CN104087684A - Kit for rapidly detecting tomato yellow leaf curl virus and application thereof - Google Patents
Kit for rapidly detecting tomato yellow leaf curl virus and application thereof Download PDFInfo
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- 241000702308 Tomato yellow leaf curl virus Species 0.000 title claims abstract description 49
- 235000007688 Lycopersicon esculentum Nutrition 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 34
- 238000001514 detection method Methods 0.000 claims abstract description 26
- 239000011536 extraction buffer Substances 0.000 claims abstract description 23
- 239000002773 nucleotide Substances 0.000 claims abstract description 10
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 10
- 241000227653 Lycopersicon Species 0.000 claims abstract 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- 239000012153 distilled water Substances 0.000 claims description 21
- 239000007853 buffer solution Substances 0.000 claims description 17
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 16
- 238000012360 testing method Methods 0.000 claims description 15
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 8
- LMBWSYZSUOEYSN-UHFFFAOYSA-N diethyldithiocarbamic acid Chemical compound CCN(CC)C(S)=S LMBWSYZSUOEYSN-UHFFFAOYSA-N 0.000 claims description 8
- 229950004394 ditiocarb Drugs 0.000 claims description 8
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 8
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 8
- 229940093916 potassium phosphate Drugs 0.000 claims description 8
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 8
- 235000011009 potassium phosphates Nutrition 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 claims description 8
- 238000011144 upstream manufacturing Methods 0.000 claims description 7
- 239000011541 reaction mixture Substances 0.000 claims description 6
- 238000012408 PCR amplification Methods 0.000 claims description 5
- 239000000499 gel Substances 0.000 claims description 5
- 238000003384 imaging method Methods 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 5
- 239000000047 product Substances 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 102000006382 Ribonucleases Human genes 0.000 claims description 3
- 108010083644 Ribonucleases Proteins 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 3
- 230000004087 circulation Effects 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 2
- 241000700605 Viruses Species 0.000 abstract description 8
- 201000010099 disease Diseases 0.000 abstract description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 241000254127 Bemisia tabaci Species 0.000 abstract description 5
- 238000012545 processing Methods 0.000 abstract description 3
- 240000000060 Tomato yellow leaf curl virus - Il Species 0.000 abstract description 2
- 239000011534 wash buffer Substances 0.000 abstract 2
- 238000002474 experimental method Methods 0.000 abstract 1
- 238000000605 extraction Methods 0.000 abstract 1
- 239000013598 vector Substances 0.000 abstract 1
- 240000003768 Solanum lycopersicum Species 0.000 description 37
- 241000196324 Embryophyta Species 0.000 description 8
- 241000702463 Geminiviridae Species 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 238000012797 qualification Methods 0.000 description 5
- 238000013016 damping Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 230000000405 serological effect Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 241000702451 Begomovirus Species 0.000 description 2
- 241000723873 Tobacco mosaic virus Species 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000011896 sensitive detection Methods 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 241000724252 Cucumber mosaic virus Species 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000709992 Potato virus X Species 0.000 description 1
- 241000710145 Tomato bushy stunt virus Species 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002887 multiple sequence alignment Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 238000010129 solution processing Methods 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses a kit for rapidly detecting tomato yellow leaf curl virus and application thereof. A pair of specific primers is designed according to the nucleotide sequence of a dominant strain of tomato yellow leaf curl virus in our country, and also an extraction buffer and a washing buffer for processing a sample are designed. By using the kit for rapid detection on tomato yellow leaf curl virus, a virus-inflected tomato sample does not need extraction of DNA, only a small amount of a disease leaf (about 10 mg) is needed, and the disease leaf only needs once PCR reaction after being processed by the extraction buffer and the washing buffer, so that identification on tomato yellow leaf curl virus is realized. Experiments prove that the method has relatively high sensitivity and the sensitivity reaches 1E4. Also, the method is capable of performing virus containing identification on virus-transmission vectors Q-biotype bemisia tabaci and B-biotype bemisia tabaci of tomato yellow leaf curl virus. Compared with traditionally biology and serology, the method is a simple, convenient, rapid, economic and sensitive method for detecting tomato yellow leaf curl virus.
