CN104141015A - Method for quickly detecting rice stripe virus and rice black-streaked dwarf virus in laodelphax striatellus - Google Patents
Method for quickly detecting rice stripe virus and rice black-streaked dwarf virus in laodelphax striatellus Download PDFInfo
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Abstract
The invention provides a method for quickly and synchronously detecting rice stripe virus (RSV) and rice black-streaked dwarf virus (RBSDV) in an insect vector laodelphax striatellus, and belongs to the field of plant protection. According to nucleotide sequences of the rice stripe virus RNA4 and the rice black-streaked dwarf virus S2, three primers are designed as follows: [A] RS-F, [B] RB-F, and [C] RSRB-R, wherein A and C are combined to detect the rice stripe virus; the B and C are combined to detect the rice black-streaked dwarf virus. After extracting total RNA of single laodelphax striatellus, only one RT-PCR is needed, so that the rice stripe virus and the rice black-streaked dwarf virus can be detected, and thus, and quick and synchronous detection for the two viruses in the laodelphax striatellus is realized. Compared with the conventional method for detecting one virus by virtue of one RT-PCR, the cost is greatly saved and the time is saved, and thus, the method is a quick, simple, flexible and specific detecting method. The method disclosed by the invention can be applied to monitoring work of a virus carried rate of a field laodelphax striatellus colony, and can also be applied to related research work of a virus and vector interaction mechanism.
Description
Technical field
The present invention relates to a kind of method to rice stripe virus in amboceptor insect small brown rice planthopper body and the detection of rice black-streaked dwarf virus Fast synchronization, platymiscium protection field.
Background technology
Rice stripe virus (Rice stripe virus, RSV) be the prototypical member of very thin Tobamovirus (Tenuivirus), its stripe disease causing is one of important disease of paddy rice, between many decades in the past, East China Dao Qu be take as main vast rice district outbreak of epidemic in Gai Bing China, and Rice Production is formed to great threat.For example, within 2004, Jiangsu Province's onset area reaches 2,355 ten thousand mu, account for 79% of Jiangsu Monitoring of Paddy Rice Plant Area, paddy rice total crop failure in flakes, within 2005, reach 2,800 ten thousand mu, and start to Zhejiang, the periphery provinces and cities such as Anhui, Henan, Shandong, Shanghai spread, and because its serious hazardness had once been called as " cancer " on paddy rice, have caused very large social repercussion.
Rice black-streaked dwarf virus (Rice black-streaked dwarf virus, RBSDV), be a kind ofly on paddy rice to endanger serious virus disease, be under the jurisdiction of Phytoreovirus section Fijivirus and belong to (Fijivirus), virion is spherical, symptom main manifestations in early stage in field is that plant stunts, leaf dark green, the later stage occurs that on blade, leaf sheath and cane wax drips shape projection, then forms secret note, very large to the yield effect of paddy rice, grave illness field even can cause without output.Except paddy rice, this virus also can infect corn and cause maize rough dwarf virus, infects wheat and cause the green short disease of wheat, in addition, also can infect multiple gramineous weeds.It is popular that black streaked dwarf virus of rice in 1963 and 1966 is with in Jiangsu Province, China, Shanghai, Zhejiang one, and in the many corns of China and paddy rice producing region outbreak of epidemic, caused heavy losses the nineties.This disease spreads generation in area, Jiangsu and Zhejiang Provinces in recent years, and is day by day serious trend, and 2008 are only Jiangsu Province's onset area just reaches 4,000,000 mu, causes local Rice Production loss serious.
RSV and RBSDV are mainly propagated by their common amboceptor insect small brown rice planthopper Laodelphax striatellus Fall é n, and difference is that RSV can be by small brown rice planthopper transovarian transmission, and RBSDV transovarian transmission not.The classical symptom of stripe disease is striped spot or the patch that occurs chlorisis, and plant height is generally normal, and being plant, the symptom of black streak dwarf stunts, and leaf dark green, visible white wax of later stage is dripped shape projection, then forms secret note.Two kinds of diseases are very easily distinguished diagnosis in field, but the band of amboceptor small brown rice planthopper poison situation cannot directly differentiate, need could realize by biology techniques.Field small brown rice planthopper colony has vital role with the prediction of malicious rate with continuing to monitor in the innoxious comprehensive prevention and control of Rice Virus disease, to the employing of follow-up anti-control techniques, can play positive directive function.
