CN103484569A - Method for quickly detecting sorghum mosaic virus by RT-LAMP (Reverse transcription loop-mediated isothermal amplification) - Google Patents
Method for quickly detecting sorghum mosaic virus by RT-LAMP (Reverse transcription loop-mediated isothermal amplification) Download PDFInfo
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- CN103484569A CN103484569A CN201310438344.6A CN201310438344A CN103484569A CN 103484569 A CN103484569 A CN 103484569A CN 201310438344 A CN201310438344 A CN 201310438344A CN 103484569 A CN103484569 A CN 103484569A
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Abstract
A method for quickly detecting a sorghum mosaic virus by RT-LAMP comprises the following steps: designing four LAMP specific primers, namely an F3, a B3, an FIP and a BIP according to the sequence of the sorghum mosaic virus provided by the NCBI (National Center of Biotechnology Information), carrying out the RT-LAMP reaction after reversely transcribing RNA (Ribonucleic Acid) extracted from a sample into cDNA, reacting for 1 h in a constant-temperature water bath through the design and the optimization of a reaction system to complete the amplification, and detecting the amplification products through electrophoresis or fluorescent dye. The method is a detection method which is simple, quick and sensitive to operate and economic.
Description
Technical field
The present invention relates to the method for rapid detection corn mosaic virus, specifically a kind of method of utilizing RT-LAMP rapid detection corn mosaic virus.
Technical background
Corn mosaic virus (Sorghum mosaic virus) is a kind of main diseases viral disease of sugarcane, belongs to the corn mosaic virus subgroup member of Potyvirus.The symptom of mosaic of sugarcane is mainly manifested on blade.But the sugarcane strain of catching an illness can make whole burst disease, and virus spreads all over complete stool.Mainly that chlorophyll is damaged or undesired development and make leaf section produce many vertical billet lines parallel with vein.It is different in size, and light yellow, the light green that has that have, be partitioned into " floral leaf " with normal part is irregular.Especially the most obvious with the young leaves symptom.The sugarcane strain of catching an illness is downgraded, the minimizing of tillering; The sick sugarcane of perennial root germinates slowly, poor growth.The loss that this disease causes be mainly the sugarcane kind susceptible after, germination rate is low; The diseased plant growth is poor, and the juice amount reduces.
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 to utilize a kind of strand displacement archaeal dna polymerase (Bst DNA polymerase) in the lower insulation of constant temperature (61-65 ℃ 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, the turbidity that perhaps by turbidimeter, detects the magnesium pyrophosphate white precipitate is judged, also can in reaction tubes, add the range estimation of fluorescent dye reagent to judge, without special instrument PCR instrument, having the advantages such as high specificity, sensitivity height, is a kind of fast and effectively detection method.Disease detection and quick diagnosis that the mankind and the various virus of animals and plants, bacterium, fungi, parasite etc. cause have been widely used at present.
Plant identification virus method commonly used has at present: the biological inoculation experiment, serology detects, electron microscopic observation and molecular Biological Detection.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 insufficient sensitivity is high, and specificity is vulnerable to restriction.Have not yet to see the method for using the LAMP method to detect corn mosaic virus.
Summary of the invention
The object of the present invention is to provide a kind of method of the RT-LAMP of utilization rapid detection corn mosaic virus, can be sensitive, special detect corn mosaic virus.
For addressing the above problem, the technical solution used in the present invention is: a kind of method of utilizing RT-LAMP rapid detection corn mosaic virus comprises the steps:
The nucleotide sequence of the CP albumen of the corn 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) utilize the M-MLV ThermoScript II that the RNA extracted is converted into to cDNA;
4) different Mg is set
2+the concentration of concentration, dNTPs and the concentration of trimethyl-glycine, determine the optimum response system; The temperature that changes reaction is respectively carried out the RT-LAMP amplified reaction, and the RT-LAMP temperature of reaction is 63 ℃, and the reaction times is 60-80min;
5) the RT-LAMP reaction system is: each 0.25 μ M of outer primer F3 and B3, each 1.5 μ M of inner primer FIP and BIP, Bst archaeal dna polymerase (8U) 1 μ L, 10 * Bst buffer2.5 μ L, trimethyl-glycine 2 μ L, 25mM MgSO41 μ L, dNTP1.4mM, masterplate 2 μ L, the aqua sterilisa that DEPC processes adds to 25 μ L, 63 ℃ of lower reaction times of condition are 60min, 80 ℃ of deactivation 5min; Using and do not infect sample and verify the specificity of this method as negative control;
6) experimental result judges by naked eyes and utilizes 1.5% electrophoresis detection to be verified or detection of fluorescent dyes can detect corn mosaic virus.
The sequence of described 2 outer primer F3/B3 and 2 inner primer FIP/BIP is:
F3:5-TGGAYGGAGATGAACAAAGA-3
B3:5-AATGCATACCGCGCTAAG-3
FIP:5-GCTGCATCACTGAAATGATGCATTA-CCRTTAAAACCAGTTA
TTGAAAACG-3
BIP:5-TCGAGTAYAGAAACTCTACAGAGCG-TATAGTCGGTGAGATTT
CGC-3。
Outstanding advantages of the present invention is:
Susceptibility and the specificity detected of having utilized the LAMP method both to guarantee, simplified again operation, and also provided cost savings, for the detection of corn mosaic virus provides a kind of sensitive, special, economical and simple method simultaneously.
