CN103243175A - Primers, kit and detection method for detecting lily symptomless virus - Google Patents
Primers, kit and detection method for detecting lily symptomless virus Download PDFInfo
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- CN103243175A CN103243175A CN2013101628136A CN201310162813A CN103243175A CN 103243175 A CN103243175 A CN 103243175A CN 2013101628136 A CN2013101628136 A CN 2013101628136A CN 201310162813 A CN201310162813 A CN 201310162813A CN 103243175 A CN103243175 A CN 103243175A
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Abstract
The invention discloses primers, a kit and a detection method for detecting a lily symptomless virus. The sequences of the primers disclosed by the invention are LSV Primer-1:5'-AAGATGAAAGTTGGCGTCGT-3', and LSV Primer-2:5'-CACAAGCATGCTGTTCCACA-3'. The kit and the detection method disclosed by the invention contain the primers including an LSV Primer-1 and an LSV Primer-2. The primers and the detection method for detecting the lily symptomless virus are high in accuracy, strong in specificity, and high in sensitivity.
Description
Technical field
The present invention relates to a kind of primer, test kit and detection method for RT-PCR detection lily asymptomatic virus, belong to biological technical field.
Background technology
Lily asymptomatic virus belongs to Dianthus caryophyllus L. Adelonosus (Carlavirus), does not temporarily have section.Mainly be distributed in Sweden, Britain, Germany, Denmark, Holland, Belgium, Italy, Latvia, the U.S., Canada, Japan and other countries.
Be limited to Liliaceae, mostly be systemic infection.LSV generally can not cause manifest symptom, shifts to an earlier date yellow but can cause lily to adopt the rear lower blade, and the influence bottle is inserted the life-span, during with compound the infecting of cucumber mosaic virus (CMV), can cause symptoms such as veinclearing, floral leaf, deformity, necrotic plaque.Propagated by the aphid perishability under the natural condition, vector aphid has cotten aphid, black peach aphid, and lily juice and seed do not pass.
The virus particle bending, size is 640X17-18nm, the shell molecular weight is 32KD, formed by 292 amino acid, nucleic acid account for plastochondria heavy 8.3%, formed by single RNA.The virion settling ratio is 172S, A260/280=1.20-1.43, and fatal temperature is 65-70 ℃, the dilution point of accumulation is 10
-5
LSV is RNA viruses, and its genome total length 8393bp has 6 open reading frame (ORF), 5 albumen of only encoding, be respectively 25KD, 12KD, 7KD, 32KD and 16KD from 5 ' end to 3 ' end, wherein the longest ORF is 876bp, coding 32KD virus capsid protein.
Its specificity of primer that the existing RT-PCR of being used for detects lily asymptomatic virus is strong relatively inadequately, and susceptibility is relatively poor relatively, and this tends to cause the detected result accuracy not high.
Summary of the invention
The objective of the invention is in order to overcome weak point of the prior art, provide a kind of and detect the primer of lily asymptomatic virus and test kit and the method that comprises this primer for RT-PCR, utilize this primer and detection method to detect lily asymptomatic virus accuracy height, high specificity, susceptibility height.
In order to achieve the above object, the present invention adopts following scheme:
A kind of primer for detection of lily asymptomatic virus is characterized in that:
Primer sequence:
LSV?Primer-1:5'-AAGATGAAAGTTGGCGTCGT-3'，
LSV?Primer-2:5'-CACAAGCATGCTGTTCCACA-3'。
A kind of test kit that detects lily asymptomatic virus is characterized in that this test kit includes:
Wherein said LSV Primer-1 sequence is 5'-AAGATGAAAGTTGGCGTCGT-3'; Described LSV Primer-2 sequence is 5'-CACAAGCATGCTGTTCCACA-3'.
Aforesaid a kind of test kit that detects lily asymptomatic virus is characterized in that Taq polysaccharase, RNA enzyme inhibitors that described Enzyme Mix comprises ThermoScript II, pcr amplification and uses; Described Buffer comprises MgCl
225mM, dNTPs10mM; Described Positive Control is the nucleic acid of lily asymptomatic virus; Described RNase Free dH2O is the deionized water of removing behind the RNA enzyme.
