CN102168151B - Reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapidly detecting rice black-streaked dwarf viruses in plant hopper - Google Patents
Reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapidly detecting rice black-streaked dwarf viruses in plant hopper Download PDFInfo
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Abstract
The invention provides a rapid detection technology for rice black-streaked dwarf viruses in plant hopper and a method for applying the rapid detection technology to disease diagnosis and prediction. By the method, the interference of southern rice black-streaked dwarf viruses can be eliminated, and whether the rice black-streaked dwarf viruses are carried in the plant hopper is rapidly identified, so that a pertinent control strategy is adopted to reduce the loss caused by diseases.
Description
Technical field
The present invention relates to the Fast Detection Technique of rice black-streaked dwarf virus in the plant hopper body and in disease screening with the application of observing and predicting, belong to the agricultural cience and farming techniques field.
Background technology
Rice black-streaked dwarf virus (Rice black-streaked dwarf virus, RBSDV), a kind ofly on the paddy rice to endanger serious virus disease, be under the jurisdiction of Phytoreovirus section Fijivirus and belong to (Fijivirus), virion is spherical, mainly propagated by small brown rice planthopper, symptom main manifestations in early stage in field stunts for planting, leaf dark green, later stage wax occurs at blade, leaf sheath and cane and drips the shape projection, then form secret note, very large to the yield effect of paddy rice, grave illness field even can cause without output.This virus also can infect corn (maize rough dwarf virus), wheat (wheat is green short) and multiple gramineous weeds except infecting paddy rice.Be with popularly in 1963 and 1966 in Jiangsu Province, China, Shanghai, Zhejiang one, caused heavy losses in the many corns of China and paddy rice producing region eruption and prevalence the nineties.Should disease spread generation in the area, Jiangsu and Zhejiang Provinces in recent years, and be day by day serious trend, 2008 are only Jiangsu Province's onset area just reaches 4,000,000 mu, causes local Rice Production loss serious.
Economized a kind of Virus Diseases of Rice of kainogenesis since 2007 in Guangdong and Hainan etc., the field symptom is extremely similar to rice black-streaked dwarf virus, its genome sequence is measured rear discovery itself and rice black-streaked dwarf virus have larger difference (homology of both S9 and S10 fragment only is 75% and 80%), name and be southern rice black-streaked dwarf virus (Southernrice black-streaked dwarfvirus, SRBSDV), this virus also belongs to Phytoreovirus section Fijivirus and belongs to (Fijivirus), virion is spherical, genome is comprised of 10 double-stranded RNAs, vector is mainly white backed planthopper, small brown rice planthopper also can be propagated, the field Symptoms is that plant stunts, dark green leaf color, strip oyster white or Vandyke brown pimple appear in blade back and stem stalk, and the host also comprises corn and multiple weeds except paddy rice.Should spread generation in disease THE LOWER YANGTZE VALLEY area in recent years, and be day by day serious trend, area occurs and reaches 3,000,000 mu in the southern rice black-streaked dwarf virus whole nation in 2009, rose to rapidly 1,900 ten thousand mu in 2010, only the Hunan Province just occurs nearly 1,000 ten thousand mu, 80,000 mu of total crop failure areas have brought huge loss to paddy rice production.
Because two-strain is closely similar at aspects such as symptom, plastochondria shape, vector and hosts, has also caused difficulty to its diagnosis and evaluation thus.The normal method that adopts has on the plant virus Identification at present: the biological inoculation experiment, serology detects, electron microscopic observation and molecular Biological Detection.Because this two-strain all has the characteristic that approaches very much aspect a lot, therefore traditional plant virus detection method identifies that such as electron microscopic observation (the virus particle size shape is close), traditional biological inoculation (symptom, amboceptor, host range are close), serology detect methods such as (sequence still have higher homology) and all be difficult to differentiation.Ring mediated constant temperature nucleic acid amplification technology (loop-mediated isothermal amplification, LAMP), that it is a kind of high-sensitive strand displacement technology, a kind of novel PCR substitute technology by the constant temperature nucleic acid amplification method in a kind of novelty of invention in 2000 such as Notomi T.The LAMP characteristics be for 6 zone design of target gene 4 special primers, utilizing a kind of strand displacement archaeal dna polymerase---Bst (Bacillus stearothermophilus) DNA polymerase reacts under the condition of constant temperature (65 ℃) can finish nucleic acid amplification reaction in 1 hour, just can obtain clearly reaction result by the direct visual colorimetry of fluorescent dye.Do not need long temperature cycle, do not need the expensive instruments such as PCR, do not need the loaded down with trivial details processes such as electrophoresis ultraviolet observation.Disease detection and quick diagnosis that the mankind and the various viruses of animals and plants, bacterium, parasite etc. cause have been widely used at present.
Summary of the invention
The invention provides the method for rice black-streaked dwarf virus in a kind of rapid detection plant hopper body, can quick diagnosis go out plant hopper by the method and whether carry rice black-streaked dwarf virus.