Description
Technical field
The present invention relates to a kind of virus detection kit, particularly a kind of quick detection kit that tomato yellow leaf curl virus is carried out and application thereof, the invention belongs to biological technical field.
Background technology
Tomato yellow leaf curl virus (Tomato yellow leaf curl virus, TYLCV) be a kind of virus of serious harm tomato production, this virus is strand ring-type DNA virus, Genome Size is 2.7～2.8kb, belong to geminivirus infection section (Geminiviridae) Begomovirus (Begomovirus), it passes virus mediator is Type B and Q type Bemisia tabaci (Bemisia tabaci).Tomato yellow leaf curl virus disease all can occur in each stage of tomato growth, after the early stage plant that grows is susceptible, plant is non-evident sympton in appearance, main manifestations is that plant strain growth is stagnated or slow, plant dwarfing afterwards, internode obviously shortens, blade thickening, diminish, blade edge is to the yellow of vein region and have rough gauffer or distortion, leaf margin upsweeps, and cannot normally yield positive results; Grow later stage disease plant tamato fruit deformity, diminish, normal annesl, output and commodity value decline to a great extent.
In China, the tomato disease being caused by geminivirus infection is only confined to the tomato main producing region in province, the west and south such as Guangxi, Yunnan and Taiwan at first.End the report that Deposits in Eastern Coastal China and hinterland, Midwest before 2005 there is no geminivirus infection harm, but, successively occur from East Coastals such as Zhejiang in 2006 and Shanghai after the report of geminivirus infection harm tomato, this disease spreads and comes at East Coastal rapidly, and cause serious harm in the tomato production in multiple provinces, become the main limiting factor that current tomato in China is produced.
At present, mainly adopt four kinds of methods for the identification qualification of plant virus: biology detection, serological detection method, electron microscopic observation method and molecular Biological Detection method.Biology detection and electron microscopic observation method are subject to interference from human factor larger, and detected result is with a low credibility; Serology detects and is mainly ELISA method, but its sensitivity and specificity are not high, are not suitable for rapid detection yet.At present the detection of TYLCV is mainly adopted to molecular biological method, but just can carry out next step detection after need to extracting total DNA of plant.
Adopt test kit of the present invention to detect TYLCV, without extracting the total DNA of disease plant, only need to adopt Extraction buffer, lavation buffer solution to carry out simple process to a small amount of tomato sample, then can realize the detection qualification to tomato yellow leaf curl virus through PCR and agarose gel electrophoresis.Feature easy, quick, highly sensitive, high specificity that test kit of the present invention has, and can be the field investigation of TYLCV, and the appearance of industrial seedling rearing detects reliable technical support is provided.
Summary of the invention
One of technical problem to be solved by this invention is to provide a kind of test kit that can rapid detection tomato yellow leaf curl virus;
Two of technical problem to be solved by this invention is to provide the application of described test kit in rapid detection tomato yellow leaf curl virus;
Three of technical problem to be solved by this invention is to provide a kind of method that uses mentioned reagent box to carry out rapid detection tomato yellow leaf curl virus.
Technical problem to be solved by this invention realizes by following technique means:
The test kit of a kind of rapid detection tomato yellow leaf curl virus of the present invention, is characterized in that comprising Extraction buffer, lavation buffer solution and the primer sets for detection of tomato yellow leaf curl virus;
Wherein, in described Extraction buffer, contain the potassiumphosphate of 0.05-0.15mol/L, the Thiovanic acid that volume percent is 0.05-0.15%, the Thiocarb of 0.005-0.015mol/L and the ethylenediamine tetraacetic acid (EDTA) of 0.001-0.005mol/L, all the other are distilled water (ddH
2o), regulate pH to 7-8;
Wherein, the KCl of NaCl, the 0.5-1g/L that contains 8-8.5g/L in described lavation buffer solution, the Na of 1-1.5g/L
2hPO
4, the KH of 0.1-0.5g/L
2pO
4and the volume percent TWEEN-20 that is 0.02-0.1%, all the other are distilled water.
