CN101691614B - Method for rapidly identifying rice black-streaked dwarf virus and southern rice black-streaked dwarf virus - Google Patents
Method for rapidly identifying rice black-streaked dwarf virus and southern rice black-streaked dwarf virus Download PDFInfo
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Abstract
The invention provides a method for rapidly identifying the rice black-streaked dwarf virus and southern rice black-streaked dwarf virus, belonging to the field of plant protection. Three primers are designed according to the nucleotide sequence of the rice black-streaked dwarf virus and southern rice black-streaked dwarf virus, which are respectively [A] RB1_S9_F, [B] RB2_S9_F and [C] RB_S9_R, wherein the combination of A and C detects the rice black-streaked dwarf virus and the combination of B and C detects the southern rice black-streaked dwarf virus. After the total RNA is extracted from the samples, the two viruses of the rice black-streaked dwarf virus and the southern rice black-streaked dwarf virus can be detected by only one RT-PCR, thus realizing the identification of two viruses. Compared with the traditional method in which one RT-PCR can only detect one virus, the invention greatly reduces cost, reduces time, and is a detection method which is special, sensitive, economic and convenient.
Description
Technical field
The present invention relates to a kind of to rice black-streaked dwarf virus and the quick method of differentiating of southern rice black-streaked dwarf virus, platymiscium protection field.
Background technology
Rice black-streaked dwarf virus (Rice black-streaked dwarf virus, RBSDV), a kind ofly on the paddy rice to endanger serious virus disease, be under the jurisdiction of Phytoreovirus section Fijivirus and belong to (Fijivirus), virion is spherical, mainly propagated by small brown rice planthopper, symptom main manifestations in early stage in field stunts for planting, leaf dark green, later stage wax occurs at blade, leaf sheath and cane and drips the shape projection, then form secret note, very large to the yield effect of paddy rice, grave illness field even can cause without output.This virus also can infect corn (maize rough dwarf virus), wheat (wheat is green short) and multiple gramineous weeds except infecting paddy rice.Be with popularly in 1963 and 1966 in Jiangsu Province, China, Shanghai, Zhejiang one, caused heavy losses in the many corns of China and paddy rice producing region eruption and prevalence the nineties.Should disease spread generation in the area, Jiangsu and Zhejiang Provinces in recent years, and be day by day serious trend, 2008 are only Jiangsu Province's onset area just reaches 4,000,000 mu, causes local Rice Production loss serious.
Find again a kind of new Virus Diseases of Rice in Guangdong Province and Hainan Province in recent years, the field symptom is extremely similar to rice black-streaked dwarf virus, its genome sequence is measured rear discovery itself and rice black-streaked dwarf virus also have larger difference (homology of both S9 and S10 fragment only is 75% and 80%), so tentative southern rice black-streaked dwarf virus (Southern rice black-streaked dwarf virus by name, SRBSDV), this virus also belongs to Phytoreovirus section Fijivirus and belongs to (Fijivirus), virion is spherical, genome is comprised of 10 double-stranded RNAs, vector is mainly white backed planthopper, small brown rice planthopper also can be propagated, the field Symptoms is that plant stunts, dark green leaf color, strip oyster white or Vandyke brown pimple appear in blade back and stem stalk, and the host also comprises corn and multiple weeds except paddy rice.
Because two-strain is closely similar at aspects such as symptom, plastochondria shape, vector and hosts, has also caused difficulty to its diagnosis and evaluation thus.
The normal method that adopts has on the plant virus Identification at present: the biological inoculation experiment, serology detects, electron microscopic observation and molecular Biological Detection.Because this two-strain all has the characteristic that approaches very much aspect a lot, therefore the detection method of traditional plant virus identifies that such as electron microscopic observation (the virus particle size shape is close), traditional biological inoculation (symptom, amboceptor, host range are close), serology detect methods such as (sequence still have higher homology) and all be difficult to differentiation.Many still molecular Biological Detection methods of using in the detection of RBSDV at present, wherein RT-PCR is the method for commonly using, also there was in the past report to use the method for RT-PCR to detect RBSDV, can detect simultaneously RBSDV and SRBSDV but not yet see, and the method for distinguishing, present method only needs a RT-PCR can realize two kinds virus discriminating, is guaranteeing under susceptibility and the specific prerequisite, simplified operation, provided cost savings.