The method of rice black-streaked dwarf virus on a kind of rapid detection plant provided by the present invention obtains by the following method:
1) the rice black-streaked dwarf virus S10 nucleotide sequence of having reported according to NCBI, analyze the rear relative conservative region 950-1467bp of selection by software DNAs tar, utilize the corresponding zone on the AY050488.1 to design 4 primers by online LAMP primer-design software primerExplorer V4, comprising 2 outer primer F3/B3 and 2 inner primer FIP/BIP.
2) Trizol reagent method is extracted the total RNA of plant;
3) different inside and outside primer concentration ratios, Mg are set
2+Concentration is determined the optimum response system; Change respectively reaction times and temperature and carry out the RT-LAMP amplified reaction, determine optimum reaction condition.
4) under the optimum response Parameter Conditions, take the specificity of southern rice black-streaked dwarf virus as this method of contrast checking;
5) compare the reliability of checking RT-LAMP with the RT-PCR method;
6) by naked eyes judgement and SYBR GreenI dyeing the RT-LAMP product is carried out visualization processing.
Primer sequence provided by the invention is as follows:
F 3GGTACTTCTACTTCTAGTCCTT
B 3CTTTTTTCATGTCCATCAACTT
FIP ACGTAAAGACGATCCTTTTTCAGT-ACAGATAAGAAATTCATTGACTGG
BIP AGTGCCACTAATACTTCAACCAC-TCGTCAAAATATTGAACAGAGA
The temperature of reaction of RT-LAMP is 61-63 ℃, and the reaction times is 60-80min.Utilize electrophoresis detection or fluorescence dye method can get rid of the interference of southern rice black-streaked dwarf disease, quick diagnosis goes out plant hopper and whether carries rice black-streaked dwarf virus.
Utilize this method can get rid of the interference of southern rice black-streaked dwarf disease, whether Rapid identification goes out in the plant hopper body rice black-streaked dwarf virus, and then takes targetedly control strategy, reduces the loss that disease is brought.
Description of drawings
Fig. 1 is that (M is standard molecular weight for the electrophorogram of RT-LAMP result under the differential responses temperature condition; 1 is 60 ℃; 2 is 61 ℃; 3 is 62 ℃; 4 is 63 ℃; 5 is 64 ℃; 6 is 65 ℃; 7 negative contrasts).
Fig. 2 be rice black-streaked dwarf virus RT-LAMP reaction specific test as a result figure (M is standard molecular weight; 1 is the small brown rice planthopper of carrying RBSDV; 2 is the white backed planthopper of carrying SRBSDV; 3 is the small brown rice planthopper of not carrying RBSDV; 4 negative contrasts).
Specific implementation method
1. the design of rice black-streaked dwarf virus RT-LAMP primer.
According to the rice black-streaked dwarf virus of having reported on the NCBI (AF227206.1, http://www.ncbi.nlm.nih.gov/nuccore/183229399; EU784843.1, http://www.ncbi.nlm.nih.gov/nuccore/209867306) the S10 nucleotide sequence, by finding out the relatively conservative regional 950-1467bp design primer of this viral nucleic acid after the software DNAstar analysis.Use online LAMP primer-design software primerExplorer V4 (http://primerexplorer.jp/elamp4.0.0/index.html) to design primer with one section sequence (950-1467bp) that this virus is relatively conservative as template.After template sequence uploaded, calculate the preliminary LAMP combination of primers (F3 of acquisition by system software, B3, FIP, BIP), corresponding 3 ' the end of the primer that provides by software again or the parameters such as 5 ' end stability and primer dimer manyly compare selection to primer to what design, finally choose one group of only primer, and sequence is as follows:
F 3GGTACTTCTACTTCTAGTCCTT
B 3CTTTTTTCATGTCCATCAACTT
FIP ACGTAAAGACGATCCTTTTTCAGT-ACAGATAAGAAATTCATTGACTGG
BIP AGTGCCACTAATACTTCAACCAC-TCGTCAAAATATTGAACAGAGA
2. the thermograde of rice black-streaked dwarf virus RT-LAMP experiment
Obtain to be with malicious white backed planthopper for examination after susceptible rice plant is raised poison, the detection of susceptible rice plant is with reference to season quintessence's etc. method (season quintessence etc., " rice in China science ", 2011).Reference
Reagent (Invitrogen) operation instruction is extracted the total RNA of sample.Although the optimal reactive temperature of Bst DNA polymerase is 65.8 ℃, but many reports have confirmed that the suitable temperature of reaction of RT-LAMP is between 60 ℃-65 ℃, simultaneously because different primers can not guarantee that when design the Tm value of all primers is in full accord, and RT-LAMP also will consider the suitable temperature of reaction of ThermoScript II, in view of this, the present invention is optimized test to the RT-LAMP temperature of reaction.The RT-LAMP system is: each 0.2 μ M of outer primer F3 and B3, each 1.2 μ M of inner primer FIP and BIP, dNTP mixture (10mM each) 2.5 μ l, 20mM Tris-HCl (pH8.8,25 ℃), 10mM KCl, 10mM (NH4)
2SO
4, 8mM MgSO
4, 0.1%TritonX-100, Bst archaeal dna polymerase, large fragment 8U, M-MuLV ThermoScript II 100U, RNase Inhibitor 20U, 0.8M trimethyl-glycine, 5.5 μ l RNA templates.The mixing reactant is at 60 ℃, and 61 ℃, 62 ℃, 63 ℃, 64 ℃, isothermal reaction 1h under 65 ℃ of six kinds of conditions, 80 ℃ of reaction 5min stop the RT-LAMP reaction, get 2 μ L amplified production loadings, carry out electrophoresis at 1.5% sepharose, electrophoresis result EB dyeing.The result is presented under the same reaction system condition, at 60 ℃, 61 ℃, 62 ℃, behind 63 ℃ of isothermal reaction 1h stepped amplified band is arranged, 61 ℃, 62 ℃, the electrophoretic band brightness of the amplified production behind 63 ℃ of isothermal reaction 1h is almost identical, and the band brightness of amplified production weakens a bit behind 60 ℃ of isothermal reaction 1h, and 64 ℃, 65 ℃ of isothermal reaction 1h and negative control are without stepped amplified band (Fig. 1).Therefore the temperature of reaction of RT-LAMP of the present invention should be 61-63 ℃.