A kind of detection method that detects lily asymptomatic virus is characterized in that may further comprise the steps:
A, testing sample RNA extract, and get template ribonucleic acid solution;
B, the template ribonucleic acid solution reverse transcription in the steps A is become cDNA, adopt primer LSV Primer-1 and LSV Primer-2 to carrying out pcr amplification again, get the PCR reaction solution; Wherein said LSV Primer-1 sequence is 5'-AAGATGAAAGTTGGCGTCGT-3'; Described LSV Primer-2 sequence is 5'-CACAAGCATGCTGTTCCACA-3';
C, get the PCR reaction solution and carry out the agarose gel electrophoresis analysis;
D, if there is the DNA band of 297bp, then proof detects and has lily asymptomatic virus in the articles for use.
The DNA band sequence of 297bp described in the present invention is:
AAGATGAAAGTTGGCGTCGTATCTAACAACATGGCAACCACTGAACAAATGGCCA?AGATAGCGTCAGACATCGCAGGGCTTGGGGTACCAACGGAGCACGTCGCGTCAGTAATA?TTGCAAATGGTCATCATGTGTGCTTGCGTGAGCAGTTCTGCGTTCCTTGACCCTGAGGG?CAGCATTGAGTTTGAGAATGGAGCGGTGCCCGTCGACTCCATCACTGCGATCATGAAGA?AGCACGCTGGACTGCGGAAAGTCTGCCGGCTCTATGCCCCTATCGTGTGGAACAGCATG?CTTGTG。
Aforesaid a kind of detection method that detects lily asymptomatic virus is characterized in that the RNA of testing sample described in the steps A extracts, and specifically comprises the steps:
A1, choose the lily leaf tissue of 0.1-0.2g performance suspect, use the equivalent health tissues simultaneously in contrast; The lily leaf tissue is added liquid nitrogen freezing, in the 1.5mL centrifuge tube of packing into after fully grinding;
A2, in centrifuge tube, add 400 ice-cold μ L RNA extracts, put upside down and mix;
4 ℃ of a3, tabletop refrigerated centrifuges, the centrifugal 10min of 12000g;
A4, get supernatant, add the mixed solution of 400 μ L phenol, chloroform, primary isoamyl alcohol, wherein the volume ratio of phenol, chloroform, primary isoamyl alcohol is 25:24:1, puts upside down to mix;
4 ℃ of a5, tabletop refrigerated centrifuges, the centrifugal 10min of 12000g;
A6, get supernatant, add the mixed solution of 400 μ L chloroforms, primary isoamyl alcohol, wherein the volume ratio of chloroform, primary isoamyl alcohol is 24:1, puts upside down to mix;
4 ℃ of a7, tabletop refrigerated centrifuges, the centrifugal 10min of 12000g;
A8, get supernatant, add 1000 μ L dehydrated alcohols and 40 μ L3M sodium-acetate pH5.2, put upside down and mix;
A9,4 ℃ of centrifugal 10min of 12000g of tabletop refrigerated centrifuge;
A10, stay precipitation, with the cold washing with alcohol of 500 μ L70% once, place on the Bechtop to dry up, being dissolved in 20 μ L does not then have in the water of RNA enzyme.
Aforesaid a kind of RT-PCR detects the detection method of lily asymptomatic virus, it is characterized in that the reverse transcription of template ribonucleic acid solution described in the step B becomes cDNA, specifically may further comprise the steps:
In the 0.5mL centrifuge tube, add deionized water 9.5 μ L, add total RNA2 μ L, Primer-2 primer 1 μ L, concentration is the dNTPs1 μ L of 10mmol/L, places 2min on ice immediately behind 70 ℃ of water-bath 5min; The centrifugal 30s of 3000rpm then; Add the synthetic damping fluid 4 μ L of 5 * cDNA, first chain again, 0.1mol/L DTT1 μ L; RNase inhibitor 1 μ L (30U); M-MLV ThermoScript II 1 μ L (20U); 37 ℃ of 60min.