At present, the authentication method of the plant virus in insect amboceptor mainly contains: biological inoculation experiment, serology detection, electron microscopic observation and molecular Biological Detection.Biological inoculation experiment is to collect small brown rice planthopper thorn to inhale susceptible rice varieties, then observes paddy rice symptom, and not only workload is large, and length consuming time, and for example the aobvious disease time of black streak dwarf often needs 1 month.The dependency that serology detects antagonist is high, needs antibody with high specificity, and the monoclonal antibody of RSV detects comparative maturity at present, and RBSDV antiserum(antisera) is also immature efficiently.Electron microscopic observation needs high value plant and instrument, and is not suitable for a large amount of sample detection, is generally applied to the research work of pathogenic characteristic.What current application was more still take RT-PCR technology as main molecular Biological Detection method, also there is report to use the method for RT-PCR to detect RSV and RBSDV before, but not yet report the technological method of energy synchronous detection RSV and RBSDV, present method only needs a RT-PCR can realize the virus discriminating of two kinds, guaranteeing under the prerequisite of reliability and sensitivity, simplified operation, provided cost savings and the time.
Summary of the invention
The present invention has filled up the blank on rice stripe virus and rice black-streaked dwarf virus Fast synchronization authentication technique in amboceptor small brown rice planthopper body, a kind of quick, easy, sensitive, special discrimination method is provided, can be applicable to field small brown rice planthopper colony with the monitoring of malicious rate and virus and amboceptor do mutually machine-processed research work.
1, design of primers:
1) NCBI (
http:// www.ncbi.nlm.nih.gov/sites/entrez? db=nucleotide) the rice stripe virus RNA4 nucleotide sequence (D10979 that reported of upper download, AF513505, EU931525, AJ871748, FJ602692, EU931521, EU931517, AF221834, AF221835, FJ602700) and rice black-streaked dwarf virus S2 nucleotide sequence (AJ409145, KC134290), use the Clustal V method of DNAstar-MegAlign software to carry out sequence alignment, find that RSV-RNA4 has a bit of sequence very similar to RBSDV-S2, the general primer that utilizes this section of similar district design RSV and RBSDV, primer sequence is as follows:
RSRB-R：5’-CCYATCACAAASAAATMAAAAT-3’
RS-F：5’-AGATCCAGAGAGAGTCACGGAAG-3’
RB-F：5’-GTTCAAAGACAATACACTCAAAA-3’
2) size of primer pairing and amplification object nucleic acid band is as follows:
Combination of primers detects Virus Name object clip size
RS-F/RSRB-R rice stripe virus 1114bp
RB-F/RSRB-R rice black-streaked dwarf virus 414bp
2, total RNA extracts:
1) get single head small brown rice planthopper and be placed in 1.5mL centrifuge tube, add 250 μ L Trizol reagent fully to grind, room temperature is placed 5min;
2) add 50 μ L chloroforms, mix rear standing 3min;
3) 4 ℃, 12000g, centrifugal 10min;
4) get upper water and move into mutually new 1.5mL centrifuge tube, add isopyknic Virahol, mix rear standing 5min;
5) 4 ℃, 12000g, centrifugal 10min, supernatant discarded;
6) add 1mL70% washing with alcohol precipitation, 12000g, centrifugal 5min, abandons supernatant;
7) dry RNA precipitation, adds 40 μ L DEPC and processes water dissolution precipitation, with concentration and the quality of NanoDrop2000C spectrophotometric determination RNA ,-70 ℃ of preservations.
