CN102559937B - Method for synchronously detecting three types of paddy viruses - Google Patents
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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 plant protection technology field, the method for three kinds of Rice Virus of especially a kind of synchronous detection.
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 Jiangsu and Zhejiang Provinces, RSV) and rice black-streaked dwarf virus (rice black-streaked dwarf virus, RBSDV) a plurality of paddy fields of You China are popular, only between 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), (the Rice black-streaked dwarf virus2 that has another name called rice black-streaked dwarf virus No. 2, RBSDV-2), it is nearest newfound a kind of important Rice Virus, from calendar year 2001, since Guangdong Yangjiang City report, within 2010, in North Vietnam and China south China, Central China, East China Deng14Ge provinces, cities and autonomous regions, occurred, onset area reaches more than 2,000 ten thousand mu.Above 3 kinds of important Rice Virus are propagated in persistence mode by insect, and all can infect some common gramineous crop and the weeds such as paddy rice, corn, jowar, barnyard grass in field.Wherein, the rice stripe virus of being propagated by small brown rice planthopper, rice black-streaked dwarf virus belong to very thin Tobamovirus (genus Tenuivirus) and Reoviridae (Reoviridae) Fijivirus belongs to (genus Fijivirus), and the southern rice black-streaked dwarf virus of being propagated by white backed planthopper is that 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 the crop yields such as paddy rice in China for these 3 kinds of Virus Diseases of Rices, though disease index is different but region all occurs throughout the year and occurs exist certain overlapping in recent years, especially in China East China, this has brought certain difficulty to diagnosis, prediction and the prevention and control of such disease, also the detection of disease has been proposed to higher technical requirements.Serological method is one of detection method of the quick cheapness commonly used, but owing to there being strong serological cross reaction between RBSDV and 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. according to S10 sequence, developed respectively nido and one-step RT-PCR method 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.Japanese scholars Le et al. adopts the isothermal amplification technique of ring mediation to set up the detection method of 9 kinds of Rice Virus, but these methods all can not be used for detecting these 3 kinds of viruses simultaneously.
Summary of the invention
The present invention will solve the shortcoming of above-mentioned prior art, and a kind of method of quick, accurate, sensitive and these 3 kinds of important Rice Virus of synchronous detection is provided.
The present invention solves the technical scheme that its technical problem adopts: the method for three kinds of Rice Virus of this synchronous detection, applying a set of virus-specific primer take the total RNA of testing sample and carries out one-step RT-PCR amplification as template, this primer has 6, and their gene order is expressed 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’。
Finally by gel electrophoresis strip with judgement rice black-streaked dwarf virus (RBSDV), rice stripe virus (RSV) and southern rice black-streaked dwarf virus (SRBSDV) the situation that infects, it specifically comprises the following steps:
1) design of primers: GenBank database 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 by analyzing, obtain 3 kinds of viruses and plant the region that is polymorphism between interior conservative species, and apply Primer6.0 programdesign for 6 Auele Specific Primers of 3 kinds of Rice Virus, these primers are synthetic in Shanghai Sheng Gong biotechnology Services Co., Ltd;
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, proceed to after 1.5mL centrifuge tube, then add 1mL TRIzol reagent (Invitrogen); For plant hopper sample, get single head polypide, be placed in 1.5mL centrifuge tube, add 500 μ L TRIzol reagent (Invitrogen), then with the glass rod through pyroprocessing, polypide is fully ground; After this operation is all extracted total RNA by TRIzol reagent working instructions: after vibration mixes, add chloroform 0.2ml, mix rear standing 3～5min; The centrifugal 10min of 12000g, gets supernatant to new centrifuge tube and adds isopyknic Virahol, mixes rear 12000g, and centrifugal 10min under 4 ℃ of conditions, abandons supernatant, and precipitation is washed 2 times with 70% ethanol, after being dried, precipitation is dissolved in to the ddH without RNase
2in O, total RNA of extraction is stored in-80 ℃ of refrigerators standby;
3) amplified reaction: adopt the precious biotechnology (Dalian) of One Step RT-PCR Kit(company limited) take the total RNA extract of sample increases as template, reaction cumulative volume 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, add ddH2O without RNase to cumulative volume 50 μ L, response procedures is 50 ℃ of reverse transcription 30min again; 94 ℃ of denaturation 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, in the sepharose loading hole of point sample to 1.2%, using DL2000DNA standard molecular weight as reference, under the voltage of 120 volts in 1 * TAE electrophoretic buffer electrophoresis 30～60min, after EB dyeing, under ultraviolet lamp, observe, record and analyze and expect the consistence of segment size.