Aforesaid a kind of RT-PCR detects the detection method of lily asymptomatic virus, it is characterized in that pcr amplification specifically may further comprise the steps described in the step B:
Add 10 * pcr amplification damping fluid, 2.5 μ L in the 0.2mL centrifuge tube, concentration is the MgCl of 25mmol/L
22 μ L, concentration is the dNTPs1 μ L of 10mmol/L, Primer-11 μ M(50pmol), Primer-21 μ M(50pmol), the cDNA first chain synthesis reaction liquid 2 μ L add water to 25 μ L, add Taq polysaccharase 0.5 μ L (1U) then; PCR cycling condition: 94 ℃ of sex change 3min; 94 ℃ of 1min then, 52 ℃ of 1min, 72 ℃ of 1min circulate 35 times; Last 72 ℃ are extended 10min; Preserve reaction product for 4 ℃.
Aforesaid a kind of detection method that detects lily asymptomatic virus is characterized in that the agarose gel electrophoresis analysis specifically may further comprise the steps described in the step C:
C1, prepare by 1.5% concentration with 1 * TAE damping fluid and agarose, fusing evenly is cooled to 50-60 ℃ in microwave oven;
C2, adding
I nucleic acid dye or concentration are the ethidium bromide of 1 μ g/mL, mix, and pour on the gel platform, plug sample comb;
C3, treat that gel solidifies after, extract comb, add an amount of TAE buffering;
C4, get 1 μ L sample loading buffer and 5 μ L PCR reaction solutions mix, respectively itself and suitable dna molecular amount standard substance are added in the sample well then;
C5, connection power supply carry out electrophoresis, voltage 5V/cm;
C6, when the tetrabromophenol sulfonphthalein in the sample loading buffer migrates to enough DNA isolation fragments, stop electrophoresis; Place gel imaging system to observe in whole glue, and the record of taking pictures.
The reagent that uses in the detection method of the present invention:
The 1PCR detection reagent
The 2RNA extraction buffer:
20mmol/L?Tris-HCl,pH8.0
1mmol/L?EDTA,pH8.0
1%SDS, 0.2% β-Mercapto base ethanol
The 3TE damping fluid
10mmol/L?Tris-HCl,pH8.0
1mmol/L?EDTA,pH8.0
The 4TAE damping fluid
40mmol/L?Tris-HAc,
1mmol/L?EDTA,pH8.0
56 * sample loading buffer
Tetrabromophenol sulfonphthalein 0.25%
6 primers are synthetic
Design Auele Specific Primer according to the LSV conserved sequence:
BSV?Primer-1:5'-AAGATGAAAGTTGGCGTCGT-3'
BSV?Primer-2:5'-CACAAGCATGCTGTTCCACA-3'
The instrument and the apparatus that use in the detection method of the present invention comprise:
1, pcr amplification instrument
2, electrophoresis apparatus
3, desk type high speed refrigerated centrifuge
4, gel imaging system
5, electronic analytical balance
6, pipettor (0.1-2 μ L, 2-10 μ L, 10-100 μ L, 100-1000 μ L)
7, mortar
8,0.2mL, 0.5mL, 1.5mL centrifuge tube
In sum, beneficial effect of the present invention:
This test kit is specifically designed to and detects banana lines virus BSV.Positive control is the carrier that contains the BSV gene that the clone comes out in the morbidity banana blade; Use this test kit, as long as add the nucleic acid of sample to be checked and a pair of primer of the BSV in this test kit, just can carry out the PCR reaction, simplified operating process widely, reduced the pollution in the PCR operating process, detection efficiency is increased substantially, and detection sensitivity is also very high, can reach 0.1pg; The PCR product 3 ' end that uses the amplification of these goods to obtain in addition also has " A " base, can directly clone in T-Vector.