3, RT-PCR amplification:
1) reverse transcription (RT) reaction:
Get PCR pipe, add sample: total RNA3 μ L of extraction, random primer (random hexamers) 1 μ L, DEPC processes water 5 μ L, is placed in sex change 5min at 70 ℃, takes out immediately ice bath 1min.Add successively more following reagent:
42 ℃ of water-bath 1h, process 5min for 70 ℃, and-20 ℃ save backup.
2) polymerase chain reaction (PCR):
The first synthetic chain cDNA of the RT of take is template, carries out pcr amplification, and reaction system is as follows:
Reaction conditions is 94 ℃ of denaturation 5min; 94 ℃ sex change 45sec, 46-48 ℃ annealing 45sec, 72 ℃ of extension 1min, 35 circulations; Last 72 ℃ are extended 10min, 4 ℃ of preservations.
4, electrophoresis detection:
Get 6 μ L PCR products through 1% sepharose electrophoresis 30min (voltage 150V) in 0.5 * tbe buffer liquid, in the ethidium bromide (EB) of 0.5 μ g/mL, dyeing 10min observes in gel imaging system.If small brown rice planthopper is only carried RSV, can observe the nucleic acid band of a 1114bp, as only carried RBSDV, can observe the nucleic acid band of a 414bp, when small brown rice planthopper is carried RSV and RBSDV two-strain simultaneously, can observe two bands of 1114bp and 414bp size, if small brown rice planthopper is not carried two-strain, can not amplify band (Fig. 1).
Accompanying drawing explanation
Accompanying drawing 1 is that single head small brown rice planthopper is with electrophoresis detection result (the M:DNA standard molecular weight of malicious situation; 1: nontoxic small brown rice planthopper sample; 2: carry rice stripe virus small brown rice planthopper sample; 3: the small brown rice planthopper sample that simultaneously carries rice stripe virus and rice black-streaked dwarf virus; 4: carry rice black-streaked dwarf virus small brown rice planthopper water sample).
Accompanying drawing 2 is that band poison (RSV) small brown rice planthopper is raised part polypide detected result (the M:DNA standard molecular weight after malicious black streaked dwarf virus of rice plant; 1-24: small brown rice planthopper numbering).
Embodiment
In embodiment, method therefor is ordinary method if no special instructions below.
Example one: band poison (RSV) small brown rice planthopper is raised the band poison of polypide after malicious black streaked dwarf virus of rice plant and detects
1, pass virus mediator
Amboceptor small brown rice planthopper is this laboratory screening preservation, screening method (the Jiangsu agricultural journal such as builds with reference to bang, 2007,23 (5): method 492-494): by the female worm of small brown rice planthopper single head, raise respectively in vial (every bottle has 3-4 two leaf phase rice seedlings, and kind is Wu-Yu-Geng 3), and every small brown rice planthopper is carried out to mark, after laying eggs 3-4 days, take out, detect band poison (RSV) situation of maternal small brown rice planthopper, its offspring continues to raise standby.Choose hatching rate family higher and that maternal band is malicious and feed 3-4 after generation, its offspring's band poison situation is detected, determine the band poison rate of family, raise standby.
2, small brown rice planthopper raises poison
In field, gather the diseased plant that manifests typical water rice black streak dwarf symptom, through Molecular Detection, being accredited as RBSDV infects, diseased plant is implanted in 3L large beaker, be with poison (RSV) small brown rice planthopper nymph (being with malicious rate approximately 40%) to move into beaker age 400 1-2, nylon gauze sealing, moves into small brown rice planthopper in healthy water rice sprouts after the 48h that feeds, spend the phase of walking around to (three weeks), when small brown rice planthopper grows for adult, draw small brown rice planthopper, extract RNA.
