CN1873019A - Quick method for testing chain reaction of multiple reverse transcription polymerase of lily virus - Google Patents
Quick method for testing chain reaction of multiple reverse transcription polymerase of lily virus Download PDFInfo
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Abstract
This invention relates to a method for rapidly detecting lily virus by multiplex RT-PCR. The method comprises: (1) synthesizing three pairs of primers of lily symptomless virus (LSV), lily mottle virus (LMoV) and Cucumber mosaic virus (CMV) sequences, and optimizing the concentrations, amplification temperatures, amplification time and cycle time; (2) introducing the three pairs of primers to a same reaction system, and performing PCR reaction. The method can detect CMV, LSV and LmoV simultaneously, and has such advantages as rapid detection, high accuracy, high efficiency, high sensitivity, high specificity.
Description
Technical field
The present invention relates to the detection method of a kind of plant virus, especially the chain reaction of multiple reverse transcription polymerase of lily virus (RT-PCR) method for quick.
Background technology
Lily is important cash crop, and at the existing long cultivation history of China, along with the continuous expansion of cultivating scale in recent years, lily suffers the phenomenon of virus infection also more and more serious.Studies show that; The virus that infects lily reaches 16 kinds, and wherein cucumber mosaic virus (CMV), lily asymptomatic virus (LSV), lily mottle virus (LMoV) are the main virus causing diseases that infects lily, and rose mosaic virus (PNRSV) also has generation on lily.Lily plant after susceptible shows as: yellow, floral leaf, downright bad streak, chlorisis streak, veinclearing, yellow keratin, deformity, chlorisis are mottled, necrotic spot, spend broken look etc., have had a strong impact on the output and the quality of fresh cutting flower.In the lily virus context of detection, comparatively common method has the RT-PCR detection method of serum detection method, Electronic Speculum detection method and single virus.Wherein serum detection method MONOCLONAL ANTIBODIES SPECIFIC FOR and purchase are difficult for, and waste time and energy, and need 2-3 days just can draw detected result at least, have therefore limited the utilization of this method; Though the Electronic Speculum detection method is with low cost, need comparatively expensive large-scale instrument-Electronic Speculum, have only few unit that purchasing power is arranged; The susceptibility of the RT-PCR detection method of single virus is higher, exact value can be realized the very viral RNA sample analysis of trace, but its detection can only be done qualitative analysis to single virus, if will detect three viruses, then can expend more human and material resources and financial resources.
Summary of the invention
The object of the present invention is to provide a kind of method for quick that can carry out RT-PCR simultaneously to 3 kinds of lily virus, when improving detection speed greatly, can save the detection cost to greatest extent, total RNA of high efficiency extraction sample and CDNA transcribe, and realize specificity, high efficiency and the stability of detection.
The present invention realizes by the following technical solutions: a kind of quick method for testing chain reaction of multiple reverse transcription polymerase of lily virus, preparation, reverse transcription and the cDNA that comprises design of primers, total RNA template is synthetic, pcr amplification, amplified production detect, the result judges, it is characterized in that:
(1), the following primer that is used for chain reaction of multiple reverse transcription polymerase of design:
LSVCP1-F：5’-ATGCAATCAAGACCAGCACA-3’
LSVCP2-R：5’-TCATCCATTATTTGCGTATC-3’
LMoV-F：5’-TGGGCACCTTGTGAATTACA-3’
LMoV-R：5’-TGCTGTATGCCTCTCCGTGT-3’
CMV-F：5’-CgTTCACATCTATCACCCTA-3’
CMV-R：5’-TACTTTCTCATgTCgCCTAT-3’
(2), reverse transcription and cDNA are synthetic
In Eppendorf PCR pipe, add following reagent successively:
Distilled water ddH
2O 8.0 μ L
50～100pmol/ μ L complementary primer, 0.5 μ L
Total RNA 2.0 μ L
70 ℃ of sex change 10min place 5min on ice
5 * M-MLV reverse transcription damping fluid, 4.0 μ L
10～20mmol/L?dNTPs 4.0μL
40～80u/ μ L ribonuclease inhibitor, 0.5 μ L
100～200u/ μ L M-MLV reversed transcriptive enzyme, 1.0 μ L