Summary of the invention
The present invention has filled up the blank on rice black-streaked dwarf virus and the southern rice black-streaked dwarf virus authentication technique, and a kind of quick, easy, sensitive, special discrimination method is provided.
1, design of primers:
(1) according to the rice black-streaked dwarf virus S9 nucleotide sequence of having reported on the NCBI and south rice black matrix dwarf virus S9 nucleotide sequence design degenerated primer, primer sequence is as follows:
RB1_S9_F：5`-GRTAGACAGGCAAAYMTAAGCGT-3`
RB2_S9_F：5`-TTACAYCAAGCACTTTGCGAGG-3`
RB_S9_R：5`-GGATTACAACAHACACAMCGAAA-3`
(2) primer pairing and amplification purpose nucleic acid band is big or small as follows:
Combination of primers RT primer detects Virus Name purpose clip size
RB1_S9_F/RB_S9_R RB_S9_R rice black-streaked dwarf virus 1119bp
RB2_S9_F/RB_S9_R RB_S9_R southern rice black-streaked dwarf virus 569bp
2, total RNA extracts:
Get plant sample and organize 0.1g, add 1mL Trizol reagent and fully grind, lapping liquid moves into a 1.5ml centrifuge tube, and room temperature is placed 5min; Add chloroform 200 μ L, leave standstill 3min behind the mixing; 12,000g, 4 ℃ of centrifugal 15min; Get upper water and move into mutually in the new 1.5ml centrifuge tube, add isopyknic Virahol, leave standstill 5min behind the mixing; 12,000g, 4 ℃, centrifugal 10min, supernatant discarded; Add 1mL 70% washing with alcohol precipitation; Dry RNA precipitation then adds 40 μ L DEPC and processes water dissolution precipitation ,-20 ℃ of preservations.
3, RT-PCR amplification:
(1) reverse transcription (RT):
Get Eppendorf pipe, add sample: total RNA 3 μ L of extraction, primer RB_S9_R 1 μ L, DEPC processes water 8.5 μ L, places 65 ℃ of lower sex change 5min, takes out to place immediately and places 1min on ice.Add successively more following reagent:
5 * M-MuLV ThermoScript II damping fluid, 4 μ L
dNTPs(10mM/dNTP) 2μL
RNase inhibitor(20U/μL) 0.5μL
M-M μ LV ThermoScript II (200U/ μ L) 1 μ L
42 ℃ of water-bath 1h process 5min for 70 ℃, and-20 ℃ save backup.
(2) polymerase chain reaction (PCR):
Take synthetic cDNA the first chain of RT as template, carry out pcr amplification, reaction system comprises:
Reverse transcription product 2 μ L
10 * PCR damping fluid, 2.5 μ L
dNTP(10mM/dNTP) 0.5μL
RB1_S9_F(10μmol/L) 1.5μL
RB2_S9_F(10μmol/L) 1.5μL
RB_S9_R(10μmol/L) 1.5μL
TaqDNA polysaccharase (5U/L) 0.5 μ L
DEPC processes water 15 μ L
Cumulative volume 25 μ L
Reaction conditions is 94 ℃ of denaturation 5min; 94 ℃ sex change 50sec, 50-55 ℃ annealing 50sec, 72 ℃ of extension 2min, 30-40 circulation; Last 72 ℃ are extended 10min, 4 ℃ of preservations.
