) detect with the arbitrary described test kit of claim 1～7, concrete operations are following:
Get the pretreated sample of 200 μ l, add 800 μ l RNA extracting solution TRIZOL, the vibration mixing, room temperature is placed 5min, adds 200 μ l chloroforms, shaken 15s, after room temperature is placed 2-3min, 12, the centrifugal 15min of 000rpm (4 ℃); Suct layer water in another new Ep pipe, add the 0.5ml Virahol, abundant mixing, behind-20 ℃ of placement 15-30min, 12, the centrifugal 10min of 000rpm (4 ℃); Supernatant discarded adds 75% ethanol 1ml, jolting mixing, thorough washing deposition, 7, the centrifugal 5min of 500rpm (4 ℃); Supernatant discarded does not have the globule on natural drying at room temperature to the tube wall, is dissolved in the 10 μ l DEPC treating water, and-70 ℃ of preservations are subsequent use;
(3) RT-PCR amplification
In a PCR reagent pipe, add the extraction of 0.5-1 μ L sample RNA template, RT-PCR reaction solution, add 0.2-1 μ L AMV ThermoScript II, RNase inhibitor enzyme and Taq enzyme respectively, instantaneous centrifugal mixing is put the PCR appearance and is increased; 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 uv lamp, observe; There is the 820bpDNA band in positive hole and under the situation that negative hole does not have; If sample well has 820 bpDNA bands, then contain the sick virus of pig lid tower in the sample, otherwise then do not have.
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| CN108950080A (en) * | 2018-08-08 | 2018-12-07 | 广东省疾病预防控制中心 | A kind of primed probe group and kit based on double fluorescent PCR method joint-detection sindbis alphavirus and getah virus |
| 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 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108950080A (en) * | 2018-08-08 | 2018-12-07 | 广东省疾病预防控制中心 | A kind of primed probe group and kit based on double fluorescent PCR method joint-detection sindbis alphavirus and getah virus |
| 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 |
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| CN102337356B (en) | 2013-02-13 |
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