With above all the components mixing, behind the low-speed centrifugal, 37 ℃ of reaction 1h synthesize cDNA in the thermal cycler, carry out pcr amplification or are stored in-20 ℃;
(3), pcr amplification
In Eppendorf PCR pipe, add following reagent successively:
Distilled water ddH
2O 31.5 μ L
10 * PCR damping fluid, 5.0 μ L
10～20mmol/L?dNTPs 2.0μL
20～25pmol/ μ L LSV upstream primer, 1.0 μ L
20～25pmol/ μ L LSV downstream primer, 1.0 μ L
25～30pmol/ μ L LMoV upstream primer, 1.25 μ L
25～30pmol/ μ L LMoV downstream primer, 1.25 μ L
40～45pmol/ μ L CMV upstream primer, 0.5 μ L
40～45pmol/ μ L CMV downstream primer, 0.5 μ L
5～10u/ μ L rTaq polysaccharase, 1.0 μ L
20～50ng?cDNA 5.0μL
Cumulative volume 50.0 μ L
Abundant mixing, of short duration low-speed centrifugal is placed in the PCR instrument and increases, and reaction pattern for the formula PCR that falls progressively, specific procedure is: 94～95 ℃ of pre-sex change 10min, preceding 10 circulations are 94 ℃ of sex change 30s, annealing (1 ℃ of every cycle down is reduced to 54 ℃ from 64 ℃) 30s, 72 ℃ are extended 60s, 20 circulations in back are 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 60s, and last 72 ℃ are extended 10min.
Difficult point of the present invention and superiority are embodied in:
1, at 3 kinds of common in the lily culture main viruses, be cucumber mosaic virus (CMV), lily asymptomatic virus (LSV), lily mottle virus (LMoV), be difficult to its problem of rapid detection simultaneously with the detection method of routine, the invention provides the multiple RT-PCR method for quick, by this detection method above-mentioned 3 kinds of main lily virus are detected in same reaction system simultaneously, effectively improved detection speed, greatly reduce the detection cost, realized the high efficiency of the multiple rapid detection of lily virus, sensitivity and specificity are for the detection technique systems of flowers provides technical support.
2, the present invention is when carrying out the multiplex PCR amplification, size at amplified fragments is determined amplification condition, be that 3 pairs of primer sizes that the present invention designs are respectively 876bp (LSV), 552bp (LMoV), 335bp (CMV), both considered and to have reduced again because the big fragment product amplification difficulty that the product difference in size obviously causes by the convenient Different Results of distinguishing of electrophoresis.
3, the present invention is when design LSV, LMoV and three pairs of primers of CMV, determined primer concentration simultaneously, because primer concentration can directly influence the expanding effect of multiplex PCR, in order to make each amplified fragments can obtain the amplification of homogeneous simultaneously, when the best primer final concentration of having determined the LSV among the PCR, LMoV and CMV is respectively 20～25pmol/ μ L, 25～30pmol/ μ L and 40～45pmol/ μ L, make three fragments increase out.
4, for guaranteeing that every pair of primer all can obtain higher specificity and amplification efficiency in the multiplex PCR, the present invention adopts the formula PCR (annealing temperature Tm falls progressively to 54 ℃ from 64 ℃) that falls progressively, annealing temperature during with beginning selects to be higher than the Tm value of three pairs of primers, along with round-robin carries out, annealing temperature reduces gradually, and finally be lower than the Tm value of every pair of primer, to guarantee that the hybridization of first primer-template occurs between the complementary reactant, be between primer and the target fragment, like this, although annealing temperature finally can be reduced to the Tm value of non-specific hybridization, but the purpose product of this moment has begun the amplification of how much multiples, is in considerably beyond the status of non-specific PCR product in remaining circulation.
5, the present invention compares with multiple detection technique and can detect and distinguish the multiple virus that exists in the lily efficiently simultaneously, particularly when detecting compound dip-dye object, can be used as best detection technique means, also can be used for the regular rapid detection of lily original silkworm egg or detoxification core seedling.From extracting RNA to the PCR detected result that draws three kinds of viruses, can in 6-7h, finish, detection time is shorter, and sensitivity is higher, detects cost and greatly reduces, and higher reliability and repeatability are arranged.