In the present invention, preferably, in described Extraction buffer, contain the potassiumphosphate of 0.1mol/L, the Thiovanic acid that volume percent is 0.1%, the Thiocarb of 0.01mol/L and the ethylenediamine tetraacetic acid (EDTA) of 0.001mol/L, all the other are distilled water, regulate pH to 7.8; In described lavation buffer solution, contain the KCl of NaCl, the 0.746g/L of 8.182g/L, the Na of 1.136g/L
2hPO
4, the KH of 0.272g/L
2pO
4and the volume percent TWEEN-20 that is 0.05%, all the other are distilled water.
In the present invention, preferred, described primer sets is made up of upstream primer and downstream primer, and wherein the nucleotide sequence of upstream primer is as shown in SEQ ID NO.1, and the nucleotide sequence of downstream primer is as shown in SEQ ID NO.2.
In the present invention, preferably, described test kit also comprises without RNase water and PCR reaction mixture, described PCR reaction mixture contains Taq archaeal dna polymerase and dNTPs, in a specific embodiment of the present invention, described PCR reaction mixture is 2xTaq MasterMix, purchased from Beijing CoWin Bioscience Co., Ltd..
Further, the invention allows for a kind of method of rapid detection tomato yellow leaf curl virus, comprise the following steps:
(1) sample preparation
Get the sick leaf of 10mg tomato in 1.5mL centrifuge tube, add 100 μ L extracting solution damping fluids fully to grind; By the centrifugal 5min of 12000rpm; Getting 50 μ L supernatant liquors adds in the PCR pipe of 0.2mL; In 37 DEG C of water-baths, hatch 10min; Outwell the liquid in PCR pipe; Washing PCR with 100 μ L washings damping fluids manages 2 times; PCR pipe is fully dry;
Wherein, in described Extraction buffer, contain the potassiumphosphate of 0.05-0.15mol/L, the Thiovanic acid that volume percent is 0.05-0.15%, the Thiocarb of 0.005-0.015mol/L and the ethylenediamine tetraacetic acid (EDTA) of 0.001-0.005mol/L, all the other are distilled water, regulate pH to 7-8;
Wherein, the KCl of NaCl, the 0.5-1g/L that contains 8-8.5g/L in described lavation buffer solution, the Na of 1-1.5g/L
2hPO
4, the KH of 0.1-0.5g/L
2pO
4and the volume percent TWEEN-20 that is 0.02-0.1%, all the other are distilled water;
(2) pcr amplification
To adding following reagent in step (1) PCR pipe after treatment:
Reaction conditions is: 94 DEG C of denaturation 10min, and 94 DEG C of sex change 45s, 54 DEG C of annealing 30s, 72 DEG C are extended 45s, 35 circulations, last 72 DEG C are extended 10min, 4 DEG C of preservations;
(3) agarose gel electrophoresis detects:
Get 5 μ L PCR products after 1.2% agarose gel electrophoresis, in gel imaging system, observe.
In the method for the invention, preferably, in described Extraction buffer, contain the potassiumphosphate of 0.1mol/L, the Thiovanic acid that volume percent is 0.1%, the Thiocarb of 0.01mol/L and the ethylenediamine tetraacetic acid (EDTA) of 0.001mol/L, all the other are distilled water, regulate pH to 7.8; In described lavation buffer solution, contain the KCl of NaCl, the 0.746g/L of 8.182g/L, the Na of 1.136g/L
2hPO
4, the KH of 0.272g/L
2pO
4and the volume percent TWEEN-20 that is 0.05%, all the other are distilled water.
In the method for the invention, preferred, the nucleotide sequence of described upstream primer is as shown in SEQ ID NO.1, and the nucleotide sequence of downstream primer is as shown in SEQ ID NO.2.
Use test kit of the present invention to carry out the detection of tomato yellow leaf curl virus, tomato sample is without extracting DNA, only need a small amount of sample (～10mg) after Extraction buffer, lavation buffer solution reason, as long as carry out a PCR reaction, can realize the qualification to tomato yellow leaf curl virus, experimental results show that the method has higher sensitivity, reach 1E4 (1:10000), there is lower detection lower limit (1:5000) than regular-PCR.Meanwhile, the method also can be carried out toxic qualification to biography virus mediator Q type and the Type B Bemisia tabaci of tomato yellow leaf curl virus.Therefore, method of the present invention, compared with traditional biology, serological method, is a kind of easy, quick, economic, sensitive detection method that is applicable to tomato yellow leaf curl virus.
