CN104141015B - A kind of method of rice stripe virus and rice black-streaked dwarf virus in rapid detection small brown rice planthopper body - Google Patents
A kind of method of rice stripe virus and rice black-streaked dwarf virus in rapid detection small brown rice planthopper body Download PDFInfo
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Abstract
The invention provides a kind of method that Fast synchronization detects rice stripe virus (RSV) and rice black-streaked dwarf virus (RBSDV) in insect amboceptor small brown rice planthopper body, platymiscium protection field.Three primers are designed: [A] RS-F, [B] RB-F, [C] RSRB-R, wherein A/C combine detection rice stripe virus, B/C combine detection rice black-streaked dwarf virus according to rice stripe virus RNA4 and rice black-streaked dwarf virus S2 nucleotide sequence.After single head small brown rice planthopper extracts total serum IgE, only need the RT-PCR of a reaction, can detect rice stripe virus and rice black-streaked dwarf virus two-strain, the Fast synchronization achieving two-strain in small brown rice planthopper body detects.Relative to the method for a traditional a kind of virus of RT-PCR reaction detection, greatly having saved cost, saved the time, is a kind of quick, easy, sensitive, special detection method.Present method can be applicable to the monitoring of field small brown rice planthopper colony with malicious rate, also can be applicable to the correlative study work of virus and amboceptor Coupling effects.
Description
Technical field
The present invention relates to a kind of method that rice stripe virus in amboceptor insect small brown rice planthopper body and rice black-streaked dwarf virus Fast synchronization are detected, platymiscium protection field.
Background technology
Rice stripe virus (Ricestripevirus, RSV) be the prototypical member of very thin Tobamovirus (Tenuivirus), its stripe disease caused is one of important disease of paddy rice, between many decades in the past, this disease based on the vast rice district outbreak of epidemic of East China Dao Qu, forms great threat to Rice Production in China.Such as, 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 blocks, within 2005, reach 2,800 ten thousand mu, and start to Zhejiang, Anhui, Henan, Shandong, the periphery provinces and cities such as Shanghai spread, be once called as " cancer " on paddy rice because of its serious hazardness, causing very large social repercussion.
Rice black-streaked dwarf virus (Riceblack-streakeddwarfvirus, RBSDV), be a kind ofly on paddy rice 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, there is wax drop-wise projection in the later stage, then form secret note on blade, leaf sheath and cane, 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.Within 1963 and 1966, black streaked dwarf virus of rice is with popular in Jiangsu Province, China, Shanghai, Zhejiang one, and the nineties, in the many corns of China and paddy rice producing region outbreak of epidemic, causes heavy losses.This disease spreads generation in area, Jiangsu and Zhejiang Provinces in recent years, and in 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.
RSV and RBSDV propagates primarily of their common amboceptor insect small brown rice planthopper LaodelphaxstriatellusFall é n, and difference is that RSV can by small brown rice planthopper transovarian transmission, and RBSDV not transovarian transmission.The classical symptom of stripe disease is the striped spot or the patch that occur chlorisis, and normally, and the symptom of black streak dwarf is plant to plant height stunts, and leaf dark green, later stage visible white wax drop-wise projection, then forms secret note.Two kinds of diseases very easily distinguish diagnosis in field, but the band of amboceptor small brown rice planthopper poison situation then cannot directly be differentiated, could need realize by biology techniques.The prediction of field small brown rice planthopper colony with malicious rate has vital role with continuing to monitor in the innoxious Synthetical prevention of Rice Virus disease, can play positive directive function to the employing of follow-up Control Technology.
At present, the authentication method of the plant virus in insect amboceptor mainly contains: biological inoculation experiment, Serologic detection, electron microscopic observation and molecular Biological Detection.Biological inoculation experiment collects small brown rice planthopper thorn to inhale susceptible rice varieties, and then observe paddy rice symptom, not only workload is large, and length consuming time, and the aobvious disease time of such as black streak dwarf often needs 1 month.The dependency of Serologic detection antagonist is high, needs antibody with high specificity, and the monoclonal antibody of current RSV detects comparative maturity, and RBSDV antiserum(antisera) is also immature efficiently.Electron microscopic observation needs high level plant and instrument, and is not suitable for a large amount of sample detection, is generally applied to the research work of pathogenic characteristic.The molecular biological assay still based on RT-PCR technology that current application is more, also there is report to use the method for RT-PCR to detect RSV and RBSDV before, but the not yet technological method of report energy synchronous detection RSV and RBSDV, the virus that present method only needs RT-PCR can realize two kinds is differentiated, under the prerequisite ensureing reliability and sensitivity, simplify operation, provide cost savings and the time.