3, design of primers:
1) NCBI (
http:// www.ncbi.nlm.nih.gov/sites/entrez? db=nucleotide) the rice stripe virus RNA4 nucleotide sequence (D10979 that reported of upper download, AF513505, EU931525, AJ871748, FJ602692, EU931521, EU931517, AF221834, AF221835, FJ602700) and rice black-streaked dwarf virus S2 nucleotide sequence (AJ409145, KC134290), use the Clustal V method of DNAstar-MegAlign software to carry out sequence alignment, find that RSV-RNA4 has a bit of sequence very similar to RBSDV-S2, the general primer that utilizes this section of similar district design RSV and RBSDV, primer sequence is as follows:
RSRB-R：5’-CCYATCACAAASAAATMAAAAT-3’
RS-F：5’-AGATCCAGAGAGAGTCACGGAAG-3’
RB-F：5’-GTTCAAAGACAATACACTCAAAA-3’
2) size of primer pairing and amplification object nucleic acid band is as follows:
Combination of primers detects Virus Name object clip size
RS-F/RSRB-R rice stripe virus 1114bp
RB-F/RSRB-R rice black-streaked dwarf virus 414bp
4, total RNA extracts:
1) get single head small brown rice planthopper and be placed in 1.5mL centrifuge tube, add 250 μ L Trizol reagent fully to grind, room temperature is placed 5min;
2) add 50 μ L chloroforms, mix rear standing 3min;
3) 4 ℃, 12000g, centrifugal 10min;
4) get upper water and move into mutually new 1.5mL centrifuge tube, add isopyknic Virahol, mix rear standing 5min;
5) 4 ℃, 12000g, centrifugal 10min, supernatant discarded;
6) add 1mL70% washing with alcohol precipitation, 12000g, centrifugal 5min, abandons supernatant;
7) dry RNA precipitation, adds 40 μ L DEPC and processes water dissolution precipitation, with concentration and the quality of NanoDrop2000C spectrophotometric determination RNA ,-70 ℃ of preservations.
5, RT-PCR amplification:
1) reverse transcription (RT) reaction:
Get PCR pipe, add sample: total RNA3 μ L of extraction, random primer (random hexamers) 1 μ L, DEPC processes water 5 μ L, is placed in sex change 5min at 70 ℃, takes out immediately ice bath 1min.Add successively more following reagent:
42 ℃ of water-bath 1h, process 5min for 70 ℃, and-20 ℃ save backup.
2) polymerase chain reaction (PCR):
The first synthetic chain cDNA of the RT of take is template, carries out pcr amplification, and reaction system is as follows:
Reaction conditions is 94 ℃ of denaturation 5min; 94 ℃ sex change 45sec, 46-48 ℃ annealing 45sec, 72 ℃ of extension 1min, 35 circulations; Last 72 ℃ are extended 10min, 4 ℃ of preservations.
6, electrophoresis detection:
Get 6 μ L PCR products through 1% sepharose electrophoresis 30min (voltage 150V) in 0.5 * tbe buffer liquid, in the ethidium bromide (EB) of 0.5 μ g/mL, dyeing 10min observes in gel imaging system.If small brown rice planthopper is only carried RSV, can observe the nucleic acid band of a 1114bp, as only carried RBSDV, can observe the nucleic acid band of a 414bp, when small brown rice planthopper is carried RSV and RBSDV two-strain simultaneously, two bands can observing 1114bp and 414bp size, if small brown rice planthopper is not carried two-strain, can not amplify band.Fig. 2 is that part small brown rice planthopper is with the electrophoresis detection result figure of malicious situation.
7, result statistics
Raise after poison, the small brown rice planthopper of survival carries out RT-PCR detection by present method, amounts to 255, by statistics, carries 93 of the small brown rice planthoppers of RSV, carries 20 of the small brown rice planthoppers of RBSDV, carries 14 of the small brown rice planthoppers of RSV and RBSDV, 128 of nontoxic small brown rice planthoppers simultaneously.This experiment has also proved that small brown rice planthopper can carry RSV and RBSDV.
Example two: field small brown rice planthopper population is with the synchronous detection of malicious situation
Spring in 2013 gathers overwinter generation small brown rice planthopper at Gaochun County, Nanjing polylith wheatland with chessboard method, amount to 2000, after stored frozen, choose at random 150, according to the method for example one, extract RNA, with 3 primers, carry out RT-PCR detection, found that 3 small brown rice planthoppers carry RSV (detecting the band of 1114bp size), 2 small brown rice planthoppers are carried RBSDV (detecting the band of 414bp size), do not have small brown rice planthopper to carry two-strain simultaneously, it is RSV:2% that area, calculating Gaochun overwinter generation small brown rice planthopper in 2013 is with malicious rate, RBSDV:1.3%, for forecast and the prevention and control of virus disease provide data supporting.