The present invention is through experimental study, and its result is reported as follows:
The compound sick sample that infects of sick sample, LSV+LmoV+CMV and the lily healthy plant that only have LSV or LmoV or CMV are carried out triple RT-PCR amplifications, the same embodiment of test method respectively.The nucleic acid samples that the result contains LSV, LmoV, CMV, LSV+LmoV+CMV all can amplify about 876bp, the 552bp that conforms to test design, the obvious band of 335bp, and healthy plant can not amplify any band, sees Fig. 1 for details.
Description of drawings
Fig. 1 is that the specificity of triple RT-PCR compares;
Fig. 2 is with the LSV of malicious lily plant, LMOV and the triple RT-PCR detected results of CMV;
Fig. 3 is LSV, LMOV and the triple RT-PCR detected results of CMV with malicious lily ball.
Among Fig. 1, M:200bp molecular weight (Marker), 1: healthy leaves, 2:LSV+LMOV+CMV, 3:CMV, 4:LMOV, 5:LSV.
Among Fig. 2, M:200bp molecular weight (Marker), 1: be with malicious plant leaf, 2: the health plant blade.
Among Fig. 3, M:200bp molecular weight (Marker), 1: the healthy ball of planting, 2: band seed culture of viruses ball.
Embodiment
Below in conjunction with specific embodiment the present invention is described further, but content of the present invention is not limited in these.
1, viral isolates is gathered the blade at susceptible naturally lily plant middle part, academy of agricultural sciences, Yunnan Province flowers institute unity base, and sample detects through DAS-ELISA and confirms by lily asymptomatic virus (LSV), lily mottle virus (LMoV), compound the infecting of cucumber mosaic virus (CMV).
2, the total RNA extraction reagent box adopts the RNAex Reagent SystemIV of Shanghai Hua Shun company; Reversed transcriptive enzyme, cDNA synthetic agent box, clone's test kit are available from Promega company; Taq enzyme, dNTP, tetrabromophenol sulfonphthalein (6 * Loading Dye Solution) are available from TaKaRa company; Other reagent and consumptive material are respectively available from Sangon company, magnificent company etc.
3, damping fluid commonly used and plant and instrument
5 * TBE electrophoretic buffer
Tris 54g
Boric acid 27.5g
0.5mol/L?EDTA(pH8.0) 20mL
Be settled to 1000mL, 4 ℃ of preservations
Plant and instrument
High speed freezing centrifuge 5417C Eppendorf
PCR thermal cycler MJ-200 Pharmacia Biotech
Disk electrophoresis instrument Beckman
The Syngene of gel imaging analysis system
Other miniature instrument MJ
4, the preparation of total RNA template
The leaf tissue of the susceptible lily plant of 0.05g and the leaf tissue of healthy plant are put into the sterilization mortar respectively, each adds 1mL RNAex Reagent solution and is ground into homogenate, homogenate is moved in the 1.5mL centrifuge tube, room temperature is placed 5min, make and organize abundant cracking, add 200 μ L chloroforms, firmly put upside down centrifuge tube with mixing, leave standstill 10min after, the centrifugal 10min of 12000rpm, draw the upper strata water, move in another 1.5mL centrifuge tube, add the Virahol of 1/2 volume, mixing, the RNA precipitation reagent that adds 1/2 volume again, abundant mixing, room temperature is placed 10min.The centrifugal 5min of 7500rpm abandons supernatant, adds 500mL75% ethanol and washes 2～3 times, and the centrifugal 30min of 7500rpm abandons supernatant carefully, and room temperature leaves standstill 5～15min, makes the RNA precipitation dry just, adds 50 μ L distilled waters, and is standby;
5, reverse transcription and cDNA are synthetic
In the centrifuge tube of 0.2mL, add following reagent successively:
Distilled water ddH
2O 8.0 μ L
50pmol/ μ L complementary primer 0.5 μ L
The total RNA 2.0 μ L of lily blade
70 ℃ of sex change 10min place 5min on ice
5 * M-MLV reverse transcription damping fluid, 4.0 μ L
10mmol/L?dNTPs 4.0μL
40u/ μ L ribonuclease inhibitor 0.5 μ L
200u/ μ L M-MLV reversed transcriptive enzyme 1.0 μ L
With above all the components mixing, behind the low-speed centrifugal, in the thermal cycler 37 ℃ the reaction 1h, transcribe out cDNA, be used to carry out pcr amplification or be stored in-20 ℃ stand-by;