Brief description of the drawings
Fig. 1 is primer validation verification result;
M:Trans2K DNA Marker; 1: distilled water; 2: the total DNA of healthy tomato plant; 3: Shandong sense TYLCV tomato sample; 4: tomato sense tobacco mosaic virus (TMV) sample; 5: tomato sense cucumber mosaic virus sample; 6: tomato sense tomato bushy stunt virus sample; 7: tomato sense potato virus X sample;
Fig. 2 is the electrophoresis detection result of tomato yellow leaf curl virus.
M:Trans2K Plus II DNA Marker; 1: Tianjin sense TYLCV tomato sample; 2: Shandong sense TYLCV tomato sample; 3: Henan sense TYLCV tomato sample; 4: Hebei sense TYLCV tomato sample; 5: Jiangsu sense TYLCV tomato sample; 6: Zhejiang sense TYLCV tomato sample; 7: healthy tomato sample.
Fig. 3 a is that susceptibility detects analytical results (control group);
M:Trans2K DNA Marker; 1: distilled water; 2: the total DNA of healthy tomato plant; 3-8: the total DNA of tomato leaf that infects TYLCV dilutes with different Dilution ratios from distilled water: 1:1,1:10,1:100,1:1000,1:5000,1:10000;
Fig. 3 b is that susceptibility detects analytical results (the inventive method);
M:Trans2K DNA Marker; 1: distilled water; 2: healthy tomato plant is through Extraction buffer supernatant liquor after treatment; 3-9: infect TYLCV tomato leaf and dilute with different Dilution ratios through Extraction buffer supernatant liquor after treatment from healthy tomato plant through Extraction buffer supernatant liquor after treatment: 1:1,1:10,1:100,1:1000,1:5000,1:10000,1:20000.
Embodiment
Further describe the present invention below in conjunction with specific embodiments and the drawings, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these amendments and replacement all fall within the scope of protection of the present invention.
The preparation of embodiment 1 test kit
1, design of primers
According to after the Chinese advantage strain of the upper logged tomato yellow leaf curl virus of NCBI nucleotide sequence Multiple Sequence Alignment, carry out design of primers, primer sequence is as follows, and primer validation verification is shown in Fig. 1:
Upstream primer (TY-V1): 5 '-AGTACTATGTCGAAGCGWCCAG-3 ' (shown in SEQ ID NO.1, W=A/T)
Downstream primer (TY-V2): 5 '-TCTAGATTAATTTKRTATTGAATCATAGA-3 ' (shown in SEQ ID NO.2, K=G/T, R=A/G)
The size of amplification object fragment is 789bp.
2, the preparation of Extraction buffer and lavation buffer solution
(1) preparation of Extraction buffer (100ml)
Add 90ml ddH
2o, regulates pH to 7.8, is settled to 100ml, room temperature preservation.
(2) preparation of lavation buffer solution (100ml)
Add 90ml ddH
2o, regulates pH to 7.4, is settled to 100ml, room temperature preservation after autoclaving.
(3) without RNase water, PCR reaction mixture 2xTaq MasterMix purchased from Beijing CoWin Bioscience Co., Ltd..
Embodiment 2 test kit of the present invention is in the application detecting in tomato yellow leaf curl virus
1, specimen material: taking the tomato sample of the 7 known infection tomato yellow leaf curl virus of provinces and cities as detected object, with the negative contrast of healthy tomato sample.
2, method
(1) sample preparation
Get the sick leaf of 10mg tomato in 1.5mL centrifuge tube, add 100 μ L extracting solution damping fluids (according to the method preparation of embodiment 1) fully to grind; By the centrifugal 5min of 12000rpm; Getting 50 μ L supernatant liquors adds in the PCR pipe of 0.2mL; In 37 DEG C of water-baths, hatch 10min, be convenient to virion and be adsorbed onto on PCR tube wall; Outwell the liquid in PCR pipe; With 100 μ L washings damping fluids (according to the method preparation of embodiment 1), washing PCR manages 2 times; PCR pipe is fully dry;
(2) pcr amplification
To adding following reagent in the PCR pipe of processing through step (1):
Reaction conditions is: 94 DEG C of denaturation 10min, and 94 DEG C of sex change 45s, 54 DEG C of annealing 30s, 72 DEG C are extended 45s, 35 circulations, last 72 DEG C are extended 10min, 4 DEG C of preservations;
(3) agarose gel electrophoresis detects:
Get 5 μ L PCR products after 1.2% agarose gel electrophoresis (0.5xTBE, 120V, 20min), in gel imaging system, observe.