4, electrophoresis detection:
Get 10 μ L PCR products through 1% sepharose electrophoresis 40min under 0.5 * tbe buffer liquid and 120V voltage conditions, dyeing 10min in the ethidium bromide (EB) of 0.5 μ g/mL, in gel imaging system, observe after the dyeing, in the sample that only infects rice black-streaked dwarf virus, can observe the nucleic acid band of a 1119bp, in the sample that only infects southern rice black-streaked dwarf virus, can observe the nucleic acid band of a 569bp, infect at the same time in the sample of rice black-streaked dwarf virus and southern rice black-streaked dwarf virus and can observe 569bp and two nucleic acid bands of 1119bp.
Description of drawings
Accompanying drawing is the electrophoresis detection result (M: standard molecular weight of rice black-streaked dwarf virus and southern rice black-streaked dwarf virus; 1: the rice black-streaked dwarf virus sample; 2: the southern rice black-streaked dwarf virus sample; 3: the composite sample of rice black-streaked dwarf virus and southern rice black-streaked dwarf virus)
Embodiment
Example one:
Take the paddy rice biased sample of known infection rice black-streaked dwarf virus and southern rice black-streaked dwarf virus and the paddy rice sample that infects respectively rice black-streaked dwarf virus and southern rice black-streaked dwarf virus as detected object, with the negative contrast of healthy paddy rice sample.
1, design of primers:
(1) according to the rice black-streaked dwarf virus S9 nucleotide sequence of having reported on the NCBI and south rice black matrix dwarf virus S9 nucleotide sequence design degenerated primer, primer sequence is as follows:
RB1_S9_F：5`-GRTAGACAGGCAAAYMTAAGCGT-3`
RB2_S9_F：5`-TTACAYCAAGCACTTTGCGAGG-3`
RB_S9_R：5`-GGATTACAACAHACACAMCGAAA-3`
(2) primer pairing and amplification purpose nucleic acid band is big or small as follows:
Combination of primers RT primer detects Virus Name purpose clip size
RB1_S9_F/RB_S9_R RB_S9_R rice black-streaked dwarf virus 1119bp
RB2_S9_F/RB_S9_R RB_S9_R southern rice black-streaked dwarf virus 569bp
2, total RNA extracts:
Get plant sample and organize 0.1g, add 1mL Trizol reagent and fully grind, lapping liquid moves into a 1.5ml centrifuge tube, and room temperature is placed 5min; Add chloroform 200 μ L, leave standstill 3min behind the mixing; 12,000g, 4 ℃ of centrifugal 15min; Get upper water and move into mutually in the new 1.5ml centrifuge tube, add isopyknic Virahol, leave standstill 5min behind the mixing; 12,000g, 4 ℃, centrifugal 10min, supernatant discarded; Add 1mL 70% washing with alcohol precipitation; Dry RNA precipitation then adds 40 μ L DEPC and processes water dissolution precipitation ,-20 ℃ of preservations.
3, RT-PCR amplification:
(1) reverse transcription (RT):
Get Eppendorf pipe, add sample: total RNA 3 μ L of extraction, primer RB_S9_R 1 μ L, DEPC processes water 8.5 μ L, places 65 ℃ of lower sex change 5min, takes out to place immediately and places 1min on ice.Add successively more following reagent:
5 * M-MuLV ThermoScript II damping fluid, 4 μ L
dNTPs(10mM/dNTP) 2μL
RNase inhibitor(20U/μL) 0.5μL
M-MuLV ThermoScript II (200U/ μ L) 1 μ L
42 ℃ of water-bath 1h process 5min for 70 ℃, and-20 ℃ save backup.