6, pcr amplification
In the Eppendorf of 0.2mL centrifuge tube, add following reagent successively:
Distilled water ddH
2O 31.5 μ L
10 * PCR damping fluid, 5.0 μ L
10mmol/L?dNTPs 2.0μL
20pmol/ μ L LSV upstream primer 1.0 μ L
20pmol/ μ L LSV downstream primer 1.0 μ L
25pmol/ μ L LMoV upstream primer 1.25 μ L
25pmol/ μ L LMoV downstream primer 1.25 μ L
40pmol/ μ L CMV upstream primer 0.5 μ L
40pmol/ μ L CMV downstream primer 0.5 μ L
5u/ μ L rTaq polysaccharase 1.0 μ L
50ng?cDNA 5.0μL
Cumulative volume 50.0 μ L
Abundant mixing, of short duration low-speed centrifugal is placed in the PCR instrument and increases.The employing formula PCR response procedures that falls progressively:
1＝95.0°for 10:00
2＝94.0°for 0:30
3＝64.0°for 0:30
-1.0°per?cycle
4＝72.0°for 1:00
5＝Goto?2， 10times
6＝94.0°for 0:30
7＝53.0°for 0:30
8＝72.0°for 1:00
9＝Goto?6， 20times
10＝72.0°for 10:00
11＝end
7, amplified production detects
5 * TBE being diluted to 1 * TBE working fluid at 1: 5, is mixed with the solution of 1.2～1.5% (W/V) with agarose (Agarose), and heating is melted agarose fully.When treating that coagulant liquid is cooled to 50 ℃～60 ℃, add an amount of ethidium bromide (final concentration is 0.5 μ g/ μ L), record platform, cooling.The PCR product is pressed 10: 1 mixing tetrabromophenol sulfonphthaleins (6 * Loading Dye Solution), point sample 20 μ L.By the voltage electrophoresis of 3～5V/cm, when tetrabromophenol sulfonphthalein migrates to 2/3 place of offset plate length, stop electrophoresis.Offset plate is taken out, be put in the gel imaging system amplification of observing, take a picture.
8, the result judges
The susceptible lily plant leaf sample amplification that contains LSV, LMoV and CMV has gone out 3 bands, and length is about 876bp, 552bp, 335bp, and healthy plant does not amplify any band (Fig. 2).
Embodiment 2
1, isolate is gathered the susceptible naturally lily ball outer scale of the Long Gelan of Yunnan Province gardening responsibility company limited, and sample detects through DAS-ELISA and confirms by lily asymptomatic virus (LSV), lily mottle virus (LMoV), compound the infecting of cucumber mosaic virus (CMV).
2, the total RNA extraction reagent box adopts the RNAex Reagent SystemIV of Shanghai Hua Shun company; Reversed transcriptive enzyme, cDNA synthetic agent box, clone's test kit are available from Promega company; Taq enzyme, dNTP, tetrabromophenol sulfonphthalein (6 * Loading Dye Solution) are available from TaKaRa company; Other reagent and consumptive material are respectively available from Sangon company, magnificent company etc.
3, damping fluid commonly used and plant and instrument
5 * TBE electrophoretic buffer
Tris 54g
Boric acid 27.5g
0.5mol/L?EDTA(PH8.0) 20mL
Be settled to 1000mL, 4 ℃ of preservations
Plant and instrument
High speed freezing centrifuge 5417C Eppendorf
PCR thermal cycler MJ-200 Pharmacia Biotech
Disk electrophoresis instrument Beckman
The Syngene of gel imaging analysis system
Other miniature instrument MJ
4, the preparation of total RNA template
The face tissue and the healthy face tissue of planting the ectosphere scale of the susceptible lily ball outer scale of 0.05g are put into the sterilization mortar respectively, each adds 1mL RNAex Reagent solution and is ground into homogenate, homogenate is moved in the 1.5mL centrifuge tube, and room temperature is placed 5min, makes and organizes abundant cracking.Add 200 μ L chloroforms, firmly put upside down centrifuge tube with mixing, leave standstill 10min after, the centrifugal 10min of 12000rpm.Draw the upper strata water, move in another 1.5mL centrifuge tube.The Virahol that adds 1/2 volume, mixing adds the RNA precipitation reagent of 1/2 volume again, abundant mixing, room temperature is placed 10min.The centrifugal 5min of 7500rpm abandons supernatant.Add 500mL 75% ethanol and wash 2～3 times, the centrifugal 30min of 7500rpm abandons supernatant carefully.Room temperature leaves standstill 5～15min, makes the RNA precipitation dry just, adds 50 μ L distilled waters, and is standby.