Summary of the invention
The present invention has filled up the blank in amboceptor small brown rice planthopper body on rice stripe virus and rice black-streaked dwarf virus Fast synchronization authentication technique, provide a kind of quick, easy, sensitive, special discrimination method, can be applicable to the monitoring of field small brown rice planthopper colony with malicious rate and the research work of virus and amboceptor Coupling effects.
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, and rice black-streaked dwarf virus S2 nucleotide sequence (AJ409145 FJ602700), KC134290), the ClustalV method of DNAstar-MegAlign software is used to carry out sequence alignment, find that RSV-RNA4 and RBSDV-S2 has a bit of sequence very similar, utilize the general primer of this section of similar district's 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 bands 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 RNAs extraction:
1) get single head small brown rice planthopper and be placed in 1.5mL centrifuge tube, add 250 μ LTrizolreagent and fully grind, room temperature places 5min;
2) add 50 μ L chloroforms, after mixing, leave standstill 3min;
3) 4 DEG C, 12000g, centrifugal 10min;
4) get upper water and move into new 1.5mL centrifuge tube mutually, add isopyknic Virahol, after mixing, leave standstill 5min;
5) 4 DEG C, 12000g, centrifugal 10min, supernatant discarded;
6) add 1mL70% washing with alcohol precipitation, 12000g, centrifugal 5min, abandons supernatant;
7) dry RNA precipitation, adds 40 μ LDEPC process water dissolution precipitations, by concentration and the quality of NanoDrop2000C spectrophotometric determination RNA, and-70 DEG C of preservations.
3, RT-PCR amplification:
1) reverse transcription (RT) is reacted:
Get PCR pipe, add sample: the total serum IgE 3 μ L of extraction, random primer (randomhexamers) 1 μ L, DEPC process water 5 μ L, sex change 5min at being placed in 70 DEG C, take out ice bath 1min immediately.Add following reagent successively again:
42 DEG C of water-bath 1h, 70 DEG C of process 5min ,-20 DEG C save backup.
2) polymerase chain reaction (PCR):
With the first chain cDNA of RT synthesis for template, carry out pcr amplification, reaction system is as follows:
Reaction conditions is 94 DEG C of denaturation 5min; 94 DEG C sex change 45sec, 46-48 DEG C annealing 45sec, 72 DEG C of extension 1min, 35 circulations; Last 72 DEG C extend 10min, 4 DEG C of preservations.
4, electrophoresis detection:
Get 6 μ LPCR 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 only carries RSV, then can observe the nucleic acid bands of a 1114bp, as only carried RBSDV, then can observe the nucleic acid bands of a 414bp, when small brown rice planthopper carries RSV and RBSDV two-strain simultaneously, two band of 1114bp and 414bp size can be observed, if small brown rice planthopper does not carry two-strain, then can not amplify band (Fig. 1).
Accompanying drawing explanation
Accompanying drawing 1 is electrophoresis detection result (the M:DNA standard molecular weight of single head small brown rice planthopper with 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 simultaneously carrying 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 part polypide detected result (the M:DNA standard molecular weights after band poison (RSV) small brown rice planthopper raises malicious black streaked dwarf virus of rice plant; 1-24: small brown rice planthopper is numbered).
Embodiment
In embodiment, method therefor is ordinary method if no special instructions below.
Example one: after band poison (RSV) small brown rice planthopper raises malicious black streaked dwarf virus of rice plant, the band poison of polypide detects
1, virus mediator is passed
Amboceptor small brown rice planthopper is this laboratory screening preservation, screening method (the Jiangsu's agriculture journal such as to build with reference to bang, 2007,23 (5): 492-494) method: by female for small brown rice planthopper single head worm, raises in vial (every bottle has 3-4 two leaf phase rice seedlings, and kind is Wu-Yu-Geng 3) respectively, and mark is carried out to every small brown rice planthopper, take out after laying eggs 3-4 days, detect band poison (RSV) situation of maternal small brown rice planthopper, it is for subsequent use that its offspring continues raising.Choose that hatching rate is higher after 3-4 generation and family that is maternal band poison is fed, to be detected the band of its offspring poison situation, determine the band poison rate of family, raise for subsequent use.