(2) polymerase chain reaction (PCR):
Take synthetic cDNA the first chain of RT as template, carry out pcr amplification, reaction system comprises:
10 * PCR damping fluid, 2.5 μ L
dNTP(10mM/dNTP) 0.5μL
RB1_S9_F(10μmol/L) 1.5μL
RB2_S9_F(10μmol/L) 1.5μL
RB_S9_R(10μmol/L) 1.5μL
TaqDNA polysaccharase (5U/L) 0.5 μ L
DEPC processes water 15 μ L
Cumulative volume 25 μ L
Reaction conditions is 94 ℃ of denaturation 5min; 94 ℃ sex change 50sec, 50-55 ℃ annealing 50sec, 72 ℃ of extension 2min, 30-40 circulation; Last 72 ℃ are extended 10min, 4 ℃ of preservations.
4, electrophoresis detection:
Get 10 μ L PCR products through 1% sepharose electrophoresis 40min under 0.5 * tbe buffer liquid and 120V voltage conditions, dyeing 10min observes in gel imaging system after the dyeing in the ethidium bromide (EB) of 0.5 μ g/mL.
5, interpretation of result:
In the sample that only infects rice black-streaked dwarf virus, can observe the nucleic acid band of a 1119bp, in the sample that only infects southern rice black-streaked dwarf virus, can observe the nucleic acid band of a 569bp, infect at the same time in the sample of rice black-streaked dwarf virus and southern rice black-streaked dwarf virus and can observe 569bp and two nucleic acid bands of 1119bp.
Example two:
Take the paddy rice biased sample of known infection rice black-streaked dwarf virus and southern rice black-streaked dwarf virus and the paddy rice sample that infects respectively rice black-streaked dwarf virus and southern rice black-streaked dwarf virus as detected object, with the negative contrast of healthy paddy rice sample.
1, design of primers:
(1) according to the rice black-streaked dwarf virus S9 nucleotide sequence of having reported on the NCBI and south rice black matrix dwarf virus S9 nucleotide sequence design degenerated primer, primer sequence is as follows:
RB1_S9_F：5`-GRTAGACAGGCAAAYMTAAGCGT-3`
RB2_S9_F：5`-TTACAYCAAGCACTTTGCGAGG-3`
RB_S9_R：5`-GGATTACAACAHACACAMCGAAA-3`
(2) primer pairing and amplification purpose nucleic acid band is big or small as follows:
Combination of primers RT primer detects Virus Name purpose clip size
RB1_S9_F/RB_S9_R RB_S9_R rice black-streaked dwarf virus 1119bp
RB2_S9_F/RB_S9_R RB_S9_R southern rice black-streaked dwarf virus 569bp
2, total RNA extracts:
Get plant sample and organize 0.1g, add 1mL Trizol reagent and fully grind, lapping liquid moves into a 1.5ml centrifuge tube, and room temperature is placed 5min; Add chloroform 200 μ L, leave standstill 3min behind the mixing; 12,000g, 4 ℃ of centrifugal 15min; Get upper water and move into mutually in the new 1.5ml centrifuge tube, add isopyknic Virahol, leave standstill 5min behind the mixing; 12,000g, 4 ℃, centrifugal 10min, supernatant discarded; Add 1mL 70% washing with alcohol precipitation; Dry RNA precipitation then adds 40 μ L DEPC and processes water dissolution precipitation ,-20 ℃ of preservations.
3, RT-PCR amplification:
(1) reverse transcription (RT):
Get Eppendorf pipe, add sample: total RNA 3 μ L of extraction, primer RB_S9_R 1 μ L, DEPC processes water 8.5 μ L, places 65 ℃ of lower sex change 5min, takes out to place immediately and places 1min on ice.Add successively more following reagent:
5 * M-MuLV ThermoScript II damping fluid, 4 μ L
dNTPs(10mM/dNTP) 2μL
RNase inhibitor(20U/μL) 0.5μL
M-MuLV ThermoScript II (200U/ μ L) 1 μ L
42 ℃ of water-bath 1h process 5min for 70 ℃, and-20 ℃ save backup.
