CN103382506A - RT-LAMP technology for rapidly detecting sorghum mosaic virus - Google Patents
RT-LAMP technology for rapidly detecting sorghum mosaic virus Download PDFInfo
- Publication number
- CN103382506A CN103382506A CN2013101860049A CN201310186004A CN103382506A CN 103382506 A CN103382506 A CN 103382506A CN 2013101860049 A CN2013101860049 A CN 2013101860049A CN 201310186004 A CN201310186004 A CN 201310186004A CN 103382506 A CN103382506 A CN 103382506A
- Authority
- CN
- China
- Prior art keywords
- mosaic virus
- sorghum mosaic
- lamp
- reaction
- sorghum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a technology for rapidly detecting Sorghum mosaic virus. By the utilization of the technology, rapid and specific diagnosis of Sorghum mosaic virus during the growth process of sugarcane can be achieved. The technology belongs to the field of agricultural science and farming techniques. According to sorghum mosaic virus sequence provided by NCBI, four RT-LAMP specific primers (F3, B3, FIP and BIP) are designed; after RNA extraction of a sample, RT-LAMP reaction is carried out; through design and optimization of the reaction system, the reaction can be carried out in a thermostat water bath for 1h so as to complete amplification; and amplification products can undergo electrophoresis or fluorochrome to achieve the detection. The invention relates to a rapid, sensitive and economic detection method which is simple to operate.
Description
Technical field
The present invention relates to a kind of method of rapid detection sorghum mosaic virus, belong to the agricultural cience and farming techniques fields.
Technical background
A kind of main diseases viral disease on sorghum mosaic virus (Sorghum mosaic virus) sugarcane belongs to the corn mosaic virus subgroup member of Potyvirus.The sugarcane producing region generally occurs in China Guangdong, Guangxi, Zhejiang, Fujian etc., causes harm seriously in partial area.General yellowish green alternate irregular embedding line, the streak or mottled of occurring of disease plant, especially obvious with new leaf base symptom, cause sugarcane yield and sugarcane stem sugar degree to reduce.
Ring mediated constant temperature nucleic acid amplification technology (Loop-mediated isothermal amplification, LAMP) by Notomi T etc. invention in 2000, it is a kind of constant temperature nucleic acid amplification method based on highly sensitive strand displacement technology, its principle is: utilize a kind of strand displacement archaeal dna polymerase (Bst DNA poly-merase) in the lower insulation of constant temperature (65 ℃ of left and right) dozens of minutes, can realize the amplification of nucleic acid.Reaction result can carry out visual inspection by the formation of byproduct of reaction magnesium pyrophosphate white precipitate, and the turbidity that perhaps detects the magnesium pyrophosphate white precipitate by turbidimeter is judged, also can add the range estimation of fluorescent dye reagent to judge in reaction tubes.The method is simple to operate, need not special instrument (PCR instrument), and this method has the advantages such as high specificity, sensitivity height, is a kind of quick and effective detection method.Disease detection and quick diagnosis that the mankind and the various viruses of animals and plants, bacterium, fungi, parasite etc. cause have been widely used at present.
The normal method that adopts has on the plant virus Identification at present: biology is learned inoculation experiments, and serology detects, and electron microscopic observation and molecules detect.Wherein molecular biology method is the most common with PCR, but PCR is time-consuming and need expensive instrument (PCR instrument); And in Serology test, comparatively common are ELISA and DIBA etc., but sensitivity is not high, specificity is also easily received restriction, and the LAMP method has well overcome the deficiency that exists in current method, has not yet to see the method for using the method to detect the sugarcane sorghum mosaic virus.The LAMP method had both guaranteed the susceptibility and the specificity that detect, had simplified again operation simultaneously, provided cost savings, for the investigation of this disease from now on provides a kind of simple effective means.
Summary of the invention
The invention provides a kind of method of rapid detection sugarcane sorghum mosaic virus, by the method can be sensitive, special diagnosis goes out the sugarcane sorghum mosaic virus.
The method of a kind of rapid detection sorghum mosaic virus provided by the present invention obtains by the following method:
The nucleotide sequence of the CP albumen of the sorghum mosaic virus of 1) having reported according to NCBI, select relative conserved sequence by Multiple Sequence Alignment, by 4 primers of online LAMP primer-design software primerExplorer V4 design, comprising 2 outer primer F3/B3 and 2 inner primer FIP/BIP.
2) utilize RNAiso Plus method to extract total RNA of sugarcane plant;
3) different Mg is set
2+The concentration ratio of concentration, interior outer primer and the concentration of trimethyl-glycine are determined the optimum response system; The temperature that changes reaction is respectively carried out the RT-LAMP amplified reaction, determines best reaction conditions.
4) under the condition of optimum response parameter, do not verify the specificity of this method as negative control to infect sample;
5) experimental result can judge and 1.5% electrophoresis detection is verified by naked eyes.
