CN102559937A - Method for simply, quickly and synchronously detecting three types of paddy viruses - Google Patents
Method for simply, quickly and synchronously detecting three types of paddy viruses Download PDFInfo
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Abstract
The invention relates to a method for simply, quickly and synchronously detecting three types of paddy viruses, and the method comprises the following step of: carrying out one-step RT-PCR (reverse transcription-polymerase chain reaction) amplification by use of a set of virus specific primers and by taking the gross RNA (ribonucleic acid) of a sample to be detected as a template, wherein the number of the primers is six, the primers are respectively DRS-1, DRS-2, DRB-1, DRB-2, DSR-1 and DSR-2, the gene sequences of the primers are shown in a sequence table, and the infection status of the rice black streaked dwarf virus (RBSDV), the rice stripe virus (RSV) and the southern rice black-streaked dwarf virus (SRBSDV) is judged by a gel electrophoresis strip. The method has the beneficial effects that aiming at the production practicality, the method for quickly, exactly, sensitively and synchronously detecting the three types of important paddy viruses is built, the method is successfully applicable to the detection and diagnosis of single-head plant hopper and field samples, and a technical support is provided for the exact diagnosis, forecast and scientific prevention and control of the RBSDV, the RSV and the SRBSDV.
Description
Technical field
The present invention relates to the plant protection technology field, the method for three kinds of paddy rice viruses of especially a kind of simple synchronous detection fast.
Background technology
In recent years; Rice stripe virus (the rice stripe virus that the beginning of the sixties in last century, one band was found in the Jiangsu and Zhejiang Provinces; RSV) and rice black-streaked dwarf virus (rice black-streaked dwarf virus; RBSDV) popular in a plurality of paddy fields of China again, only in the period of the 2004-2005 rice stripe virus at the onset area in Jiangsu Province just more than 2,000 ten thousand mu.Southern rice black-streaked dwarf virus (Southern rice black-streaked dwarf virus; SRBSDV), have another name called rice black-streaked dwarf virus No. 2 (Rice black-streaked dwarf virus 2, RBSDV-2); Be recently newfound a kind of important paddy rice virus; From calendar year 2001 since Guangdong Yangjiang City report, 14 provinces, cities and autonomous regions' generations in North Vietnam and China south China, Central China, East China etc. in 2010, onset area reaches more than 2,000 ten thousand mu.More than 3 kinds of important paddy rice viruses propagate with the persistence mode by insect, and all can infect paddy rice in the field, some common gramineous crop and weeds such as corn, jowar, barnyard grass grass.Wherein, Rice stripe virus, the rice black-streaked dwarf virus propagated by small brown rice planthopper belong to very thin Tobamovirus (genus Tenuivirus) and Reoviridae (Reoviridae) Fijivirus genus (genus Fijivirus), and the southern rice black-streaked dwarf virus of being propagated by white backed planthopper is that the Reoviridae Fijivirus belongs to second group of tentative newcomer.From epidemiologies such as host range, epidemic characteristic, regional distributions; All once big area was popular and caused the heavy losses of crop yields such as paddy rice in China for these 3 kinds of paddy rice virus diseases; Though disease index is different but the region all takes place throughout the year and takes place exist certain overlapping in recent years; Especially in China East China, this has brought certain difficulty for diagnosis, prediction and the prevention and control of such disease, and also the detection to disease has proposed higher technical requirements.Serological method is one of quick cheap detecting method of using always, but owing to have the intensive serological cross reaction between RBSDV and the SRBSDV, prepared polyclonal antibody still can not be used for distinguishing these 2 kinds of viruses at present.The foundation of the molecular detecting method that has promoted that these are viral is measured and logined and disclose to a large amount of RSV, RBSDV, SRBSDV genom sequence in succession; The Zhou Guohui of south China agricultural university etc.] and the Zhou Qian of Hunan agricultural university etc. has developed nido respectively according to the S10 sequence and an one step RT-PCR method is used for detecting specifically SRBSDV, the season quintessences of academy of agricultural sciences, Jiangsu etc. have been developed the method that detects RBSDV and SRBSDV according to the more S9 sequence of sequence difference.Japan scholar Le et al. adopts the isothermal amplification technique of ring mediation to set up the detection method of 9 kinds of paddy rice viruses, but these methods all can not be used for detecting simultaneously these 3 kinds of viruses.
Summary of the invention
The present invention will solve the shortcoming of above-mentioned prior art, provide a kind of fast, accurately, the methods of sensitivity and these 3 kinds important paddy rice viruses of synchronous detection.
