CN1563411A - Kit of testing garlic virus and testing method - Google Patents
Kit of testing garlic virus and testing method Download PDFInfo
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- CN1563411A CN1563411A CN 200410014721 CN200410014721A CN1563411A CN 1563411 A CN1563411 A CN 1563411A CN 200410014721 CN200410014721 CN 200410014721 CN 200410014721 A CN200410014721 A CN 200410014721A CN 1563411 A CN1563411 A CN 1563411A
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Abstract
This invention relates to garlic virus RT-PCR test reagent box and its test method specially used in the test of GMV and GCLV, among which, the box includes: an inverse transcription reagent box, a PCR box and a positive control. The test method includes reaction of the inverse transcription, PCR reaction and electrophoresis detection, the electrophoresis containing GMV test result is the amplified 32Tbp strip and the result with GCLV is the amplified 469bp strip. The PCR part of the reagent box adopts small reaction system.
Description
One, technical field
The invention belongs to garlic virus RT-PCR detection kit and detection method thereof, be exclusively used in the detection of garlic mosaic virus (GMV), garlic cryptovirus (GCLV).
Two, technical background
Garlic (Alli μ m sativum L.) is a kind of important export vegetables of China.But garlic belongs to pollen abortion type plant, mainly carries out vegetative propagation by bulb in the production.Therefore, virus constantly accumulation propagation in the garlic nutrient organ causes deterioration of strains, makes output and downgrade, has a strong impact on production and the outlet of China garlic.The garlic virus disease belongs to refractory and treats disease, does not also have a kind of effective medicine so far.The garlic mosaic virus disease is garlic mosaic virus (GMV, Garlic mosaic virus) infects the disease that the garlic plant causes, its field plant symptom presents remarkable yellow cord floral leaf, blade is distortion, folding and atrophing state, the virion daughter is 700～800 μ m, passes poison with bulb, juice, aphid, thrips and nematode etc.Garlic cryptovirus disease is that garlic cryptovirus (GCLV, Garlic commonlatent virus) infects the disease that the garlic plant causes, its field plant does not show any band toxication shape, does not promptly have disease band poison, does not influence output; But under field conditions (factors), the most and garlic mosaic virus mixed infection garlic plant of this virus, the diseased plant of being infected usually presents system's flower leaf paresthesia.The virion daughter is 580～720 μ m, passes poison with bulb, juice and aphid etc.At present, solving garlic virus mainly is to come production detoxification garlic by tissue culture method, becomes the problem that should urgently solve in the production of detoxification garlic so set up sensitive, quick, easy, business-like detection technique.
Since 30 generations of 20th century, the detection of garlic virus mainly is that outward appearance symptom and the plant indicator according to plant itself carries out, and these methods take longer, and sensitivity is lower, and is subject to seasonal restrictions.The seventies in 20th century, along with the development of immunological technique, its application in the detection of garlic virus also more and more widely to the initial stage eighties.The susceptibility that immunological method detects garlic virus improves greatly, and specificity also strengthens, and detection speed also is greatly improved, and has become the detection method of the most frequently used garlic virus at present.
After 20th century the mid-80s, molecular biological technology has obtained development fast, for the detection of garlic virus provide a kind of higher than above-mentioned several method sensitivity, specificity is stronger, speed method faster.
The ultimate principle of RT-PCR technology for detection virus is at first to understand fully all or part of sequence of viral RNA to be checked, and with this synthetic a pair of Auele Specific Primer (also available random primer); Then viral RNA is carried out reverse transcription and pcr amplification; Amplified production is carried out electrophoresis, dyeing, produce the specific DNA bands of a spectrum, can detect virus in view of the above.The RT-PCR technology relatively has the following advantages as a kind of international virus detection techniques and other detection method: 1. sensitiveer than ELISA, specificity is better, and the result is more reliable; 2. compare "dead" danger with the making nucleic acid molecular hybridization technology; 3. compare the plant virus and the extensive sample that can detect the fg order of magnitude with the dsRNA electrophoretic technique.Therefore this technology has broad application prospects in garlic virus context of detection.
Particularly Japan has begun to adopt Protocols in Molecular Biology to detect garlic virus in recent years both at home and abroad, and is respond well, and report utilizes the garlic virus of RT-PCR technology for detection that garlic cryptovirus (ArchVirol.1998,143:1093～1107) such as (GCLV) is arranged.But, so far there is not garlic RT-PCR virus detection kit to occur, an one step RT-PCR test kit of selling on the market, reverse transcription reaction and one step of PCR reaction carry out, the time that just must start anew and cause and the waste of medicine in case a certain link is made mistakes, Chang Yong RT-PCR reaction system is the big system of 100 μ l simultaneously, causes the waste of a large amount of medicines, and complicated operation pollutes easily, and causes test failure.