Description of drawings
Fig. 1 is for detecting the gel electrophoresis figure of lily asymptomatic virus with the RT-PCR method among the present invention.
Wherein, 1: molecular weight standard;
2: positive control;
3-7: sample detection;
8: negative control.
Embodiment
Below in conjunction with embodiment the present invention is described further:
Test kit of the present invention:
1, purposes: the PCR method detects the LSV in the lily plant.
2, Packing Unit: 50 amounts of the RT-PCR reaction system of 50 μ l.
3, goods explanation
This test kit adopts step (One Step) RT-PCR method, and the LSV in the lily plant is detected.This test kit comprises enzyme mix reagent, damping fluid, LSV detection primer and positive control.Experimental implementation is simple and convenient, prevents crossed contamination.
This test kit has following characteristics:
3.1 carry out RT-PCR amplification efficiently, detection sensitivity is 0.1pg.
3.2 the attached A base of purpose product 3 ' end of using the amplification of this test kit to obtain can directly be cloned in T-Vector.
4, goods content (50 amounts)
5, preserve :-20 ℃
6, detection method
6.1 by following composition preparation RT-PCR reaction solution.
6.2 carry out the RT-PCR reaction by following condition.
At first 50 ℃ of 30min reverse transcriptions; 94 ℃ of sex change 3min then; 94 ℃ of 1min again, 52 ℃ of 1min,
72 ℃ of 1min, 35 circulations; Last 72 ℃ of 10min.
6.3 reaction is got PCR reaction solution 5 μ l and carried out agarose gel electrophoresis after finishing, positive findings should be 297bp.
7, remarks
7.1 when needing to detect a plurality of sample simultaneously, should prepare the mixed solution of all ingredients earlier, and then branch installs to each reaction tubes
7.2 when using Enzyme Mix, mixing is avoided bubbling gently; The centrifugal reaction tubes bottom branch of collecting in ground of being careful before branch is got should slowly be drawn when getting.
7.3Buffer with the abundant mixing of vortex, use centrifugal back before using.
7.4 enzyme preparation should just take out from-20 ℃ before detection, also should put back to immediately after the use in-20 ℃ and preserve.
7.5 must use new rifle head (Tip) when dividing installed reagents, pollute to prevent sample room.
RT-PCR of the present invention detects the detection method of lily asymptomatic virus, may further comprise the steps:
A, testing sample RNA extract, and get template ribonucleic acid solution;
A1, choose the lily leaf tissue of 0.1-0.2g performance suspect, use the equivalent health tissues simultaneously in contrast; The lily leaf tissue is added liquid nitrogen freezing, in the 1.5mL centrifuge tube of packing into after fully grinding;
A2, in centrifuge tube, add 400 ice-cold μ L RNA extracts, put upside down and mix;
4 ℃ of a3, tabletop refrigerated centrifuges, the centrifugal 10min of 12000g;
A4, get supernatant, add the mixed solution of 400 μ L phenol, chloroform, primary isoamyl alcohol, wherein the volume ratio of phenol, chloroform, primary isoamyl alcohol is 25:24:1, puts upside down to mix;
4 ℃ of a5, tabletop refrigerated centrifuges, the centrifugal 10min of 12000g;
A6, get supernatant, add the mixed solution of 400 μ L chloroforms, primary isoamyl alcohol, wherein the volume ratio of chloroform, primary isoamyl alcohol is 24:1, puts upside down to mix;
4 ℃ of a7, tabletop refrigerated centrifuges, the centrifugal 10min of 12000g;
A8, get supernatant, add 1000 μ L dehydrated alcohols and 40 μ L3M sodium-acetate pH5.2, put upside down and mix;
A9,4 ℃ of centrifugal 10min of 12000g of tabletop refrigerated centrifuge;
A10, stay precipitation, with the cold washing with alcohol of 500 μ L70% once, place on the Bechtop to dry up, being dissolved in 20 μ L does not then have in the water of RNA enzyme.