The present invention solves the technical scheme that its technical problem adopts: the method for three kinds of paddy rice viruses of this simple synchronous detection fast; Using a cover virus-specific primer is that template is carried out one step RT-PCR amplification with the total RNA of testing sample; This primer has 6, and their gene order is represented as follows:
DRS-1，5’-CACTCTAGCTGATTTGCAGAAGGCA-3’；
DRS-2，5’-GGTCTTCACTTTCCCATTGGTGATG-3；
DRB-1，5’-ACTAAGCTTATTTGCTACCTCCAAAC-3’；
DRB-2，5’-ATTAGTRCGCAAMGTGGACAAACTG-3’(R＝A，G；M＝A，C)；
DSR-1，5’-CGCTTTAGATGCTGACAAATCACTTTTA-3’；
DSR-2，5’-CTCCTTTTCTAAGTGCAGACAGTCC-3’。
Through the infect situation of gel electrophoresis strip with judgement rice black-streaked dwarf virus (RBSDV), rice stripe virus (RSV) and southern rice black-streaked dwarf virus (SRBSDV), it specifically may further comprise the steps at last:
1) design of primers: the GenBank DB is announced and the gene of rice black-streaked dwarf virus, rice stripe virus and the southern rice black-streaked dwarf virus coat protein of this experimental determination through analyzing; Obtain 3 kinds of viruses and plant the zone that is polymorphum between interior conservative species; And Using P rimer6.0 programdesign is to 6 Auele Specific Primers of 3 kinds of paddy rice virus, and these primers are given birth to worker's biotechnology Services Co., Ltd in Shanghai synthetic;
2) the total RNA of sample extracts: for plant samples such as paddy rice, get 0.1g diseased plant or healthy tree blade and in mortar, add the liquid nitrogen shape of pulverizing, change the 1.5mL centrifuge tube over to after, add 1mL TRIzol reagent (Invitrogen) again; For the plant hopper sample, get the single head polypide, place the 1.5mL centrifuge tube, add 500 μ L TRIzol reagent (Invitrogen), with glass rod polypide is fully ground again through pyroprocessing; After this operation is all extracted total RNA by TRIzol reagent working instructions: behind the vibration mixing, add chloroform 0.2ml, leave standstill 3～5min behind the mixing; The centrifugal 10min of 12000g gets supernatant to new centrifuge tube and add isopyknic Virahol, 12000g behind the mixing, and centrifugal 10min under 4 ℃ of conditions abandons supernatant, and deposition is washed 2 times with 70% ethanol, after the drying deposition is dissolved in the ddH of no RNase
2Among the O, total RNA of extraction is stored in-80 ℃ of refrigerators subsequent use;
3) amplified reaction: adopting One Step RT-PCRKit (precious biotechnology (Dalian) ltd) is that template increases with the total RNA extract of sample, and reaction TV 50 μ L include the total RNA extract 5 μ L of sample; Other each component reference reagent box explanations; Comprise: 2 * reaction buffer, 25 μ L, 6 each 1 μ L of primer, enzyme mixture 2 μ L; The ddH2O that adds no RNase again is to TV 50 μ L, and response procedures is 50 ℃ of reverse transcription 30min; 94 ℃ of preparatory sex change 2min; 94 ℃ of sex change 30s, 63 ℃ of annealing 30s, 72 ℃ are extended 30～60s, 30 circulations; 72 ℃ are extended 10min;
4) electrophoretic analysis: get amplified production 5 μ L; On the sepharose of point sample to 1.2% appearance hole in; With the DL2000DNA standard molecular weight as reference; Under 120 volts the voltage in 1 * TAE electrophoretic buffer electrophoresis 30～60min, EB dyeing back is observed under uv lamp, record is also analyzed and the consistence of expecting that segment is big or small.
The synthetic back of primer confirms that through grads PCR the righttest annealing temperature of primer is 63 ℃, and is as shown in Figure 1; DRS-1 and the DRS-2 nucleocapsid protein gene of RSV that increases specifically wherein, the amplified production size is 757bp; Increase the specifically main outer capsid protein gene of RBSDV of DRB-1 and DRB-2, the amplified production size is 592bp; Increase the specifically main outer capsid protein gene of SRBSDV of DSR-1 and DSR-2, the amplified production size is 142bp.