After primer is synthetic, by grads PCR, determine that the suitableeest annealing temperature of primer is 63 ℃, as shown in Figure 1; DRS-1 and the DRS-2 Nucleocapsid Protein Gene of RSV that increases specifically wherein, amplified production size is 757bp; Increase the specifically main outer capsid protein gene of RBSDV of DRB-1 and DRB-2, amplified production size is 592bp; Increase the specifically main outer capsid protein gene of SRBSDV of DSR-1 and DSR-2, amplified production size is 142bp.
Inventing useful effect is: the present invention is directed to and produce reality, a kind of method of quick, accurate, sensitive and these 3 kinds of important Rice Virus of synchronous detection has been set up in development, and be successfully applied to the diagnosis and detection of single head plant hopper and field sample, for Accurate Diagnosis, prediction and the science bridle of black streaked dwarf virus of rice, stripe disease, southern black streak dwarf provides technical support.
Accompanying drawing explanation
Fig. 1 is presented at the amplification efficiency of RT-PCR under Gradient annealing temperature (Tm) condition.Wherein M represents 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 and sensitive poison are analyzed.Wherein M represents 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 amplifications RBSDV, SRBSDV, RSV biased sample; Swimming lane 17～28 is that three pairs of mix primer amplifications are in the RBSDV of 1:10 ratio gradient dilution, SRBSDV and RSV biased sample;
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 paddy rice diseased plant sample; Swimming lane 9-10 is corn diseased plant sample; Swimming lane 11 is healthy nontoxic small brown rice planthopper sample; Swimming lane 12-14 is small brown rice planthopper sample; Swimming lane 15-16 is white backed planthopper sample; Swimming lane 17 is healthy nontoxic white backed planthopper sample.
Embodiment
Below in conjunction with accompanying drawing, the invention will be further described:
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 mainly gather from provinces and cities and North Vietnam provinces and cities such as China Zhejiang, Shandong, Hubei, Hunan, Guangdong, Shanghai between 2008-2011, and are stored in-80 ℃ of refrigerators.
Design of primers: GenBank database 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 by analyzing, obtain 3 kinds of viruses and plant the region that is polymorphism between interior conservative species, and apply Primer6.0 programdesign for 6 Auele Specific Primers of 3 kinds of Rice Virus, these primers are synthetic in Shanghai Sheng Gong biotechnology Services Co., Ltd.
The total RNA of vegetable material extracts: for plant samples such as paddy rice, corns, get 0.1g blade and in mortar, add the liquid nitrogen shape of pulverizing, proceed to after 1.5mL centrifuge tube, add again 1mL TRIzol reagent (Invitrogen), after vibration mixes, add chloroform 0.2ml, mix rear standing 3-5min; The centrifugal 10min of 12000g, gets supernatant to new centrifuge tube and adds isopyknic Virahol, mixes rear 12000g, and centrifugal 10min under 4 ℃ of conditions, abandons supernatant, and precipitation is washed 2 times with 70% ethanol, after being dried, precipitation is dissolved in to the ddH without RNase
2in O, total RNA of extraction is stored in-80 ℃ of refrigerators standby.
Amplified reaction: reaction cumulative volume 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, then add the ddH without RNase
2o is to cumulative volume 50 μ L.Response procedures is 50 ℃ of reverse transcription 30min; 94 ℃ of denaturation 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, in the sepharose loading hole of point sample to 1.2%, using DL2000DNA standard molecular weight as reference, under the voltage of 120 volts in 1 * TAE electrophoretic buffer electrophoresis 30～60min, after EB dyeing, under ultraviolet lamp, observe, record and analyze and expect that the consistence of segment size, part electrophoresis identify that picture is shown in Fig. 3 swimming lane 1-10.