B, the template ribonucleic acid solution reverse transcription in the steps A is become cDNA, adopt primer LSV Primer-1 and LSV Primer-2 to carrying out pcr amplification again, get the PCR reaction solution; Wherein said LSV Primer-1 sequence is 5'-AAGATGAAAGTTGGCGTCGT-3'; Described LSV Primer-2 sequence is 5'-CACAAGCATGCTGTTCCACA-3';
In the 0.5mL centrifuge tube, add deionized water 9.5 μ L, add total RNA2 μ L, Primer-2 primer 1 μ L, concentration is the dNTPs1 μ L of 10mmol/L, places 2min on ice immediately behind 70 ℃ of water-bath 5min; The centrifugal 30s of 3000rpm then; Add the synthetic damping fluid 4 μ L of 5 * cDNA, first chain again, 0.1mol/L DTT1 μ L; RNase inhibitor 1 μ L (30U); M-MLV ThermoScript II 1 μ L (20U); 37 ℃ of 60min.
Add 10 * pcr amplification damping fluid, 2.5 μ L in the 0.2mL centrifuge tube, concentration is the MgCl of 25mmol/L
22 μ L, concentration is the dNTPs1 μ L of 10mmol/L, Primer-11 μ M(50pmol), Primer-21 μ M(50pmol), the cDNA first chain synthesis reaction liquid 2 μ L add water to 25 μ L, add Taq polysaccharase 0.5 μ L (1U) then; PCR cycling condition: 94 ℃ of sex change 3min; 94 ℃ of 1min then, 52 ℃ of 1min, 72 ℃ of 1min circulate 35 times; Last 72 ℃ are extended 10min; Preserve reaction product for 4 ℃.
C, get PCR reaction solution 5 μ l and carry out the agarose gel electrophoresis analysis;
C1, prepare by 1.5% concentration with 1 * TAE damping fluid and agarose, fusing evenly is cooled to 50-60 ℃ in microwave oven;
C2, adding
I nucleic acid dye or concentration are the ethidium bromide of 1 μ g/mL, mix, and pour on the gel platform, plug sample comb;
C3, treat that gel solidifies after, extract comb, add an amount of TAE buffering;
C4, get 1 μ L sample loading buffer and 5 μ L PCR reaction solutions mix, respectively itself and suitable dna molecular amount standard substance are added in the sample well then;
C5, connection power supply carry out electrophoresis, voltage 5V/cm;
C6, when the tetrabromophenol sulfonphthalein in the sample loading buffer migrates to enough DNA isolation fragments, stop electrophoresis; Place gel imaging system to observe in whole glue, and the record of taking pictures.
D, if there is the DNA band of 297bp, then proof detects and has lily asymptomatic virus in the articles for use.
Claims (8)
1. primer for detection of lily asymptomatic virus is characterized in that:
Primer sequence:
LSV?Primer-1:5'-AAGATGAAAGTTGGCGTCGT-3'，
LSV?Primer-2:5'-CACAAGCATGCTGTTCCACA-3'。
2. test kit that detects lily asymptomatic virus is characterized in that this test kit includes:
Wherein said LSV Primer-1 sequence is 5'-AAGATGAAAGTTGGCGTCGT-3'; Described LSV Primer-2 sequence is 5'-CACAAGCATGCTGTTCCACA-3'.
3. a kind of test kit that detects lily asymptomatic virus according to claim 2 is characterized in that Taq polysaccharase, RNA enzyme inhibitors that described Enzyme Mix comprises ThermoScript II, pcr amplification and uses; Described Buffer comprises MgCl
225mM, dNTPs10mM; Described Positive Control is the nucleic acid of lily asymptomatic virus; Described RNase Free dH2O is the deionized water of removing behind the RNA enzyme.