Three, summary of the invention
Technical problem: the object of the present invention is to provide a kind of garlic virus RT-PCR detection kit and detection method thereof, set up the RT-PCR detection kit and the using method of garlic GMV, GCLV virus, for garlic virus quarantine and the detection of garlic detoxic seedling virus provide a kind of quick, accurate, easy to operate, sensitive, detection method that specificity is good.
Technical scheme:
1 one kinds of garlic virus RT-PCR detection kit comprise: the reverse transcription test kit, and PCR test kit and positive control three parts:
1) reverse transcription test kit contains the water of PCR reagent pipe 1 and no RNA enzyme, and wherein PCR reagent pipe 1 includes 10 * buffer, dNTP, MgCl
2, RNA enzyme inhibition factor, oligo-dT primer, ThermoScript II;
Per 1 component, 7.5～15 μ l reverse transcription reagent preparation:
Composition reagent concentration consumption
10×buffer 15mmol/μl 1～3μl
dNTP 10mmol/μl 1～3μl
MgCl
2 25mmol/l 3～4μl
RNA enzyme inhibition factor 40U/ μ l 0.5～1 μ l
Oligo-dT primer 0.1ug/ μ l 1～2 μ l
ThermoScript II 200U/ μ l 1～2 μ l
2) PCR test kit contains PCR reagent pipe 2, reagent A and reagent B, and wherein PCR reagent pipe 2 includes
10 * PCR buffer, MgCl
2, dNTP, the Taq enzyme,
Per 1 component, 2.7～8.5 μ l PCR reagent preparation:
Composition reagent concentration consumption
10×PCR?buffer 15mmol/μl 1～3μl
MgCl
2 25mmol/μl 1～3μl
dNTP 10mmol/μl 0.5～2μl
Taq enzyme 5U/ μ l 0.2～0.5 μ l
The reagent A preparation:
Primer GMVP1:5 '-TGGAAGGCAAAGTTACAGGAGG-3 ' and
Primer GMVP2:5 '-GTCAAATGCGTACCGTGCGAGA-3 '
Be dissolved into 5～20pmol/ μ l with pure water;
Reagent B preparation:
Primer GCLVP1:5 '-AGTTGCGACTGCTGAAGACC-3 ' and
Primer GCLVP2:5 '-GCCATTACGCCTATTCCTAC-3 '
Be dissolved into 5～20pmol/ μ l with pure water;
3) positive control for what extract, contains the RNA of GMV, two kinds of viral RNAs of GCLV from the sick leaf of garlic.The detection method of 2 garlic virus RT-PCR detection kit:
(1) reverse transcription reaction: in PCR reagent pipe 1, add the water of total RNA sample of 0.1～1 μ g garlic and no RNA enzyme, make the total reaction system reach 20 μ l, carry out reverse transcription reaction then, obtain cDNA, its condition is 42 ℃, 30 minutes, 95 ℃, 5 minutes, and 4 ℃, 5 minutes;
(2) PCR reaction: the cDNA that obtains is transcribed in negate, adds in the PCR reagent pipe 2, adds reagent A or reagent B again, add pure water then and make the total reaction system reach 20 μ l, carry out the PCR reaction then, its parameter is 94 ℃ of sex change 1 minute, annealed 1 minute for 57 ℃, 72 ℃ were extended 1 minute, and carried out 32 circulations;
(3) electrophoresis detection: in the PCR reagent pipe 2 that the PCR reaction finishes, add the electrophoresis sample-loading buffer, extract reaction solution and on the gel that contains agarose and ethidium bromide (EB), carry out electrophoresis, the observation of taking pictures on gel imaging system then: the electrophoresis detection result who contains GMV virus garlic samples is the electrophoretic band that amplifies 327bp, and the electrophoresis detection result who contains GCLV virus garlic samples is the electrophoretic band that amplifies 469bp.
Beneficial effect: garlic virus RT-PCR detection kit provided by the present invention and detection method thereof compared with prior art, have following advantage and positively effect:
1) test kit of the present invention is identified through specificity and sensitivity, proves that specificity and sensitivity are higher.
2) reagent is sub-divided in two PCR reagent pipes, has avoided the pollution in the operating process, and is easy to use.
3) test kit reverse transcription and PCR carry out in two steps, compare with an one step RT-PCR test kit of selling on the market, avoided in case because a part of the make mistakes time that just must start anew to cause and the waste of medicine, and the laggard performing PCR of reverse transcription can be provided with repetition when reacting, compare with an one step RT-PCR test kit, increased result's reliability.