The accompanying drawing explanation
The electrophorogram that Fig. 1 is RT-LAMP result under condition of different temperatures (M: standard molecular weight; 1,61 ℃; 2,62 ℃; 3,63 ℃; 4,64 ℃; 5,65 ℃; 6 negative controls).；
The result that Fig. 2 is the electrophoresis detection corn mosaic virus (M: standard molecular weight; 1, corn mosaic virus; 2, negative control) embodiment
The method of utilizing RT-LAMP rapid detection corn mosaic virus of the present invention, comprise the steps:
The optimization of corn mosaic virus RT-LAMP reaction conditions
1, the design of primer:
F3:5-TGGAYGGAGATGAACAAAGA-3
B3:5-AATGCATACCGCGCTAAG-3
FIP:5-GCTGCATCACTGAAATGATGCATTA-CCRTTAAAACCAGTTATTGAA
AACG-3
BIP:5-TCGAGTAYAGAAACTCTACAGAGCG-TATAGTCGGTGAGATTTCGC-3
The thermograde experiment of corn mosaic virus RT-LAMP
The RT-LAMP reaction system is: each 0.25 μ M of outer primer F3 and B3, each 1.5 μ M of inner primer FIP and BIP, Bst archaeal dna polymerase (8U) 1 μ L, 10 * Bst buffer2.5 μ L, trimethyl-glycine 2 μ L, 25mM MgSO41 μ L, dNTP1.4mM, masterplate 2 μ L, the aqua sterilisa that DEPC processes adds to 25 μ L.Mix reactant at 61 ℃, 62 ℃, 63 ℃, 64 ℃, isothermal reaction 1h under 65 ℃ of five kinds of conditions, 80 ℃ of reaction 5min stop the RT-LAMP reaction, experiment is got 10 μ L amplified production loadings after finishing, and carries out electrophoresis on 1.5% agarose gel, and result is presented at 61 ℃, 62 ℃, under 63 ℃ of isothermal reaction conditions, stepped amplified band is arranged, but 63 ℃ of bands are very clear, and 64 ℃, 65 ℃ isothermal reactions and negative control are without stepped amplified band, as shown in Figure 1, therefore, the temperature of reaction of the RT-LAMP that the present invention adopts is 63 ℃.
The specific test of corn mosaic virus RT-LAMP reaction
In order to verify the specificity of RT-LAMP, select not infect the material of corn mosaic virus in contrast, result shows that stepped amplified band does not appear in negative control, and the sample that corn mosaic virus is arranged amplifies the purpose product, as shown in Figure 2, this specificity that shows this RT-LAMP reaction is better.
Claims (2)
1. utilize the method for RT-LAMP rapid detection corn mosaic virus, it is characterized in that, comprise the steps:
The nucleotide sequence of the CP albumen of the corn 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) utilize the M-MLV ThermoScript II that the RNA extracted is converted into to cDNA;
4) different Mg is set
2+the concentration of concentration, dNTPs and the concentration of trimethyl-glycine, determine the optimum response system; The temperature that changes reaction is respectively carried out the RT-LAMP amplified reaction, and the RT-LAMP temperature of reaction is 63 ℃, and the reaction times is 60-80min;
5) the RT-LAMP reaction system is: each 0.25 μ M of outer primer F3 and B3, each 1.5 μ M of inner primer FIP and BIP, Bst archaeal dna polymerase (8U) 1 μ L, 10 * Bst buffer2.5 μ L, trimethyl-glycine 2 μ L, 25mM MgSO41 μ L, dNTP1.4mM, masterplate 2 μ L, the aqua sterilisa that DEPC processes adds to 25 μ L, 63 ℃ of lower reaction times of condition are 60min, 80 ℃ of deactivation 5min; Using and do not infect sample and verify the specificity of this method as negative control;
6) experimental result judges by naked eyes and utilizes 1.5% electrophoresis detection to be verified or detection of fluorescent dyes can detect corn mosaic virus.
2. the method for utilizing RT-LAMP rapid detection corn mosaic virus according to claim 1, is characterized in that, the sequence of described 2 outer primer F3/B3 and 2 inner primer FIP/BIP is:
F3:5-TGGAYGGAGATGAACAAAGA-3
B3:5-AATGCATACCGCGCTAAG-3
FIP:5-GCTGCATCACTGAAATGATGCATTA-CCRTTAAAACCAGTTA
TTGAAAACG-3
BIP:5-TCGAGTAYAGAAACTCTACAGAGCG-TATAGTCGGTGAGATTT
CGC-3。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755570A (en) * | 2016-11-29 | 2017-05-31 | 山东农业大学 | The foundation and application of corn mosaic virus Group IV separator PCR detection architectures |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906483A (en) * | 2010-07-20 | 2010-12-08 | 云南省农业科学院甘蔗研究所 | Quarantine method of sugarcane seedlings to be exported |
| CN103146847A (en) * | 2013-03-22 | 2013-06-12 | 南京农业大学 | RT-LAMP (loop-mediated isothermal amplification) rapid detection kit for cucumber green mottle mosaic virus (CGMMV) and detection method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906483A (en) * | 2010-07-20 | 2010-12-08 | 云南省农业科学院甘蔗研究所 | Quarantine method of sugarcane seedlings to be exported |
| CN103146847A (en) * | 2013-03-22 | 2013-06-12 | 南京农业大学 | RT-LAMP (loop-mediated isothermal amplification) rapid detection kit for cucumber green mottle mosaic virus (CGMMV) and detection method |
Non-Patent Citations (1)
| Title |
|---|
| 李利君等: "利用斑点杂交法和RT-PCR 技术检测甘蔗花叶病毒", 《福建农业大学学报》, vol. 29, no. 3, 31 December 2000 (2000-12-31) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755570A (en) * | 2016-11-29 | 2017-05-31 | 山东农业大学 | The foundation and application of corn mosaic virus Group IV separator PCR detection architectures |
| CN106755570B (en) * | 2016-11-29 | 2021-03-23 | 山东农业大学 | Establishment and application of sugarcane mosaic virus IV group isolate PCR detection system |
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