4. detection method that detects lily asymptomatic virus is characterized in that may further comprise the steps:
A, testing sample RNA extract, and get template ribonucleic acid solution;
B, the template ribonucleic acid solution reverse transcription in the steps A is become cDNA, adopt primer LSVPrimer-1 and LSV Primer-2 to carrying out pcr amplification again, get the PCR reaction solution; Wherein said LSV Primer-1 sequence is 5'-AAGATGAAAGTTGGCGTCGT-3'; Described LSV Primer-2 sequence is 5'-CACAAGCATGCTGTTCCACA-3';
C, get the PCR reaction solution and carry out the agarose gel electrophoresis analysis;
D, if there is the DNA band of 297bp, then proof detects and has lily asymptomatic virus in the articles for use.
5. a kind of detection method that detects lily asymptomatic virus according to claim 4 is characterized in that the RNA of testing sample described in the steps A extracts, and specifically comprises the steps:
A1, choose the lily leaf tissue of 0.1-0.2g performance suspect, use the equivalent health tissues simultaneously in contrast; The lily leaf tissue is added liquid nitrogen freezing, in the 1.5mL centrifuge tube of packing into after fully grinding;
A2, in centrifuge tube, add 400 ice-cold μ L RNA extracts, put upside down and mix;
4 ℃ of a3, tabletop refrigerated centrifuges, the centrifugal 10min of 12000g;
A4, get supernatant, add the mixed solution of 400 μ L phenol, chloroform, primary isoamyl alcohol, wherein the volume ratio of phenol, chloroform, primary isoamyl alcohol is 25:24:1, puts upside down to mix;
4 ℃ of a5, tabletop refrigerated centrifuges, the centrifugal 10min of 12000g;
A6, get supernatant, add the mixed solution of 400 μ L chloroforms, primary isoamyl alcohol, wherein the volume ratio of chloroform, primary isoamyl alcohol is 24:1, puts upside down to mix;
4 ℃ of a7, tabletop refrigerated centrifuges, the centrifugal 10min of 12000g;
A8, get supernatant, add 1000 μ L dehydrated alcohols and 40 μ L3M sodium-acetate pH5.2, put upside down and mix;
A9,4 ℃ of centrifugal 10min of 12000g of tabletop refrigerated centrifuge;
A10, stay precipitation, with the cold washing with alcohol of 500 μ L70% once, place on the Bechtop to dry up, being dissolved in 20 μ L does not then have in the water of RNA enzyme.
6. a kind of detection method that detects lily asymptomatic virus according to claim 4 is characterized in that the reverse transcription of template ribonucleic acid solution described in the step B becomes cDNA, specifically may further comprise the steps:
In the 0.5mL centrifuge tube, add deionized water 9.5 μ L, add total RNA2 μ L, Primer-2 primer 1 μ L, concentration is the dNTPs1 μ L of 10mmol/L, places 2min on ice immediately behind 70 ℃ of water-bath 5min; The centrifugal 30s of 3000rpm then; Add the synthetic damping fluid 4 μ L of 5 * cDNA, first chain again, 0.1mol/L DTT1 μ L; RNase inhibitor 1 μ L (30U); M-MLV ThermoScript II 1 μ L (20U); 37 ℃ of 60min.
7. a kind of detection method that detects lily asymptomatic virus according to claim 4 is characterized in that pcr amplification specifically may further comprise the steps described in the step B:
Add 10 * pcr amplification damping fluid, 2.5 μ L in the 0.2mL centrifuge tube, concentration is the MgCl of 25mmol/L
22 μ L, concentration is the dNTPs1 μ L of 10mmol/L, Primer-11 μ M(50pmol), Primer-21 μ M(50pmol), the cDNA first chain synthesis reaction liquid 2 μ L add water to 25 μ L, add Taq polysaccharase 0.5 μ L (1U) then; PCR cycling condition: 94 ℃ of sex change 3min; 94 ℃ of 1min then, 52 ℃ of 1min, 72 ℃ of 1min circulate 35 times; Last 72 ℃ are extended 10min; Preserve reaction product for 4 ℃.