Inventing useful effect is: the present invention is directed to and produce reality; Development set up a kind of fast, accurately, the methods of sensitivity and these 3 kinds important paddy rice viruses of synchronous detection; And be successfully applied to the detection and the diagnosis of single head plant hopper and field sample, for accurate diagnosis, prediction and the science prevention and control of black streaked dwarf virus of rice, stripe disease, southern black streak dwarf provide technical support.
Description of drawings
Fig. 1 is presented at the amplification efficiency of RT-PCR under gradient annealing temperature (Tm) condition.Wherein M representes DNA standard molecular weight DL2000; Swimming lane 1-12 is respectively the amplified production under ℃ condition of Tm=59.8～72.0;
Fig. 2 is that this detection method specificity is analyzed with sensitive poison.Wherein M representes DNA standard molecular weight DL2000; Swimming lane 1～3 is the primer DRB-1/DRB-2 RBSDV that increases successively, SRBSDV and RSV standard model; Swimming lane 4～6 is the primer DRZ-1/DRZ-2 RBSDV that increases successively, SRBSDV and RSV standard model; Swimming lane 7～9 is the primer DRS-1/DRS-2 RBSDV that increases successively, SRBSDV and RSV standard model; Swimming lane 10～12 is three pairs of mix primer RBSDV that increase successively, SRBSDV and RSV standard model; Swimming lane 13-16 is three couples of mix primer amplification RBSDV, SRBSDV, RSV biased sample; Swimming lane 17～28 is RBSDV, SRBSDV and the RSV biased samples of three pairs of mix primer amplifications in 1: 10 ratio gradient dilution;
Fig. 3 is the application of present method in the sample detection of different sources.Wherein M is DNA standard molecular weight DL2000; Swimming lane 1 is healthy paddy rice sample; Swimming lane 2-8 is a paddy rice diseased plant sample; Swimming lane 9-10 is a corn diseased plant sample; Swimming lane 11 is healthy nontoxic small brown rice planthopper sample; Swimming lane 12-14 is the small brown rice planthopper sample; Swimming lane 15-16 is the white backed planthopper sample; Swimming lane 17 is healthy nontoxic white backed planthopper sample.
Embodiment
Below in conjunction with accompanying drawing the present invention is described further:
Embodiment 1: the detection of plant sample.
Plant sample is chosen: the paddy rice of the doubtful virus infection in field, corn diseased plant sample are mainly gathered from provinces and cities and North Vietnam provinces and cities such as China Zhejiang, Shandong, Hubei, Hunan, Guangdong, Shanghai in the period of 2008-2011, and are stored in-80 ℃ of refrigerators.
Design of primers: the GenBank DB is announced and the gene of rice black-streaked dwarf virus, rice stripe virus and the southern rice black-streaked dwarf virus coat protein of this experimental determination through analyzing; Obtain 3 kinds of viruses and plant the zone that is polymorphum between interior conservative species; And Using P rimer6.0 programdesign is to 6 Auele Specific Primers of 3 kinds of paddy rice virus, and these primers are given birth to worker's biotechnology Services Co., Ltd in Shanghai synthetic.
The total RNA of vegetable material extracts: for plant samples such as paddy rice, corns, get the 0.1g blade and in mortar, add the liquid nitrogen shape of pulverizing, change the 1.5mL centrifuge tube over to after; Add 1mL TRIzol reagent (Invitrogen) again; Behind the vibration mixing, add chloroform 0.2ml, leave standstill 3-5min behind the mixing; The centrifugal 10min of 12000g gets supernatant to new centrifuge tube and add isopyknic Virahol, 12000g behind the mixing, and centrifugal 10min under 4 ℃ of conditions abandons supernatant, and deposition is washed 2 times with 70% ethanol, after the drying deposition is dissolved in the ddH of no RNase
2Among the O, total RNA of extraction is stored in-80 ℃ of refrigerators subsequent use.
Amplified reaction: reaction TV 50 μ L include the total RNA extract 5 μ L of sample, 2 * reaction buffer, 25 μ L, 6 each 1 μ L of primer, enzyme mixture 2 μ L, the ddH of the no RNase of interpolation again
2O is to TV 50 μ L.Response procedures is 50 ℃ of reverse transcription 30min; 94 ℃ of preparatory sex change 2min; 94 ℃ of sex change 30s, 63 ℃ of annealing 30s, 72 ℃ are extended 30～60s, 30 circulations; 72 ℃ are extended 10min.