5, reverse transcription and cDNA are synthetic
Get the Eppendorf centrifuge tube of 0.2mL, add following reagent successively:
Distilled water ddH
2O 8.0 μ L
100pmol/ μ L complementary primer 0.5 μ L
The total RNA 2.0 μ L of lily ball
70 ℃ of sex change 10min place 5min on ice
5 * M-MLV reverse transcription damping fluid, 4.0 μ L
20mmol/L?dNTPs 4.0μL
80u/ μ L ribonuclease inhibitor 0.5 μ L
100u/ μ L M-MLV reversed transcriptive enzyme 1.0 μ L
With above all the components mixing, behind the low-speed centrifugal, 37 ℃ of reaction 1h transcribe out eDNA in the thermal cycler, carry out pcr amplification or are stored in-20 ℃.
6, pcr amplification
In the Eppendorf of 0.2mL centrifuge tube, add following reagent successively:
Distilled water ddH
2O 31.5 μ L
10 * PCR damping fluid, 5.0 μ L
20mmol/L?dNTPs 2.0μL
25pmol/ μ L LSV upstream primer 1.0 μ L
25pmol/ μ L LSV downstream primer 1.0 μ L
30pmol/ μ L LMoV upstream primer 1.25 μ L
30pmol/ μ L LMoV downstream primer 1.25 μ L
45pmol/ μ L CMV upstream primer 0.5 μ L
45pmol/ μ L CMV downstream primer 0.5 μ L
10u/ μ L rTaq polysaccharase 1.0 μ L
20ng?cDNA 5.0μL
Cumulative volume 50.0 μ L
Abundant mixing, of short duration low-speed centrifugal is placed in the PCR instrument and increases.The employing formula PCR response procedures that falls progressively:
1＝95.0°for 10:00
2＝94.0°for 0:30
3＝64.0°for 0:30
-1.0°per?cycle
4＝72.0°for 1:00
5＝Goto?2， 10times
6＝94.0°for 0:30
7＝53.0°for 0:30
8＝72.0°for 1:00
9＝Goto?6， 20times
10＝72.0°for 10:00
11＝end
7, amplified production detects
5 * TBE being diluted to 1 * TBE working fluid at 1: 5, is mixed with the solution of 1.2～1.5% (W/V) with agarose (Agarose), and heating is melted agarose fully.When treating that coagulant liquid is cooled to 50 ℃～60 ℃, add an amount of ethidium bromide (final concentration is 0.5 μ g/ μ L), record platform, cooling.The PCR product is pressed 10: 1 mixing tetrabromophenol sulfonphthaleins (6 * Loading Dye Solution), point sample 10～20 μ L.By the voltage electrophoresis of 3～5V/cm, when tetrabromophenol sulfonphthalein migrates to 2/3 place of offset plate length, stop electrophoresis.Offset plate is taken out, be put in the gel imaging system amplification of observing, take a picture.
8, the result judges
The susceptible lily ball sample amplification that contains LSV, LMoV and CMV has gone out 3 bands, and length is about 876bp, 552bp, 335bp, and the healthy ball sample of planting does not amplify any band (Fig. 3).
The result shows: add 3 pairs of designed primers by the present invention in same reaction system, only through a reaction system of overlapping 50 μ L, just can from identical or different template, amplify LSV, LMoV and 3 kinds of lily virus bands of CMV respectively, reach the purpose that pcr amplification just can detect these 3 kinds of cause of diseases, both simplified the detection formality, saved the detection cost again, for molecular disease virus detection method field opened up one accurately, efficiently, new way efficiently.