(2) polymerase chain reaction (PCR):
Take synthetic cDNA the first chain of RT as template, carry out pcr amplification, reaction system comprises:
10 * PCR damping fluid, 2.5 μ L
dNTP(10mM/dNTP) 0.5μL
RB1_S9_F(10μmol/L) 1.5μL
RB2_S9_F(10μmol/L) 1.5μL
RB_S9_R(10μmol/L) 1.5μL
TaqDNA polysaccharase (5U/L) 0.5 μ L
DEPC processes water 15 μ L
Cumulative volume 25 μ L
Reaction conditions is 94 ℃ of denaturation 5min; 94 ℃ sex change 50sec, 50-55 ℃ annealing 50sec, 72 ℃ of extension 2min, 30-40 circulation; Last 72 ℃ are extended 10min, 4 ℃ of preservations.
4, electrophoresis detection:
Get 10 μ L PCR products through 1% sepharose electrophoresis 40min under 0.5 * tbe buffer liquid and 120V voltage conditions, dyeing 10min observes in gel imaging system after the dyeing in the ethidium bromide (EB) of 0.5 μ g/mL.
5, interpretation of result:
In the sample that only infects rice black-streaked dwarf virus, can observe the nucleic acid band of a 1119bp, in the sample that only infects southern rice black-streaked dwarf virus, can observe the nucleic acid band of a 569bp, infect at the same time in the sample of rice black-streaked dwarf virus and southern rice black-streaked dwarf virus and can observe 569bp and two nucleic acid bands of 1119bp.
Claims (3)
1. method of differentiating fast rice black-streaked dwarf virus and southern rice black-streaked dwarf virus may further comprise the steps:
(1) design of primers:
1. according to the rice black-streaked dwarf virus S9 nucleotide sequence of having reported on the NCBI and south rice black matrix dwarf virus S9 nucleotide sequence design degenerated primer, primer sequence is as follows:
RB1_S9_F：5`-GRTAGACAGGCAAAYMTAAGCGT-3`
RB2_S9_F：5`-TTACAYCAAGCACTTTGCGAGG-3`
RB_S9_R：5`-GGATTACAACAHACACAMCGAAA-3`
2. primer pairing and amplification purpose nucleic acid band is big or small as follows:
Combination of primers RT primer detects Virus Name purpose clip size
RB1_S9_F/RB_S9_R RB_S9_R rice black-streaked dwarf virus 1119bp
RB2_S9_F/RB_S9_R RB_S9_R southern rice black-streaked dwarf virus 569bp
(2) total RNA extracts:
Get plant sample and organize 0.1g, add 1mL Trizol reagent and fully grind, lapping liquid moves into a 1.5ml centrifuge tube, and room temperature is placed 5min; Add chloroform 200 μ L, leave standstill 3min behind the mixing; 12,000g, 4 ℃ of centrifugal 15min; Get upper water and move into mutually in the new 1.5ml centrifuge tube, add isopyknic Virahol, leave standstill 5min behind the mixing; 12,000g, 4 ℃, centrifugal 10min, supernatant discarded; Add 1mL 70% washing with alcohol precipitation; Dry RNA precipitation then adds 40 μ L DEPC and processes water dissolution precipitation ,-20 ℃ of preservations.
(3) RT-PCR amplification:
1. reverse transcription (RT):
Get Eppendorf pipe, add sample: total RNA 3 μ L of extraction, primer RB_S9_R1 μ L, DEPC processes water 8.5 μ L, places 65 ℃ of lower sex change 5min, takes out to place immediately and places 1min on ice.Add successively more following reagent:
42 ℃ of water-bath 1h process 5min for 70 ℃, and-20 ℃ save backup.
2. polymerase chain reaction (PCR):
Take synthetic cDNA the first chain of RT as template, carry out pcr amplification, reaction system comprises:
Reaction conditions is 94 ℃ of denaturation 5min; 94 ℃ sex change 50sec, 50-55 ℃ annealing 50sec, 72 ℃ of extension 2min, 30-40 circulation; Last 72 ℃ are extended 10min, 4 ℃ of preservations.