Electrophoresis is identified amplified production: get amplified production 5 μ L; On the sepharose of point sample to 1.2% appearance hole in; With the DL2000DNA standard molecular weight as reference, under 120 volts the voltage in 1 * TAE electrophoretic buffer electrophoresis 30～60min, EB dyeing back is observation under uv lamp; Record is also analyzed and the big or small consistence of expection segment, and part electrophoresis evaluation picture is seen Fig. 3 swimming lane 1-10.
Interpretation of result: which kind of virus infection can judge according to electrophoresis result whether plant sample exists infecting of above-mentioned 3 kinds of viruses, if infected, be.For example: only amplify the band of 592bp, show that this plant sample exists RBSDV to infect (seeing Fig. 3, swimming lane 2,8-10); Only amplify the sample of 142bp band, show that this plant sample exists SRBSDV to infect (seeing Fig. 3, swimming lane 3); Only amplify the sample of 757bp band, show that this plant sample exists RSV to infect (seeing Fig. 3, swimming lane 4).And the like, amplify the sample of 592bp and 142bp band, showing that there is RBSDV in this plant sample and SRBSDV is compound infects (seeing Fig. 3, swimming lane 5); Amplify the sample of 757bp and 142bp band, showing that there is RSV in this plant sample and SRBSDV is compound infects (seeing Fig. 3, swimming lane 6-7).
Embodiment 2: the detection of single head plant hopper sample.
The plant hopper sample: provinces and cities such as small brown rice planthopper and white backed planthopper sample are mainly gathered from Zhejiang in the period of 2008-2011, Hainan, Hunan, Shanghai, Shandong, the part plant hopper is raised on the paddy rice seedling, and remainder is stored in 70% ethanol subsequent use.
Design of primers: the GenBank DB is announced and the gene of rice black-streaked dwarf virus, rice stripe virus and the southern rice black-streaked dwarf virus coat protein of this experimental determination through analyzing; Obtain 3 kinds of viruses and plant the zone that is polymorphum between interior conservative species; And Using P rimer6.0 programdesign is to 6 Auele Specific Primers of 3 kinds of paddy rice virus, and these primers are given birth to worker's biotechnology Services Co., Ltd in Shanghai synthetic.
The total RNA of plant hopper extracts: get the single head polypide, place the 1.5mL centrifuge tube, add 500 μ L TRIzol reagent (Invitrogen), with the glass rod through pyroprocessing polypide is fully ground; Behind the vibration mixing, add chloroform 0.1ml, leave standstill 3-5min behind the mixing; The centrifugal 10min of 12000g gets the extremely new centrifuge tube of supernatant and adds isopyknic Virahol, 12000g behind the mixing; Centrifugal 10min under 4 ℃ of conditions abandons supernatant, and deposition is washed 2 times with 70% ethanol; After the drying deposition is dissolved among the ddH2O of no RNase, total RNA of extraction is stored in-80 ℃ of refrigerators subsequent use.
Amplified reaction: reaction TV 50 μ L, include the total RNA extract 5 μ L of sample, 2 * reaction buffer, 25 μ L, 6 each 1 μ L of primer, enzyme mixture 2 μ L, the ddH2O that adds no RNase again is to TV 50 μ L.Response procedures is 50 ℃ of reverse transcription 30min; 94 ℃ of preparatory sex change 2min; 94 ℃ of sex change 30s, 63 ℃ of annealing 30s, 72 ℃ are extended 30～60s, 30 circulations; 72 ℃ are extended 10min.
Electrophoresis is identified amplified production: get amplified production 5 μ L; On the sepharose of point sample to 1.2% appearance hole in; With the DL2000DNA standard molecular weight as reference; Under 120 volts the voltage in 1 * TAE electrophoretic buffer electrophoresis 30～60min, EB dyeing back is observed under uv lamp, record is also analyzed and the consistence of expecting that segment is big or small.Part electrophoresis evaluation picture is seen Fig. 3 swimming lane 11-17.
Interpretation of result: but according to whether carrying above-mentioned 3 kinds of viruses in the electrophoresis result single head plant hopper matter sample,, carry which kind of virus infection again if carry.For example: only amplify the small brown rice planthopper sample of 757bp band, show that this small brown rice planthopper carries RSV (seeing Fig. 3, swimming lane 12); Only amplify the small brown rice planthopper and the white backed planthopper sample of 142bp band, show that this small brown rice planthopper and white backed planthopper all carry SRBSDV (seeing Fig. 3, swimming lane 14,16); Only amplify the white backed planthopper sample of 592bp band, show that this white backed planthopper sample carries RBSDV (seeing Fig. 3, swimming lane 15).And the like, amplify the small brown rice planthopper sample of 592bp and 757bp band, show that this small brown rice planthopper carried RBSDV and 2 kinds of viruses of RSV (seeing Fig. 3, swimming lane 13) simultaneously.