Example three: the detection of the sick sample of paddy rice
Take and infect respectively the paddy rice sample of RSV and RBSDV and infect RSV and the biased sample of RBSDV is detected object, with the negative contrast of healthy paddy rice sample.According to the method for example one, extract RNA, with 3 primers, carry out RT-PCR detection, the sample of result infection RSV can detect the band of 1114bp size, the sample that infects RBSDV can detect the band of 414bp size, the biased sample that infects two-strain can detect the band of 1114bp and 414bp size simultaneously, healthy paddy rice sample does not have band to detect, and this illustrates that this detection method is equally applicable to the detection to paddy rice sample.Certainly, owing to infecting the paddy rice sample of RSV and RBSDV, in field symptom, very easily distinguish, therefore the detection of the sick sample of paddy rice be can be used for to the susceptible initial stage detection before of the aobvious disease of paddy rice, after aobvious disease, be of little use.
Claims (2)
1. the method for rice stripe virus and rice black-streaked dwarf virus in a rapid detection small brown rice planthopper body, it is characterized in that: utilize one section of similar sequences appropriate design primer between RSV-RNA4 fragment and RBSDV-S2 fragment, RSRB-R:5 '-CCYATCACAAASAAATMAAAAT-3 ', RS-F:5 '-AGATCCAGAGAGAGTCACGGAAG-3 ', RB-F:5 '-GTTCAAAGACAATACACTCAAAA-3 ', wherein RS-F/RSRB-R pairing detects rice stripe virus (1114bp), RB-F/RSRB-R pairing detects rice black-streaked dwarf virus (414bp), to realize the Fast synchronization of rice stripe virus and rice black-streaked dwarf virus in small brown rice planthopper body, detect.
2. the method for rice stripe virus and rice black-streaked dwarf virus in Rapid ash plant hopper body according to claim 1, it is characterized in that using random primer (random hexamers) in reverse transcription (RT) reaction, the annealing temperature in pcr amplification is 46-48 ℃.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104830997A (en) * | 2015-05-20 | 2015-08-12 | 安徽袁粮水稻产业有限公司 | Molecular identification method of rice black-streaked dwarf virus resistance |
| CN106906218A (en) * | 2017-04-25 | 2017-06-30 | 中国科学院动物研究所 | A kind of method for controlling rice stripe virus to propagate |
| CN113684314A (en) * | 2021-08-27 | 2021-11-23 | 中国农业科学院作物科学研究所 | Kit and method for detecting RBSDV (radial basis sequence-associated protein sequence) based on CRISPR-Cas (clustered regularly interspaced short palindromic repeats) system |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830997A (en) * | 2015-05-20 | 2015-08-12 | 安徽袁粮水稻产业有限公司 | Molecular identification method of rice black-streaked dwarf virus resistance |
| CN106906218A (en) * | 2017-04-25 | 2017-06-30 | 中国科学院动物研究所 | A kind of method for controlling rice stripe virus to propagate |
| CN106906218B (en) * | 2017-04-25 | 2019-08-20 | 中国科学院动物研究所 | A method of control rice stripe virus is propagated |
| CN113684314A (en) * | 2021-08-27 | 2021-11-23 | 中国农业科学院作物科学研究所 | Kit and method for detecting RBSDV (radial basis sequence-associated protein sequence) based on CRISPR-Cas (clustered regularly interspaced short palindromic repeats) system |
| CN113684314B (en) * | 2021-08-27 | 2022-10-28 | 中国农业科学院作物科学研究所 | Kit and method for detecting RBSDV (radial basis sequence-associated protein sequence) based on CRISPR-Cas (clustered regularly interspaced short palindromic repeats) system |
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