(4) electrophoresis detection:
Get 10 μ LPCR products through 1% sepharose electrophoresis 40min under 0.5 * tbe buffer liquid and 120V voltage conditions, 10min dyes in the ethidium bromide of 0.5 μ g/mL, in gel imaging system, observe after the dyeing, in the sample that only infects rice black-streaked dwarf virus, can observe the nucleic acid band of a 1119bp, in the sample that only infects southern rice black-streaked dwarf virus, can observe the nucleic acid band of a 569bp, infect at the same time in the sample of rice black-streaked dwarf virus and southern rice black-streaked dwarf virus and can observe 569bp and two nucleic acid bands of 1119bp.
2. the method for quick discriminating rice black-streaked dwarf virus according to claim 1 and southern rice black-streaked dwarf virus is characterized in that the annealing temperature in the pcr amplification is 52-53 ℃.
3. the method for quick discriminating rice black-streaked dwarf virus according to claim 1 and southern rice black-streaked dwarf virus, the cycle index that it is characterized in that pcr amplification is 30-35 time.
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Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101967491B (en) * | 2010-11-01 | 2013-02-20 | 中国农业科学院植物保护研究所 | Rice black-streaked dwarf virus (RBSDV) RNA interference vectors, construction method and application |
| CN102168150B (en) * | 2011-03-24 | 2013-04-10 | 江苏省农业科学院 | Reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapidly detecting southern rice black-streaked dwarf viruses in plant hopper |
| CN102168151B (en) * | 2011-03-24 | 2013-01-09 | 江苏省农业科学院 | Reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapidly detecting rice black-streaked dwarf viruses in plant hopper |
| CN102154518B (en) * | 2011-03-24 | 2012-11-28 | 江苏省农业科学院 | Technology for rapidly detecting rice black-streaked dwarf viruses in plants by reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method |
| CN102286634B (en) * | 2011-03-24 | 2013-03-06 | 江苏省农业科学院 | Technology for quickly detecting southern rice black-streaked stunt virus of plant by RT-LAMP (Reverse Transcription-Loop-mediated Isothermal Amplification) method |
| CN102660653B (en) * | 2012-05-29 | 2013-12-04 | 湖南农业大学 | PCR (polymerase chain reaction) primer and method for detecting southern rice black-streaked dwarf virus in bodies of plant hoppers |
| CN103103288B (en) * | 2013-01-05 | 2015-03-11 | 江苏省农业科学院 | Method for rapidly and synchronously detecting wheat yellow mosaic virus and Chinese wheat mosaic virus |
| CN103103292A (en) * | 2013-01-31 | 2013-05-15 | 江苏省农业科学院 | Method for rapidly detecting in-vivo rice black streaked dwarf virus of single-head small brown rice planthopper |
| CN104357580A (en) * | 2014-10-09 | 2015-02-18 | 江苏省农业科学院 | Multiplex RT-PCR (reverse transcription-polymerase chain reaction) method for detecting various viruses of cucurbit plant with two-step method as well as special primer group for method |
| CN104450960B (en) * | 2014-11-24 | 2017-02-01 | 浙江省农业科学院 | Method for rapidly detecting pathogen of rice black-streaked dwarf |
| CN104651537B (en) * | 2015-03-11 | 2017-04-12 | 华南农业大学 | One-step multiplex-PCR detection method capable of detecting four rice viruses at same time |
| CN104830998B (en) * | 2015-05-20 | 2018-10-09 | 江苏焦点农业科技有限公司 | A kind of method for identifying molecules of black streaked dwarf virus of rice resistance |
| CN107130057B (en) * | 2016-05-26 | 2018-02-09 | 东北农业大学 | Detect rice black-streaked dwarf virus and the RT PCR of southern rice black-streaked dwarf virus and its application |
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| 杨杰等.江苏水稻黑条矮缩病病毒的RT-PCR分析和快速检测.《华北农学报》.2008,第23卷(第6期),87-92. * |
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