Detection specificity and sensitivity analysis: (result sees Fig. 2 to utilize above-mentioned 3 pairs of primers respectively 3 kinds of viral standard models to be carried out single stage method RT-PCR amplification earlier; Swimming lane 1-9), primer is to all only can in corresponding viral standard model, the increase band of expection size of DRB-1/DRB-2, DRZ-1/DRZ-2, DRS-1/DRS-2.(result sees Fig. 2 then to utilize 3 pairs of primers of equal proportion blended also to carry out pcr amplification with RSV, RBSDV, SRBSDV standard model and composition thereof as template respectively; Swimming lane 10-16), the big or small segment of expection of utilizing 3 pairs of mix primer can increase special equally at RSV, RBSDV, SRBSDV sample; In biased sample, no matter be 2 kinds of sample mix or 3 kinds of sample mix, all only can detect the corresponding segment of expection size.And utilize these primers at other plant virus all the same specific band that detects less than any expection size in RGDV, RRSV, TMV, PVY sample with healthy paddy rice sample.In order to analyze specificity more accurately, we have also carried out sequencing analysis to the amplified production of each primer in part paddy rice diseased plant sample, and the sequence homology of each amplified production sequence of comparison result and corresponding viral respective regions is more than 98%.These results show that present method can be used to detect RSV, RBSDV, SRBSDV specifically.On this basis, we adopt the mode of gradient dilution standard model that the sensitivity of this method has been carried out analyzing (result sees Fig. 2, swimming lane 17-28) again, when being diluted to 1/10000, still can effectively detect, and show that present method has higher sensitivity.
Except that the foregoing description, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of requirement of the present invention.
Claims (6)
1. the method for three kinds of paddy rice viruses of a simple synchronous detection fast; It is characterized in that: it is that template is carried out one step RT-PCR amplification with the total RNA of testing sample that this detection method is used a cover virus-specific primer; This primer has 6; Be respectively DRS-1, DRS-2, DRB-1, DRB-2, DSR-1 and DSR-2; Their gene order is judged the situation that infects of rice black-streaked dwarf virus (RBSDV), rice stripe virus (RSV) and southern rice black-streaked dwarf virus (SRBSDV) at last through gel electrophoresis strip shown in sequence table.
2. the method for three kinds of paddy rice viruses of simple synchronous detection fast according to claim 1 specifically may further comprise the steps:
1) design of primers: through the gene of compare of analysis RBSDV, RSV and SRBSDV coat protein, obtain 3 kinds of viruses and plant the zone that is polymorphum between interior conservative species, design is to 6 Auele Specific Primers of 3 kinds of paddy rice viruses;
2) the total RNA of sample extracts: testing sample is placed the 1.5mL centrifuge tube, add TRIzol reagent, extract total RNA and be stored in-80 ℃ of refrigerators subsequent use;
3) amplified reaction: adopting One Step RT-PCR Kit is that template increases with the total RNA extract of sample, and reaction TV 50 μ L include the total RNA extract 5 μ L of sample; Other each component reference reagent box explanations; Comprise 2 * reaction buffer, 25 μ L, 6 each 1 μ L of primer, enzyme mixture 2 μ L; The ddH2O that adds no RNase again is to TV 50 μ L, and response procedures is 50 ℃ of reverse transcription 30min; 94 ℃ of preparatory sex change 2min; 94 ℃ of sex change 30s, 63 ℃ of annealing 30s, 72 ℃ are extended 30～60s, 30 circulations; 72 ℃ are extended 10min;
4) electrophoretic analysis: get amplified production 5 μ L; On the sepharose of point sample to 1.2% appearance hole in; With DL2000 DNA standard molecular weight as reference; Under 120 volts the voltage in 1 * TAE electrophoretic buffer electrophoresis 30～60min, EB dyeing back is observed under uv lamp, record is also analyzed and the consistence of expecting that segment is big or small.