3, interpretation of result
In the tomato sample lane that only infects tomato yellow leaf curl virus, can observe the band of a 789bp, and not infect in the tomato sample lane of tomato yellow leaf curl virus, not occur any band, result as shown in Figure 2.
The sensitivity of embodiment 3 the inventive method detects analyzes (detectability analysis)
Control group: get the tomato leaf that infects TYLCV, after conventional CTAB method is extracted DNA, with distilled water carry out 1:1,1:10,1:100,1:1000,1:5000,1:10000 doubly dilute, pcr amplification, product is through 1.2% agarose gel electrophoresis, EB dyeing, takes pictures in BioRad gel imaging system, as shown in Figure 3 a.
The inventive method: get the tomato leaf that infects TYLCV, after processing, Extraction buffer gets 50 μ L supernatant liquors, with healthy tomato plant (not infecting TYLCV) vanes Extraction buffer supernatant liquor after treatment, carry out 1:1,1:10,1:100,1:1000,1:5000,1:10000,1:20000 doubly dilutes, by the supernatant liquor of different extension rates respectively according to the method for embodiment 2 through hatching, lavation buffer solution processing, pcr amplification, product is through 1.2% agarose gel electrophoresis, EB dyeing, take pictures in BioRad gel imaging system, as Fig. 3 b.
Can find out from the above results, method of the present invention has higher sensitivity, reaches 1E4 (1:10000), has lower detection lower limit (1:5000) than regular-PCR (control group).Meanwhile, tomato sample, without extracting DNA, only needs a small amount of sample (～10mg) after Extraction buffer, lavation buffer solution reason, as long as carry out a PCR reaction, can realize the qualification to tomato yellow leaf curl virus.Therefore, method of the present invention, compared with traditional biology, serological method, is a kind of easy, quick, economic, sensitive detection method that is applicable to tomato yellow leaf curl virus.
Claims (8)
1. a test kit for rapid detection tomato yellow leaf curl virus, is characterized in that comprising Extraction buffer, lavation buffer solution and the primer sets for detection of tomato yellow leaf curl virus;
Wherein, in described Extraction buffer, contain the potassiumphosphate of 0.05-0.15mol/L, the Thiovanic acid that volume percent is 0.05-0.15%, the Thiocarb of 0.005-0.015mol/L and the ethylenediamine tetraacetic acid (EDTA) of 0.001-0.005mol/L, all the other are distilled water, regulate pH to 7-8;
Wherein, the KCl of NaCl, the 0.5-1g/L that contains 8-8.5g/L in described lavation buffer solution, the Na of 1-1.5g/L
2hPO
4, the KH of 0.1-0.5g/L
2pO
4and the volume percent TWEEN-20 that is 0.02-0.1%, all the other are distilled water.
2. test kit as claimed in claim 1, it is characterized in that, in described Extraction buffer, contain the potassiumphosphate of 0.1mol/L, the Thiovanic acid that volume percent is 0.1%, the Thiocarb of 0.01mol/L and the ethylenediamine tetraacetic acid (EDTA) of 0.001mol/L, all the other are distilled water, regulate pH to 7.8; In described lavation buffer solution, contain the KCl of NaCl, the 0.746g/L of 8.182g/L, the Na of 1.136g/L
2hPO
4, the KH of 0.272g/L
2pO
4and the volume percent TWEEN-20 that is 0.05%, all the other are distilled water.
3. test kit as claimed in claim 1, is characterized in that, described primer sets is made up of upstream primer and downstream primer, and wherein the nucleotide sequence of upstream primer is as shown in SEQ ID NO.1, and the nucleotide sequence of downstream primer is as shown in SEQ ID NO.2.
4. the test kit as described in claim 1-3 any one, characterized by further comprising without RNase water and PCR reaction mixture, and described PCR reaction mixture contains Taq archaeal dna polymerase and dNTPs.