Claims (1)
1, a kind of quick method for testing chain reaction of multiple reverse transcription polymerase of lily virus comprises that preparation, reverse transcription and the cDNA of design of primers, total RNA template is synthetic, pcr amplification, amplified production detect, the result judges, it is characterized in that:
(1), the following primer that is used for chain reaction of multiple reverse transcription polymerase of design:
LSVCP1-F：5’-ATGCAATCAAGACCAGCACA-3’
LSVCP2-R：5’-TCATCCATTATTTGCGTATC-3’
LMoV-F：5’-TGGGCACCTTGTGAATTACA-3’
LMoV-R：5’-TGCTGTATGCCTCTCCGTGT-3’
CMV-F：5’-CgTTCACATCTATCACCCTA-3’
CMV-R：5’-TACTTTCTCATgTCgCCTAT-3’
(2), reverse transcription and cDNA are synthetic
In Eppendorf PCR pipe, add following reagent successively:
Distilled water ddH
2O 8.0 μ L
50～100pmol/ μ L complementary primer, 0.5 μ L
Total RNA 2.0 μ L
70 ℃ of sex change 10min place 5min on ice
5 * M-MLV reverse transcription damping fluid, 4.0 μ L
10～20mmol/L?dNTPs 4.0μL
40～80u/ μ L ribonuclease inhibitor, 0.5 μ L
100～200u/ μ L M-MLV reversed transcriptive enzyme, 1.0 μ L
With above all the components mixing, behind the low-speed centrifugal, 37 ℃ of reaction 1h synthesize cDNA in the thermal cycler, carry out pcr amplification or are stored in-20 ℃;
(3), pcr amplification
In Eppendorf PCR pipe, add following reagent successively:
Distilled water ddH
2O 31.5 μ L
10 * PCR damping fluid, 5.0 μ L
10～20mmol/L?dNTPs 2.0μL
20～25pmol/ μ L LSV upstream primer, 1.0 μ L
20～25pmol/ μ L LSV downstream primer, 1.0 μ L
25～30pmol/ μ L LMoV upstream primer, 1.25 μ L
25～30pmol/ μ L LMoV downstream primer, 1.25 μ L
40～45pmol/ μ L CMV upstream primer, 0.5 μ L
40～45pmol/ μ L CMV downstream primer, 0.5 μ L
5～10u/ μ L rTaq polysaccharase, 1.0 μ L
20～50ng?cDNA 5.0μL
Cumulative volume 50.0 μ L
Abundant mixing, of short duration low-speed centrifugal is placed in the PCR instrument and increases, and reaction pattern is the formula PCR that falls progressively, specific procedure is: 94～95 ℃ of pre-sex change 10min, preceding 10 circulations are 94 ℃ of sex change 30s, annealing 30s, 1 ℃ of promptly every cycle down, reduce to 54 ℃ from 64 ℃, 72 ℃ are extended 60s, and back 20 circulations are 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 60s, and last 72 ℃ are extended 10min.