3. the method for three kinds of paddy rice viruses of simple synchronous detection fast according to claim 2, it is characterized in that: said step 2), testing sample is plant samples such as paddy rice, corn; Get the 0.1g blade and in mortar, add the liquid nitrogen shape of pulverizing; After changing the 1.5mL centrifuge tube over to, add 1mL TRIzol reagent again, behind the vibration mixing; Add chloroform 0.2ml, leave standstill 3～5min behind the mixing; The centrifugal 10min of 12000g gets supernatant to new centrifuge tube and add isopyknic Virahol, 12000g behind the mixing, and centrifugal 10min under 4 ℃ of conditions abandons supernatant, and deposition is washed 2 times with 70% ethanol, after the drying deposition is dissolved in the ddH of no RNase
2Among the O, total RNA of extraction is stored in-80 ℃ of refrigerators subsequent use.
4. the method for three kinds of paddy rice viruses of simple synchronous detection fast according to claim 2; It is characterized in that: said step 2); Testing sample is the plant hopper sample, gets the single head polypide, places the 1.5mL centrifuge tube; Add 500 μ L TRIzol reagent, with glass rod polypide is fully ground again through pyroprocessing; Behind the vibration mixing, add chloroform 0.1ml, leave standstill 3～5min behind the mixing; The centrifugal 10min of 12000g gets supernatant to new centrifuge tube and add isopyknic Virahol, 12000g behind the mixing, and centrifugal 10min under 4 ℃ of conditions abandons supernatant, and deposition is washed 2 times with 70% ethanol, after the drying deposition is dissolved in the ddH of no RNase
2Among the O, total RNA of extraction is stored in-80 ℃ of refrigerators subsequent use.
5. according to the method for three kinds of paddy rice viruses of any described simple synchronous detection fast of claim 1-4, it is characterized in that: DRS-1 and the DRS-2 nucleocapsid protein gene of RSV that increases specifically in the designed primer, the amplified production size is 757bp; Increase the specifically main outer capsid protein gene of RBSDV of DRB-1 and DRB-2, the amplified production size is 592bp; Increase the specifically main outer capsid protein gene of SRBSDV of DSR-1 and DSR-2, the amplified production size is 142bp.
6. according to the method for three kinds of paddy rice viruses of any described simple synchronous detection fast of claim 1-4, it is characterized in that: confirm that through grads PCR the righttest annealing temperature of primer is 63 ℃.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104141015A (en) * | 2014-07-09 | 2014-11-12 | 江苏省农业科学院 | Method for quickly detecting rice stripe virus and rice black-streaked dwarf virus in laodelphax striatellus |
| CN104450960A (en) * | 2014-11-24 | 2015-03-25 | 浙江省农业科学院 | Method for rapidly detecting pathogen of rice black-streaked dwarf |
| CN104651537A (en) * | 2015-03-11 | 2015-05-27 | 华南农业大学 | One-step multiplex-PCR detection method capable of detecting four rice viruses at same time |
| CN107130057A (en) * | 2016-05-26 | 2017-09-05 | 东北农业大学 | Detect RT-PCR and its application of rice black-streaked dwarf virus and southern rice black-streaked dwarf virus |
-
2012
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Non-Patent Citations (4)
| Title |
|---|
| ANH TA HOANG ET AL.: "identification,characterization,and distribution of southern rice black-streaked dwarf virus in vietnam", 《PLANT DISEASE》 * |
| 季英华等: "一种快速同步检测水稻黑条矮缩病毒和南方水稻黑条矮缩病毒的方法", 《中国水稻科学》 * |
| 杜振国: "三种水稻病毒基因沉默抑制子的鉴定和研究", 《中国博士学位论文全文数据库 农业科技辑》 * |
| 章松柏等: "2010年福建省水稻病毒病流行分布研究", 《中国植物病理学会2011年学术年会论文集》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104141015A (en) * | 2014-07-09 | 2014-11-12 | 江苏省农业科学院 | Method for quickly detecting rice stripe virus and rice black-streaked dwarf virus in laodelphax striatellus |
| CN104450960A (en) * | 2014-11-24 | 2015-03-25 | 浙江省农业科学院 | Method for rapidly detecting pathogen of rice black-streaked dwarf |
| 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 |
| CN104651537A (en) * | 2015-03-11 | 2015-05-27 | 华南农业大学 | One-step multiplex-PCR detection method capable of detecting four rice viruses at same time |
| CN107130057A (en) * | 2016-05-26 | 2017-09-05 | 东北农业大学 | Detect RT-PCR and its application of rice black-streaked dwarf virus and southern rice black-streaked dwarf virus |
| 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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| CN102559937B (en) | 2014-04-16 |
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