8. a kind of detection method that detects lily asymptomatic virus according to claim 4 is characterized in that the agarose gel electrophoresis analysis specifically may further comprise the steps described in the step C:
C1, prepare by 1.5% concentration with 1 * TAE damping fluid and agarose, fusing evenly is cooled to 50-60 ℃ in microwave oven;
C2, adding
I nucleic acid dye or concentration are the ethidium bromide of 1 μ g/mL, mix, and pour on the gel platform, plug sample comb;
C3, treat that gel solidifies after, extract comb, add an amount of TAE buffering;
C4, get 1 μ L sample loading buffer and 5 μ L PCR reaction solutions mix, respectively itself and suitable dna molecular amount standard substance are added in the sample well then;
C5, connection power supply carry out electrophoresis, voltage 5V/cm;
C6, when the tetrabromophenol sulfonphthalein in the sample loading buffer migrates to enough DNA isolation fragments, stop electrophoresis; Place gel imaging system to observe in whole glue, and the record of taking pictures.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114717359A (en) * | 2022-03-28 | 2022-07-08 | 华中农业大学 | Lily virus LCrV-1 specificity detection target sequence, kit and detection method |
| CN116479185A (en) * | 2023-05-16 | 2023-07-25 | 南昌师范学院 | Method for detecting lily asymptomatic virus by RT-qPCR |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1793858A (en) * | 2005-12-22 | 2006-06-28 | 云南农业大学 | Method of synchronous detecting lily mottle virus, lily symptom less virus and cucumbus flomer leaf virus |
| CN101532064A (en) * | 2009-04-21 | 2009-09-16 | 中山出入境检验检疫局检验检疫技术中心 | A PCR primer used by a lily symptomless virus test method and a test kit thereof |
| CN101871018A (en) * | 2010-06-24 | 2010-10-27 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Real-time fluorescent RT-PCR method for detecting whether arabis mosaic virus, bean yellow mosaic virus and lily symptomless virus infect host |
| CN102031313A (en) * | 2010-08-16 | 2011-04-27 | 深圳出入境检验检疫局动植物检验检疫技术中心 | RT-PCR (reverse transcription-polymerase chain reaction) method for detecting the infection reality of host by arabis mosaic virus, bean yellow mosaic virus and lily symptomless virus |
-
2013
- 2013-05-06 CN CN201310162813.6A patent/CN103243175B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1793858A (en) * | 2005-12-22 | 2006-06-28 | 云南农业大学 | Method of synchronous detecting lily mottle virus, lily symptom less virus and cucumbus flomer leaf virus |
| CN101532064A (en) * | 2009-04-21 | 2009-09-16 | 中山出入境检验检疫局检验检疫技术中心 | A PCR primer used by a lily symptomless virus test method and a test kit thereof |
| CN101871018A (en) * | 2010-06-24 | 2010-10-27 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Real-time fluorescent RT-PCR method for detecting whether arabis mosaic virus, bean yellow mosaic virus and lily symptomless virus infect host |
| CN102031313A (en) * | 2010-08-16 | 2011-04-27 | 深圳出入境检验检疫局动植物检验检疫技术中心 | RT-PCR (reverse transcription-polymerase chain reaction) method for detecting the infection reality of host by arabis mosaic virus, bean yellow mosaic virus and lily symptomless virus |
Non-Patent Citations (1)
| Title |
|---|
| 徐榕雪等: "百合三种病毒的多重RT-PCR检测", 《园艺学报》, vol. 34, no. 2, 30 April 2007 (2007-04-30), pages 443 - 448 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114717359A (en) * | 2022-03-28 | 2022-07-08 | 华中农业大学 | Lily virus LCrV-1 specificity detection target sequence, kit and detection method |
| CN114717359B (en) * | 2022-03-28 | 2023-10-27 | 华中农业大学 | Lily virus LCrV-1 specific detection target sequence, kit and detection method |
| CN116479185A (en) * | 2023-05-16 | 2023-07-25 | 南昌师范学院 | Method for detecting lily asymptomatic virus by RT-qPCR |
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| CN103243175B (en) | 2014-12-24 |
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