5. the application of the test kit described in claim 1-4 any one in rapid detection tomato yellow leaf curl virus.
6. a method for rapid detection tomato yellow leaf curl virus, is characterized in that comprising the following steps:
(1) sample preparation
Get the sick leaf of 10mg tomato in 1.5mL centrifuge tube, add 100 μ L Extraction buffers fully to grind, by the centrifugal 5min of 12000rpm, get 50 μ L supernatant liquors and add in the PCR pipe of 0.2mL, in 37 DEG C of water-baths, hatch 10min, outwell the liquid in PCR pipe; Washing PCR with 100 μ L lavation buffer solutions manages 2 times; PCR pipe is fully dry;
Wherein, in described Extraction buffer, contain the potassiumphosphate of 0.05-0.15mol/L, the Thiovanic acid that volume percent is 0.05-0.15%, the Thiocarb of 0.005-0.015mol/L and the ethylenediamine tetraacetic acid (EDTA) of 0.001-0.005mol/L, all the other are distilled water, regulate pH to 7-8;
Wherein, the KCl of NaCl, the 0.5-1g/L that contains 8-8.5g/L in described lavation buffer solution, the Na of 1-1.5g/L
2hPO
4, the KH of 0.1-0.5g/L
2pO
4and the volume percent TWEEN-20 that is 0.02-0.1%, all the other are distilled water;
(2) pcr amplification
To adding following reagent in step (1) PCR pipe after treatment:
Reaction conditions is: 94 DEG C of denaturation 10min, and 94 DEG C of sex change 45s, 54 DEG C of annealing 30s, 72 DEG C are extended 45s, 35 circulations, last 72 DEG C are extended 10min, 4 DEG C of preservations;
(3) agarose gel electrophoresis detects:
Get 5 μ L PCR products after 1.2% agarose gel electrophoresis, in gel imaging system, observe.
7. method as claimed in claim 6, it is characterized in that, in described Extraction buffer, contain the potassiumphosphate of 0.1mol/L, the Thiovanic acid that volume percent is 0.1%, the Thiocarb of 0.01mol/L and the ethylenediamine tetraacetic acid (EDTA) of 0.001mol/L, all the other are distilled water, regulate pH to 7.8; In described lavation buffer solution, contain the KCl of NaCl, the 0.746g/L of 8.182g/L, the Na of 1.136g/L
2hPO
4, the KH of 0.272g/L
2pO
4and the volume percent TWEEN-20 that is 0.05%, all the other are distilled water.
8. method as claimed in claim 6, is characterized in that, the nucleotide sequence of described upstream primer is as shown in SEQ ID NO.1, and the nucleotide sequence of downstream primer is as shown in SEQ ID NO.2.
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| CN107385109A (en) * | 2017-07-28 | 2017-11-24 | 陈定虎 | Primer, kit and the detection method of tomato yellow leaf curl virus identification |
| CN108070636A (en) * | 2016-11-16 | 2018-05-25 | 天津安必森生物技术有限公司 | A kind of processing method and kit of fluorescent PCR amplified sample |
| CN114196789A (en) * | 2021-12-24 | 2022-03-18 | 青岛农业大学 | Method for non-invasively and rapidly determining whether plants are infected with tomato yellow leaf curl virus |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108070636A (en) * | 2016-11-16 | 2018-05-25 | 天津安必森生物技术有限公司 | A kind of processing method and kit of fluorescent PCR amplified sample |
| CN106834536A (en) * | 2016-12-30 | 2017-06-13 | 四川师范大学 | Method using organizing the direct RT LAMP of juice to detect corium solani |
| CN107385109A (en) * | 2017-07-28 | 2017-11-24 | 陈定虎 | Primer, kit and the detection method of tomato yellow leaf curl virus identification |
| CN114196789A (en) * | 2021-12-24 | 2022-03-18 | 青岛农业大学 | Method for non-invasively and rapidly determining whether plants are infected with tomato yellow leaf curl virus |
| CN114196789B (en) * | 2021-12-24 | 2023-04-25 | 青岛农业大学 | Method for noninvasively and rapidly determining whether plants are infected with tomato yellow leaf curl virus |
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