6) primer sequence provided by the invention is as follows:
F3：5-GACCATGATGGATGGAGAW-3
B3：5-AAGTCGGTCWAGTTTCGC-3
FIP：5-TCACTTTAGTGATGCAGCTGAAG-TGAAGTCCGTATCTTGGC-3
BIP：5-TGCATAATCTGCCTAAATGRTGGAG-GAACAAAGAACATTTCCTTTAAAGC-3
The temperature of reaction of RT-LAMP is 61 ℃-63 ℃, and the reaction times is 60-90min.Utilize electrophoresis detection or detection of fluorescent dyes can detect sorghum mosaic virus.
Benefit of the present invention is the sorghum mosaic virus on can the quick diagnosis sugarcane, is a kind of simple and effective method.
Description of drawings
Accompanying drawing 2 is the result (M: standard molecular weight of electrophoresis detection sorghum mosaic virus; 1, corn mosaic virus; 2, negative control);
Specific implementation method
1, the design of primer:
F3：5-GACCATGATGGATGGAGAW-3
B3：5-AAGTCGGTCWAGTTTCGC-3
FIP：5-TCACTTTAGTGATGCAGCTGAAG-TGAAGTCCGTATCTTGGC-3
BIP：5-TGCATAATCTGCCTAAATGRTGGAG-GAACAAAGAACATTTCCTTTAAAGC-3
The thermograde experiment of sorghum mosaic virus RT-LAMP
The RT-LAMP reaction 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 1.4mM, Bst archaeal dna polymerase (8U) 1 μ L, 10 * Bst buffer1 μ L, the M-MuLV ThermoScript II, RNase Inhibitor40U, trimethyl-glycine 2.5 μ L, 25mM MgSO42.5 μ L, RNA masterplate 1 μ L, the aqua sterilisa that DEPC processes adds to 25 μ L.The mixing reactant is isothermal reaction 1h under 61 ℃, 63 ℃, 65 ℃ three kinds of conditions, 80 ℃ of reaction 5min stop the RT-LAMP reaction, after finishing, experiment gets 10 μ L amplified production loadings, carry out electrophoresis on 1.5% agarose gel, result is presented under 61 ℃, 63 ℃ isothermal reaction conditions stepped amplified band, and 65 ℃ of isothermal reactions and negative control are without stepped amplified band (Fig. 1). and therefore the temperature of reaction of RT-LAMP of the present invention is 61 ℃-63 ℃.
In order to verify the specificity of RT-LAMP, select not infect the RNA of sorghum mosaic virus in contrast, result shows that stepped amplified band does not appear in negative control, and has the RNA sample of sorghum mosaic virus to amplify purpose product (Fig. 2). this specificity that shows this RT-LAMP reaction is better.
A kind of method of rapid detection sorghum mosaic virus can rapid detection go out sorghum mosaic virus by the method.
Claims (1)
1. utilize RT-LAMP rapid detection sorghum mosaic virus, it is characterized in that: totally 2 pairs of the Auele Specific Primers that detects according to the conserved sequence of sorghum mosaic virus design RT-LAMP: a pair of outer primer F3, B3 and inner primer FIP, BIP, isothermal reaction 60-80min under 61-63 ℃ of condition, utilize the method for electrophoresis or fluorescence dye can detect sorghum mosaic virus, " Auele Specific Primer " sequence of described RT-LAMP reaction is following 4 primers:
F3：5-GACCATGATGGATGGAGAW-3
B3：5-AAGTCGGTCWAGTTTCGC-3
FIP：5-TCACTTTAGTGATGCAGCTGAAG-TGAAGTCCGTATCTTGGC-3
BIP：5-TGCATAATCTGCCTAAATGRTGGAG-GAACAAAGAACATTTCCTTTAAAGC-3。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013101860049A CN103382506A (en) | 2013-05-20 | 2013-05-20 | RT-LAMP technology for rapidly detecting sorghum mosaic virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013101860049A CN103382506A (en) | 2013-05-20 | 2013-05-20 | RT-LAMP technology for rapidly detecting sorghum mosaic virus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103382506A true CN103382506A (en) | 2013-11-06 |
Family
ID=49490432
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013101860049A Pending CN103382506A (en) | 2013-05-20 | 2013-05-20 | RT-LAMP technology for rapidly detecting sorghum mosaic virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103382506A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105969912A (en) * | 2016-06-29 | 2016-09-28 | 临沂大学 | Zucchini yellow mosaic virus RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection kit and detection method |
| CN106011311A (en) * | 2016-06-28 | 2016-10-12 | 临沂大学 | Kit and method for detecting watermelon mosaic virus RT-LAMP |
| CN106167835A (en) * | 2016-06-29 | 2016-11-30 | 临沂大学 | Cucumber green mottle viral RT LAMP detection kit and detection method |
| CN112904002A (en) * | 2021-02-01 | 2021-06-04 | 广西大学 | Method for detecting sorghum mosaic virus in sugarcane based on stern-volmer principle |
| WO2023082713A1 (en) * | 2021-11-09 | 2023-05-19 | 中国科学院西北生态环境资源研究院 | Gica-rt-lamp kit for alfafa mosaic virus and detection card |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286637A (en) * | 2011-07-21 | 2011-12-21 | 陕西省烟草研究所 | Method for detecting tobacco viruses by reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technology |