2, small brown rice planthopper raise poison
The diseased plant manifesting typical water rice black streak dwarf symptom is gathered in field, be accredited as RBSDV through Molecular Detection to infect, diseased plant is implanted in 3L large beaker, be with poison (RSV) small brown rice planthopper nymph (being with malicious rate about 40%) to move into beaker age 400 1-2, nylon gauze seals, and is moved into by 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, and rice black-streaked dwarf virus S2 nucleotide sequence (AJ409145 FJ602700), KC134290), the ClustalV method of DNAstar-MegAlign software is used to carry out sequence alignment, find that RSV-RNA4 and RBSDV-S2 has a bit of sequence very similar, utilize the general primer of this section of similar district's 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 bands 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 RNAs extraction:
1) get single head small brown rice planthopper and be placed in 1.5mL centrifuge tube, add 250 μ LTrizolreagent and fully grind, room temperature places 5min;
2) add 50 μ L chloroforms, after mixing, leave standstill 3min;
3) 4 DEG C, 12000g, centrifugal 10min;
4) get upper water and move into new 1.5mL centrifuge tube mutually, add isopyknic Virahol, after mixing, leave standstill 5min;
5) 4 DEG C, 12000g, centrifugal 10min, supernatant discarded;
6) add 1mL70% washing with alcohol precipitation, 12000g, centrifugal 5min, abandons supernatant;
7) dry RNA precipitation, adds 40 μ LDEPC process water dissolution precipitations, by concentration and the quality of NanoDrop2000C spectrophotometric determination RNA, and-70 DEG C of preservations.
5, RT-PCR amplification:
1) reverse transcription (RT) is reacted:
Get PCR pipe, add sample: the total serum IgE 3 μ L of extraction, random primer (randomhexamers) 1 μ L, DEPC process water 5 μ L, sex change 5min at being placed in 70 DEG C, take out ice bath 1min immediately.Add following reagent successively again:
42 DEG C of water-bath 1h, 70 DEG C of process 5min ,-20 DEG C save backup.
2) polymerase chain reaction (PCR):
With the first chain cDNA of RT synthesis for template, carry out pcr amplification, reaction system is as follows:
Reaction conditions is 94 DEG C of denaturation 5min; 94 DEG C sex change 45sec, 46-48 DEG C annealing 45sec, 72 DEG C of extension 1min, 35 circulations; Last 72 DEG C extend 10min, 4 DEG C of preservations.
6, electrophoresis detection:
Get 6 μ LPCR 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 only carries RSV, then can observe the nucleic acid bands of a 1114bp, as only carried RBSDV, then can observe the nucleic acid bands of a 414bp, when small brown rice planthopper carries RSV and RBSDV two-strain simultaneously, two band of 1114bp and 414bp size can be observed, if small brown rice planthopper does not carry two-strain, then can not amplify band.Fig. 2 is the electrophoresis detection result figure of part small brown rice planthopper with malicious situation.
7, result statistics
After raising poison, the small brown rice planthopper of survival carries out RT-PCR detection by present method, amounts to 255, through statistics, carries the small brown rice planthopper 93 of RSV, carries the small brown rice planthopper 20 of RBSDV, carry the small brown rice planthopper 14 of RSV and RBSDV, nontoxic small brown rice planthopper 128 simultaneously.This experiment also demonstrates small brown rice planthopper can carry RSV and RBSDV.
Example two: the synchronous detection of field small brown rice planthopper population with malicious situation
Spring in 2013 gathers overwinter generation small brown rice planthopper at Gaochun County, Nanjing polylith wheatland with chessboard method, amount to 2000, random selecting 150 after stored frozen, RNA is extracted according to the method for example one, RT-PCR detection is carried out with 3 primers, found that 3 small brown rice planthoppers carry RSV (detecting the band of 1114bp size), 2 small brown rice planthoppers carry RBSDV (detecting the band of 414bp size), do not have small brown rice planthopper to carry two-strain simultaneously, calculating area, Gaochun overwinter generation small brown rice planthopper in 2013 is with malicious rate to be RSV:2%, RBSDV:1.3%, for the forecast of virus disease and prevention and control provide data supporting.
Example three: the detection of the sick sample of paddy rice
With the biased sample of the paddy rice sample and infection RSV and RBSDV that infect RSV and RBSDV respectively for detected object, with healthy paddy rice sample for negative control.RNA is extracted according to the method for example one, RT-PCR detection is carried out with 3 primers, the sample of result infection RSV can detect the band of 1114bp size, the sample infecting RBSDV can detect the band of 414bp size, the biased sample infecting 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, because the paddy rice sample infecting RSV and RBSDV is very easily distinguished in field symptom, therefore the susceptible initial stage that the detection of the sick sample of paddy rice can be used for before paddy rice shows disease is detected, be then of little use after aobvious disease.
Claims (1)
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, RB-F/RSRB-R pairing detects rice black-streaked dwarf virus, detect with the Fast synchronization realizing rice stripe virus and rice black-streaked dwarf virus in small brown rice planthopper body.
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| 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 |
| 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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| 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 |
| CN102559937B (en) * | 2012-02-29 | 2014-04-16 | 浙江省农业科学院 | Method for synchronously detecting three types of paddy viruses |
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