In order to verify the specificity of RT-LAMP method, the southern rice black-streaked dwarf virus (SRBSDV) of selection and the same genus of RBSDV in contrast.Take the small brown rice planthopper of carrying RBSDV and the total RNA of white backed planthopper that carries SRBSDV as template, react by the RT-LAMP reaction system in the example 1 respectively.The result shows that white backed planthopper and the total RNA template of nontoxic small brown rice planthopper of carrying SRBSDV do not have amplified production; And the small brown rice planthopper of carrying RBSDV can amplify purpose product (Fig. 2).This specificity that shows this RT-LAMP primer is better.
The method of rice black-streaked dwarf virus in a kind of rapid detection plant hopper body can quick diagnosis goes out plant hopper by the method and whether carries rice black-streaked dwarf virus.
Above-mentioned enforcement does not limit the present invention in any form.
Claims (1)
1. the method for rice black-streaked dwarf virus in the rapid detection plant hopper body, it is characterized in that: the Auele Specific Primer that utilizes the RT-LAMP reaction, isothermal reaction 40-80min under 60-63 ℃ of condition, utilize electrophoresis detection or fluorescence dye method can get rid of the interference of southern rice black-streaked dwarf disease, identify whether carry rice black-streaked dwarf virus in the plant hopper body, described " Auele Specific Primer of RT-LAMP reaction " refers to following 4 primers:
F3 GGTACTTCTACTTCTAGTCCTT
B3 CTTTTTTCATGTCCATCAACTT
FIP ACGTAAAGACGATCCTTTTTCAGT-ACAGATAAGAAATTCATTGACTGG
BIP AGTGCCACTAATACTTCAACCAC-TCGTCAAAATATTGAACAGAGA。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101430335A (en) * | 2008-11-13 | 2009-05-13 | 江苏省农业科学院 | Immunity detection reagent kit for rice black-streaked dwarf fijivirus in single-head small brown rice planthopper |
| CN101691614A (en) * | 2009-10-09 | 2010-04-07 | 江苏省农业科学院 | Method for rapidly identifying rice black-streaked dwarf virus and southern rice black-streaked dwarf virus |
| CN101713000A (en) * | 2009-12-09 | 2010-05-26 | 湖南农业大学 | RT-PCR detection method of ordinary rice black streaked dwarf virus (RBSDV) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101430335A (en) * | 2008-11-13 | 2009-05-13 | 江苏省农业科学院 | Immunity detection reagent kit for rice black-streaked dwarf fijivirus in single-head small brown rice planthopper |
| CN101691614A (en) * | 2009-10-09 | 2010-04-07 | 江苏省农业科学院 | Method for rapidly identifying rice black-streaked dwarf virus and southern rice black-streaked dwarf virus |
| CN101713000A (en) * | 2009-12-09 | 2010-05-26 | 湖南农业大学 | RT-PCR detection method of ordinary rice black streaked dwarf virus (RBSDV) |
Non-Patent Citations (4)
| Title |
|---|
| Dung Tien Le et al..Molecular detection of nine rice viruses by a reverse-transcription loop-mediated isothermal amplification assay.《Journal of Virological Methods》.2010,90-93. |
| Molecular detection of nine rice viruses by a reverse-transcription loop-mediated isothermal amplification assay;Dung Tien Le et al.;《Journal of Virological Methods》;20100915;90-93 * |
| 周彤等.水稻黑条矮缩病毒RT-LAMP 快速检测方法的建立.《中国农业科学》.2012,第45卷(第7期),1285-1292. |
| 水稻黑条矮缩病毒RT-LAMP 快速检测方法的建立;周彤等;《中国农业科学》;20120430;第45卷(第7期);1285-1292 * |
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