-
2013
- 2013-05-20 CN CN2013101860049A patent/CN103382506A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286637A (en) * | 2011-07-21 | 2011-12-21 | 陕西省烟草研究所 | Method for detecting tobacco viruses by reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technology |
Non-Patent Citations (2)
| Title |
|---|
| 秦碧霞等: "侵染广西甘蔗的高粱花叶病毒分子鉴定初报", 《中国糖料》 * |
| 许东林等: "侵染甘蔗的高粱花叶病毒遗传多样性分析", 《作物学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011311A (en) * | 2016-06-28 | 2016-10-12 | 临沂大学 | Kit and method for detecting watermelon mosaic virus RT-LAMP |
| CN105969912A (en) * | 2016-06-29 | 2016-09-28 | 临沂大学 | Zucchini yellow mosaic virus RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection kit and detection method |
| CN106167835A (en) * | 2016-06-29 | 2016-11-30 | 临沂大学 | Cucumber green mottle viral RT LAMP detection kit and detection method |
| CN112904002A (en) * | 2021-02-01 | 2021-06-04 | 广西大学 | Method for detecting sorghum mosaic virus in sugarcane based on stern-volmer principle |
| WO2023082713A1 (en) * | 2021-11-09 | 2023-05-19 | 中国科学院西北生态环境资源研究院 | Gica-rt-lamp kit for alfafa mosaic virus and detection card |
| GB2618759A (en) * | 2021-11-09 | 2023-11-15 | Northwest Inst Of Eco Environment And Resources | GICA-RT-LAMP kit for Alfafa mosaic virus and detection card |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103382506A (en) | RT-LAMP technology for rapidly detecting sorghum mosaic virus | |
| CN101880711A (en) | Nucleic acid screening method of staphylococcus aureus, salmonella, shigella and listeria monocytogenes | |
| CN103642936B (en) | The specific detection of 55 type adenoviruss primed probe combination and test kit | |
| CN101613766B (en) | Peste des petits ruminants virus (PPRV) RT-LAMP kit | |
| CN101624636A (en) | LAMP-LFD detection method of infectious spleen and kidney necrosis virus (ISKNV) | |
| CN103667530B (en) | A kind of reverse transcription loop-mediated isothermal detection method of tomato chlorisis virus | |
| CN105986042A (en) | Method for quickly detecting nucleic acid of H9 subtype avian influenza virus | |
| CN105986043A (en) | Method for quickly detecting nucleic acid of H5 subtype highly pathogenic avian influenza virus | |
| Hassan et al. | Loop-mediated isothermal amplification (LAMP): Comparative advances over conventional PCR and other molecular techniques | |
| CN101845519B (en) | Loop-mediated lsothermal amplification (LAMP) kit for quick detection of prawn taura syndrome viruses (TSV) | |
| CN101245395B (en) | Ring mediated isothermality amplification detection method for turbot reddish body iridovirus | |
| CN102534038A (en) | High-sensitivity rapid detection kit for campylobacter jejuni | |
| CN106434916A (en) | LAMP primer group and kit for enterobacter sakazakii, and use method of kit | |
| CN105695584A (en) | GyrB primer combination for detecting staphylococcus aureus of experimental animals | |
| CN105624286A (en) | Legionella pneumophila fluorescence PCR detection kit | |
| Guo et al. | Rapid detection of mud crab dicistrovirus-1 using loop-mediated isothermal amplification | |
| CN102154518A (en) | Technology for rapidly detecting rice black-streaked dwarf viruses in plants by reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method | |
| CN102876808B (en) | Muscovy duckling parvovirus LAMP (loop-mediated isothermal amplification) detection reagent kit | |
| CN103509881A (en) | Method for quickly detecting sugarcane yellow leaf virus by using RT-LAMP | |
| CN111187814B (en) | Buffer solution for nucleic acid amplification colorimetric reaction and application thereof | |
| Kampliw et al. | Loop mediated isothermal amplification (LAMP) for Nosema bombycis diagnosis by small subunit ribosomal RNA (SSU rRNA) gene | |
| CN102168150A (en) | Reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapidly detecting southern rice black-streaked dwarf viruses in plant hopper | |
| CN105238878A (en) | Sugarcane streak mosaic virus RT-LAMP (reverse transcription and loop-mediated isothermal amplification) primer set and detection method and application of thereof | |
| CN103484569A (en) | Method for quickly detecting sorghum mosaic virus by RT-LAMP (Reverse transcription loop-mediated isothermal amplification) | |
| CN106011307A (en) | Nucleic acid rapid detection method for H7 subtype avian influenza virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20131106 |