4) test kit PCR partly adopts the little reaction system of 20 μ l, compares the saving medicine with the big reaction system of 100 μ l that RT-PCR is commonly used, can reduce cost.
The present invention provides a kind of quick, accurate, easy to operate, sensitive, test kit that specificity is good for garlic virus detects, can quicken the promotion and application of detoxification garlic greatly.
Four, description of drawings
The RT-PCR amplified band of Fig. 1 garlic GMV virus
M is molecular weight marker, and 1,2,3,4 is the RT-PCR amplified band that contains garlic samples Fig. 2 garlic GCLV virus of garlic GMV virus
M is molecular weight marker, and 1,2,3,4 is that the RT-PCR susceptibility that contains garlic samples Fig. 3 garlic GMV virus of garlic GCLV virus is identified
M is molecular weight marker, and 1,2,3,4,5 are respectively by 1: 10: 20: the RT-PCR susceptibility of 50: 100 dilution proportion Fig. 4 garlic GCLV virus is identified
M is molecular weight marker, and 1,2,3,4,5 are respectively by 1: 10: 20: 50: 100 dilution proportion
Five, embodiment
1, the PCR primer design is with synthetic
According to the coat protein gene of GMV (Genbank database numbering: AF500074) and the coat protein gene of GCLV (Genbank database numbering: AF228416) design primer, utilize the dna synthesizer Synthetic 2 to primer GMVP1, GMVP2 and GCLVP1, GCLVP2.The nucleotide sequence of synthetic primer GMVP1, GMVP2 amplification between the 277bp～603bp of GMV coat protein gene, length is 327bp; The nucleotide sequence of synthetic primer GCLVP1, GCLVP2 amplification between the 402bp～870bp of GCLV coat protein gene, length is 469bp.
2, the assembling of RT-PCR test kit
(1) assembling of reverse transcription test kit
Reverse transcription reagent is pressed table 1 preparation, and is divided in the PCR reagent pipe 1 (handling and sterilization with DEPC).
(2) assembling of PCR test kit
1. PCR reagent is pressed table 2 preparation, and is divided in the PCR reagent pipe 2.
2. reagent A preparation, primer GMVP1 (5 '-TGGAAGGCAAAGTTACAGGAGG-3 ') and primer GMVP2 (5 '-GTCAAATGCGTACCGTGCGAGA-3 ') be dissolved into 10pmol/ μ l with pure water.
3. reagent B preparation, primer GCLVP1 (5 '-AGTTGCGACTGCTGAAGACC-3 ') and primer GCLVP2 (5 '-GCCATTACGCCTATTCCTAC-3 ') be dissolved into 10pmol/ μ l with pure water.
(3) positive control extracts from the sick leaf of garlic, contains the RNA of GMV, two kinds of viral RNAs of GCLV.
3, the detection method of garlic virus RT-PCR detection kit
(1) reverse transcription reaction adds the water of the total RNA sample of 0.1～1 μ g garlic and no RNA enzyme in PCR reagent pipe 1, make the total reaction system reach 20 μ l, carries out reverse transcription reaction then, and its condition is 42 ℃, 30 minutes, and 95 ℃, 5 minutes, 4 ℃, 5 minutes.
(2) PCR reaction, get the cDNA that 1 μ l reverse transcription obtains, add in the PCR reagent pipe 2, add 5 μ l reagent A or reagent B again, add pure water then and make the total reaction system reach 20 μ l, carry out the PCR reaction then, its parameter is 94 ℃ of sex change 1 minute, annealed 1 minute for 57 ℃, 72 ℃ were extended 1 minute, and carried out 32 circulations.
(3) electrophoresis detection, in the PCR reagent pipe B that the PCR reaction finishes, add 6 times of electrophoresis sample-loading buffers 0.25% of 3 μ l (w/w) bromjophenol blue solution, get 10 μ l and on the glue that contains 2.0% (w/w) agarose and 10 μ l/100ml ethidium bromides (EB), carry out electrophoresis, the observation of on gel imaging system, taking pictures then.
Result of implementation is as follows:
1 specificity is identified
Utilize the compound garlic blade that infects of multiple virus (OYDV, GCLV, GMV, SLV) to detect the specificity of this test kit, the result shows, using method according to test kit, that GCLV and GMV have obtained is clear, amplified band accurately, utilize sequence length that Labwork4.0 measures according to molecular weight marker and desired design length 327bp (Fig. 1) and 469bp (Fig. 2) to match, prove that this test kit has higher specificity.