Interpretation of result: which kind of virus infection can judge according to electrophoresis result whether plant sample exists above-mentioned 3 kinds of viral infecting, 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).The like, amplify the sample of 592bp and 142bp band, show that this plant sample exists RBSDV and SRBSDV Combined Infection (seeing Fig. 3, swimming lane 5); Amplify the sample of 757bp and 142bp band, show that this plant sample exists RSV and SRBSDV Combined Infection (seeing Fig. 3, swimming lane 6-7).
Embodiment 2: the detection of single head plant hopper sample.
Plant hopper sample: the provinces and cities such as small brown rice planthopper and white backed planthopper sample mainly gather from Zhejiang between 2008-2011, Hainan, Hunan, Shanghai, Shandong, part plant hopper is raised on rice seedlings, and remainder is stored in 70% ethanol standby.
Design of primers: GenBank database 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 by analyzing, obtain 3 kinds of viruses and plant the region that is polymorphism between interior conservative species, and apply Primer6.0 programdesign for 6 Auele Specific Primers of 3 kinds of Rice Virus, these primers are synthetic in Shanghai Sheng Gong biotechnology Services Co., Ltd.
The total RNA of plant hopper extracts: get single head polypide, be placed in 1.5mL centrifuge tube, add 500 μ L TRIzol reagent (Invitrogen), then with the glass rod through pyroprocessing, polypide is fully ground; After vibration mixes, add chloroform 0.1ml, mix rear standing 3-5min; The centrifugal 10min of 12000g, gets supernatant to new centrifuge tube and adds isopyknic Virahol, mixes rear 12000g, centrifugal 10min under 4 ℃ of conditions, abandons supernatant, and precipitation is washed 2 times with 70% ethanol, after dry, precipitation is dissolved in the ddH2O without RNase, total RNA of extraction is stored in-80 ℃ of refrigerators standby.
Amplified reaction: reaction cumulative volume 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, then add ddH2O without RNase to cumulative volume 50 μ L.Response procedures is 50 ℃ of reverse transcription 30min; 94 ℃ of denaturation 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, in the sepharose loading hole of point sample to 1.2%, using DL2000DNA standard molecular weight as reference, under the voltage of 120 volts in 1 * TAE electrophoretic buffer electrophoresis 30～60min, after EB dyeing, under ultraviolet lamp, observe, record and analyze and expect the consistence of segment size.Fig. 3 swimming lane 11-17 is shown in by part electrophoresis evaluation picture.
Interpretation of result: can whether carry above-mentioned 3 kinds of viruses in single head plant hopper matter sample according to electrophoresis result, if carry, carry again which kind of virus infection.For example: only amplify the small brown rice planthopper sample of 757bp band, show that this small brown rice planthopper carries RSV(and see Fig. 3, swimming lane 12); Only amplify 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(and see 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(and sees Fig. 3, swimming lane 15).The like, amplify the small brown rice planthopper sample of 592bp and 757bp band, show that this small brown rice planthopper carried RBSDV and RSV2 kind virus (seeing Fig. 3, swimming lane 13) simultaneously.
Detection specificity and sensitivity analysis: first utilize above-mentioned 3 pairs of primers respectively 3 kinds of viral standard models to be carried out to single stage method RT-PCR amplification and (the results are shown in Figure 2, swimming lane 1-9), primer pair DRB-1/DRB-2, DRZ-1/DRZ-2, DRS-1/DRS-2 all only can increase and arrive the big or small band of expection in corresponding viral standard model.Then utilize 3 pairs of primers that equal proportion mixes and using respectively RSV, RBSDV, SRBSDV standard model and composition thereof and as template, carry out pcr amplification and (the results are shown in Figure 2, swimming lane 10-16), utilize the segment of the expection size that 3 pairs of mix primer can increase special at RSV, RBSDV, SRBSDV sample equally; In biased sample, no matter be 2 kinds of sample mix or 3 kinds of sample mix, the corresponding segment of expection size all only can be detected.And utilize these primers at other plant virus equal specific bands that can't detect any expection size the same as healthy paddy rice sample in RGDV, RRSV, TMV, PVY sample.In order to analyze more accurately specificity, also to each primer, the amplified production in part paddy rice diseased plant sample has carried out sequencing analysis for we, each amplified production sequence of comparison result with the sequence homology of corresponding viral respective regions more than 98%.These results show, present method can be specifically for detection of RSV, RBSDV, SRBSDV.On this basis, we adopt again the mode of gradient dilution standard model to carry out analyzing (the results are shown in Figure 2, swimming lane 17-28) to the sensitivity of the method, when being diluted to 1/10000, still can effectively detect, and show that present method has higher sensitivity.