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| CN101200770B (en) * | 2006-12-13 | 2010-09-15 | 中华人民共和国上海出入境检验检疫局 | Detection method of tomato black ring virus |
| CN101915835A (en) * | 2010-07-22 | 2010-12-15 | 中国科学院寒区旱区环境与工程研究所 | Lily mottle virus double-antibody sandwich enzyme-linked immunosorbent assay kit and preparation method |
| CN105695636A (en) * | 2016-04-07 | 2016-06-22 | 中国科学院寒区旱区环境与工程研究所 | Method for synchronously detecting lily symptomless virus (LSV), lily mottle virus (LMoV) and cucumber mosaic virus (CMV) |
| CN105713994A (en) * | 2016-04-07 | 2016-06-29 | 中国科学院寒区旱区环境与工程研究所 | Method for synchronously detecting LSV (lily symptomless viruse) and LMoV (lily mottle viruse) with immuno-capture double RT-PCR (reverse transcription-polymerase chain reaction) techniques |
| CN105755173A (en) * | 2016-04-07 | 2016-07-13 | 中国科学院寒区旱区环境与工程研究所 | Method for synchronously detecting three viruses of lily by immunocapture triple RT-PCR (reverse transcription-polymerase chain reaction) |
| CN105803113A (en) * | 2016-04-07 | 2016-07-27 | 中国科学院寒区旱区环境与工程研究所 | Method for synchronously detecting LSV (lily symptomless virus) and LMoV (lilymottle virus) |
| CN107164566A (en) * | 2017-07-19 | 2017-09-15 | 上海市农业科学院 | A kind of LAMP primer group, kit and detection method for detecting lily mottle virus |
| CN112458214A (en) * | 2020-12-28 | 2021-03-09 | 甘肃省农业科学院生物技术研究所 | Multiplex PCR (polymerase chain reaction) primer for detecting lily virus and application of multiplex PCR primer in RNA (ribonucleic acid) -extraction-free quick detection method of lily virus |
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- 2005-06-03 CN CN200510010835A patent/CN100582238C/en not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101200770B (en) * | 2006-12-13 | 2010-09-15 | 中华人民共和国上海出入境检验检疫局 | Detection method of tomato black ring virus |
| CN101915835A (en) * | 2010-07-22 | 2010-12-15 | 中国科学院寒区旱区环境与工程研究所 | Lily mottle virus double-antibody sandwich enzyme-linked immunosorbent assay kit and preparation method |
| CN101915835B (en) * | 2010-07-22 | 2013-06-19 | 中国科学院寒区旱区环境与工程研究所 | Lily mottle virus double-antibody sandwich enzyme-linked immunosorbent assay kit and preparation method |
| CN105803113A (en) * | 2016-04-07 | 2016-07-27 | 中国科学院寒区旱区环境与工程研究所 | Method for synchronously detecting LSV (lily symptomless virus) and LMoV (lilymottle virus) |
| CN105713994A (en) * | 2016-04-07 | 2016-06-29 | 中国科学院寒区旱区环境与工程研究所 | Method for synchronously detecting LSV (lily symptomless viruse) and LMoV (lily mottle viruse) with immuno-capture double RT-PCR (reverse transcription-polymerase chain reaction) techniques |
| CN105755173A (en) * | 2016-04-07 | 2016-07-13 | 中国科学院寒区旱区环境与工程研究所 | Method for synchronously detecting three viruses of lily by immunocapture triple RT-PCR (reverse transcription-polymerase chain reaction) |
| CN105695636A (en) * | 2016-04-07 | 2016-06-22 | 中国科学院寒区旱区环境与工程研究所 | Method for synchronously detecting lily symptomless virus (LSV), lily mottle virus (LMoV) and cucumber mosaic virus (CMV) |
| CN105755173B (en) * | 2016-04-07 | 2019-10-25 | 中国科学院寒区旱区环境与工程研究所 | Three kinds of viral methods of RT-PCR synchronous detecting lily are captured with triple complex immunities |
| CN105803113B (en) * | 2016-04-07 | 2020-02-18 | 中国科学院寒区旱区环境与工程研究所 | Method for synchronously detecting lily latent virus and lily mottle virus by double composite immunocapture RT-PCR reaction |
| CN105713994B (en) * | 2016-04-07 | 2020-02-18 | 中国科学院寒区旱区环境与工程研究所 | Method for synchronously detecting lily latent virus and lily mottle virus by using double composite immunocapture RT-PCR (reverse transcription-polymerase chain reaction) |
| CN107164566A (en) * | 2017-07-19 | 2017-09-15 | 上海市农业科学院 | A kind of LAMP primer group, kit and detection method for detecting lily mottle virus |
| CN112458214A (en) * | 2020-12-28 | 2021-03-09 | 甘肃省农业科学院生物技术研究所 | Multiplex PCR (polymerase chain reaction) primer for detecting lily virus and application of multiplex PCR primer in RNA (ribonucleic acid) -extraction-free quick detection method of lily virus |
| CN112458214B (en) * | 2020-12-28 | 2024-04-12 | 甘肃省农业科学院生物技术研究所 | Multiplex PCR primer for detecting lily virus and application of multiplex PCR primer in RNA extraction-free lily virus rapid detection method |
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