2 sensitivity are identified
The total RNA extracting solution of garlic that will contain GCLV, GMV viral RNA was by 1: 10: 20: 50: 100 dilution proportion, be equivalent to 0.8 μ g, 0.08 μ g, 0.04 μ g, 0.016 μ g, 0.008 μ g utilizes this test kit and increases through RT-PCR by using method, the result as shown in Figure 3, Figure 4, by Fig. 3, Fig. 4 as can be known, except the RNA that dilutes 100 times can not amplify the band, other can increase out, shows that detecting garlic virus with RT-PCR has higher sensitivity.
In addition, reverse transcription of this test kit and PCR carry out in two steps, compare with an one step RT-PCR test kit of selling on the market, avoided in case because a part of the make mistakes time that just must start anew to cause and the waste of medicine, and the laggard performing PCR of reverse transcription can be provided with repetition when reacting, compare with an one step RT-PCR test kit, increased result's reliability.Simultaneously, this test kit PCR partly adopts the little reaction system of 20 μ l, compare the saving medicine with the big reaction system of 100 μ l that RT-PCR is commonly used, can reduce cost, all reagent of this test kit are sub-divided in two PCR reagent pipes, compare with commonly used RT-PCR and to have avoided the pollution in the operating process, easy to use.
Table 1 reverse transcription reagent
1 of composition reagent concentration (μ l)
10×buffer 15mmol/μl 2
dNTP 10mmol/μl 2
MgCl
2 25mmol/l 4
RNA enzyme inhibition factor 40U/ μ l 0.5
Oligo-dT primer 0.1ug/ μ l 1
MMLV ThermoScript II 200U/ μ l 1
Cumulative volume 10.5
Table 2 PCR reagent
1 of composition reagent concentration (μ l)
10×PCR?buffer 15mmol/μl 2
MgCl
2 25mmol/μl 1.6
dNTP 10mmol/μl 0.5
Taq enzyme 5U/ μ l 0.2
Cumulative volume 4.3
Claims (3)
1, a kind of garlic virus RT-PCR detection kit comprises: the reverse transcription test kit, and PCR test kit and positive control three parts:
1) reverse transcription test kit contains the water of PCR reagent pipe 1 and no RNA enzyme, and wherein PCR reagent pipe 1 includes 10 * buffer, dNTP, MgCl
2, RNA enzyme inhibition factor, oligo-dT primer, ThermoScript II;
2) PCR test kit contains PCR reagent pipe 2, reagent A and reagent B, and wherein PCR reagent pipe 2 includes 10 * PCR buffer, MgCl
2, dNTP, the Taq enzyme, reagent A includes primer GMVP1:5 '-TGGAAGGCAAAGTTACAGGAGG-3 '
Primer GMVP2:5 '-GTCAAATGCGTACCGTGCGAGA-3 ' reagent B includes primer GCLVP1:5 '-AGTTGCGACTGCTGAAGACC-3 ',
Primer GCLVP2:5 '-GCCATTACGCCTATTCCTAC-3 '
3) positive control for what extract, contains the RNA of GMV, two kinds of viral RNAs of GCLV from the sick leaf of garlic.
2, garlic virus RT-PCR detection kit according to claim 1 is characterized in that,
1) per 1 component, 7.5～15 μ l reverse transcription reagent preparation:
Composition reagent concentration consumption
10×buffer 15mmol/μl 1～3μl
dNTP 10mmol/μl 1～3μl
MgCl
2 25mmol/l 3～4μl
RNA enzyme inhibition factor 40U/ μ l 0.5～1 μ l
Oligo-dT primer 0.1ug/ μ l 1～2 μ l
ThermoScript II 200U/ μ l 1～2 μ l
2) per 1 component, 2.7～8.5 μ lPCR reagent preparation:
Composition reagent concentration consumption
10×PCR?buffer?15mmol/μl 1～3μl
MgCl
2 25mmol/μl 1～3μl
dNTP 10mmol/μl 0.5～2μl
Taq enzyme 5U/ μ l 0.2～0.5 μ l
The reagent A preparation:
Primer GMVP1:5 '-TGGAAGGCAAAGTTACAGGAGG-3 ' and
Primer GMVP2:5 '-GTCAAATGCGTACCGTGCGAGA-3 '
Be dissolved into 5～20pmol/ μ l with pure water;
Reagent B preparation:
Primer GCLVP1:5 '-AGTTGCGACTGCTGAAGACC-3 ' and
Primer GCLVP2:5 '-GCCATTACGCCTATTCCTAC-3 '
Be dissolved into 5～20pmol/ μ l with pure water;
3, the detection method of the described garlic virus RT-PCR of claim 1 detection kit comprises,