In addition to the implementation, 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 (4)
1. the method for three kinds of Rice Virus of a synchronous detection, it is characterized in that: this detection method is applied a set of virus-specific primer and be take the total RNA of testing sample and carry out one-step RT-PCR amplification as template, this primer has 6, be respectively DRS-1, DRS-2, DRB-1, DRB-2, DSR-1 and DSR-2, their gene order is as shown in sequence table, finally by gel electrophoresis strip, judge the situation that infects of rice black-streaked dwarf virus (RBSDV), rice stripe virus (RSV) and southern rice black-streaked dwarf virus (SRBSDV), specifically comprise the following steps:
1) design of primers: by the gene of compare of analysis RBSDV, RSV and SRBSDV coat protein, obtain 3 kinds of viruses and plant the region that is polymorphism between interior conservative species, design is for 6 Auele Specific Primers of 3 kinds of Rice Virus;
2) the total RNA of sample extracts: testing sample is placed in to 1.5mL centrifuge tube, adds TRIzol reagent, extract total RNA and be stored in-80 ℃ of refrigerators standby;
3) amplified reaction: adopt One Step RT-PCR Kit to take the total RNA extract of sample and increase as template, reaction cumulative volume 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, add ddH2O without RNase to cumulative volume 50 μ L, response procedures is 50 ℃ of reverse transcription 30min again; 94 ℃ of denaturation 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, in the sepharose loading hole of point sample to 1.2%, using DL2000DNA standard molecular weight as reference, under the voltage of 120 volts in 1 * TAE electrophoretic buffer electrophoresis 30～60min, after EB dyeing, under ultraviolet lamp, observe, record and analyze and expect the consistence of segment size.
2. the method for three kinds of Rice Virus of synchronous detection according to claim 1, it is characterized in that: described step 2), testing sample is paddy rice, maize plant sample, get 0.1g blade and in mortar, add the liquid nitrogen shape of pulverizing, proceed to after 1.5mL centrifuge tube, then add 1mL TRIzol reagent, after vibration mixes, add chloroform 0.2ml, mix rear standing 3～5min; The centrifugal 10min of 12000g, gets supernatant to new centrifuge tube and adds isopyknic Virahol, mixes rear 12000g, and centrifugal 10min under 4 ℃ of conditions, abandons supernatant, and precipitation is washed 2 times with 70% ethanol, after being dried, precipitation is dissolved in to the ddH without RNase
2in O, total RNA of extraction is stored in-80 ℃ of refrigerators standby.
3. the method for three kinds of Rice Virus of synchronous detection according to claim 1, it is characterized in that: described step 2), testing sample is plant hopper sample, get single head polypide, be placed in 1.5mL centrifuge tube, add 500 μ L TRIzol reagent, then with the glass rod through pyroprocessing, polypide is fully ground; After vibration mixes, add chloroform 0.1ml, mix rear standing 3～5min; The centrifugal 10min of 12000g, gets supernatant to new centrifuge tube and adds isopyknic Virahol, mixes rear 12000g, and centrifugal 10min under 4 ℃ of conditions, abandons supernatant, and precipitation is washed 2 times with 70% ethanol, after being dried, precipitation is dissolved in to the ddH without RNase
2in O, total RNA of extraction is stored in-80 ℃ of refrigerators standby.
4. according to the method for the three kinds of Rice Virus of synchronous detection described in any one of claim 1-3, it is characterized in that: DRS-1 and the DRS-2 Nucleocapsid Protein Gene of RSV that increases specifically in the primer of design, amplified production size is 757bp; Increase the specifically main outer capsid protein gene of RBSDV of DRB-1 and DRB-2, amplified production size is 592bp; Increase the specifically main outer capsid protein gene of SRBSDV of DSR-1 and DSR-2, amplified production size is 142bp.
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| 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 |
| 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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