(1) reverse transcription reaction: in PCR reagent pipe 1, add the water of total RNA sample of 0.1～1 μ g garlic and no RNA enzyme, make the total reaction system reach 20 μ l, carry out reverse transcription reaction then, obtain cDNA, its condition is 42 ℃, 30 minutes, 95 ℃, 5 minutes, and 4 ℃, 5 minutes;
(2) PCR reaction: the cDNA that obtains is transcribed in negate, adds in the PCR reagent pipe 2, adds reagent A or reagent B again, add pure water then and make the total reaction system reach 20 μ l, carry out the PCR reaction then, its parameter is 94 ℃ of sex change 1 minute, annealed 1 minute for 57 ℃, 72 ℃ were extended 1 minute, and carried out 32 circulations;
(3) electrophoresis detection: in the PCR reagent pipe 2 that the PCR reaction finishes, add the electrophoresis sample-loading buffer, extract reaction solution and on the gel that contains agarose and ethidium bromide, carry out electrophoresis, the observation of taking pictures on gel imaging system then: the electrophoresis detection result who contains GMV virus garlic samples is the electrophoretic band that amplifies 327bp, and the electrophoresis detection result who contains GCLV virus garlic samples is the electrophoretic band that amplifies 469bp.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100538339C (en) * | 2005-12-22 | 2009-09-09 | 云南农业大学 | The method of synchronous detecting lily mottle virus, lily asymptomatic virus and cucumber mosaic virus |
| CN103911461A (en) * | 2014-03-13 | 2014-07-09 | 湖南农业大学 | A method of simultaneously detecting a plurality of garlic viruses by quadruple RT-PCR and a primer composition thereof |
| ES2469948R1 (en) * | 2012-12-19 | 2014-11-17 | Universidad Politécnica de Madrid | PROCEDURE TO DETECT IN PLANTS THE INFECTION BY SIMPLE CHAIN VIRUSES INFECTING GARLIC OYDV, LYSV, GCLV, SLV, IYSV and ALLEXIVIRUS. |
| CN104988245A (en) * | 2015-07-28 | 2015-10-21 | 中华人民共和国北京出入境检验检疫局 | RT-qPCR detection kit and oligonucleotides for detecting dahlia latent viroid |
| CN110872590A (en) * | 2019-11-14 | 2020-03-10 | 南京农业大学 | Application of transcription factor OsTBP2.1 |
| WO2023071370A1 (en) * | 2021-10-27 | 2023-05-04 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Multiplex pcr detection kit for garlic virus |
-
2004
- 2004-04-22 CN CN 200410014721 patent/CN1233843C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100538339C (en) * | 2005-12-22 | 2009-09-09 | 云南农业大学 | The method of synchronous detecting lily mottle virus, lily asymptomatic virus and cucumber mosaic virus |
| ES2469948R1 (en) * | 2012-12-19 | 2014-11-17 | Universidad Politécnica de Madrid | PROCEDURE TO DETECT IN PLANTS THE INFECTION BY SIMPLE CHAIN VIRUSES INFECTING GARLIC OYDV, LYSV, GCLV, SLV, IYSV and ALLEXIVIRUS. |
| CN103911461A (en) * | 2014-03-13 | 2014-07-09 | 湖南农业大学 | A method of simultaneously detecting a plurality of garlic viruses by quadruple RT-PCR and a primer composition thereof |
| CN103911461B (en) * | 2014-03-13 | 2015-11-18 | 湖南农业大学 | A kind of quadruple RT-PCR detects method and the combination of primers thereof of multiple Garlic Virus simultaneously |
| CN104988245A (en) * | 2015-07-28 | 2015-10-21 | 中华人民共和国北京出入境检验检疫局 | RT-qPCR detection kit and oligonucleotides for detecting dahlia latent viroid |
| CN104988245B (en) * | 2015-07-28 | 2018-05-08 | 中华人民共和国北京出入境检验检疫局 | Detection dahlia hides the RT-qPCR detection kits and oligonucleotides of viroid |
| CN110872590A (en) * | 2019-11-14 | 2020-03-10 | 南京农业大学 | Application of transcription factor OsTBP2.1 |
| CN110872590B (en) * | 2019-11-14 | 2022-03-29 | 南京农业大学 | Application of transcription factor OsTBP2.1 |
| WO2023071370A1 (en) * | 2021-10-27 | 2023-05-04 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Multiplex pcr detection kit for garlic virus |
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