CN102154526A - Method for quickly detecting Chinese papaya leaf curl virus by LAMP (loop mediated isothermal amplification) - Google Patents
Method for quickly detecting Chinese papaya leaf curl virus by LAMP (loop mediated isothermal amplification) Download PDFInfo
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Abstract
The invention provides a method for quickly detecting Chinese papaya leaf curl virus by LAMP (loop mediated isothermal amplification), belonging to the field of plant protection. The method comprises the following steps of: designing an LAMP primer (P-F3, P-B3, P-FIP (feline infectious peritonitis) and P-BIP (bit interleaved parity)) according to the nucleotide sequence of the Chinese papaya leaf curl virus; performing the LAMP reaction after total DNA (deoxyribonucleic acid) is extracted from a sample; and performing the electrophoresis or the fluorescent dye to a reaction product. The method is simple to operate, some special apparatuses such as a PCR apparatus and the like are avoided, and the problems of the conventional methods such as the biology, the serology and the like are overcome, so that the detection method is simple, fast, sensitive and economical.
Description
Technical field
The present invention relates to the method for the Chinese papaya curve leaf disease virus of a kind of rapid detection, platymiscium protection field.
Background technology
(Papaya leaf curl china virus is a kind of important plant virus PaLCCNV) to China's papaya curve leaf disease virus, belongs to geminivirus infection section bean golden mosaic virus and belongs to, and vector is a Bemisia tabaci.This virus is detected on the papaya in China Guangxi the earliest, so be called Chinese papaya curve leaf disease virus, except that harm papaya, this virus also endangers crops such as tomato, main performance blade diminishes last volume, yellow between leaf margin and the vein, symptoms such as plant dwarfing are serious to output and the quality influence of tomato.Should disease big area on the tomato of Henan endanger in 2007, only the Kaifeng County, Kaifeng just has 2000 heliogreenhouses to be subjected to destructive harm, nearly 2,000 ten thousand yuan of direct economic loss.Also detecting on ground crops such as Jiangsu in 2009, and showed that this virus also had the sign that spreads diffusion, is that a potential threatens.
Ring mediated constant temperature nucleic acid amplification technology (loop-mediated isothermal amplification, LAMP) the earliest 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 of work is: at 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, reaction product both can be passed through the electrophoresis ultraviolet visualization, also can directly estimate by fluorescent dye.This method is easy and simple to handle, need not specific apparatus (PCR instrument), and detected result is accurate, is a kind of fast and effectively detection method.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.
In the detection of China's kind art melon curve leaf disease virus, method commonly used has two kinds at present, and a kind of is the molecular Biological Detection method, wherein common with PCR, but PCR needs 30-35 circulating reaction (more time-consuming) and special instrument (PCR instrument); Another kind is the serology detection method, and common have ELISA and a DIBA etc., and the serology detection method is except existing in sensitivity the deficiency, and the specificity of its detection also is restricted (nucleotide homology that bean golden mosaic virus belongs between the member is higher relatively).And the LAMP method has well overcome the deficiency (reaction times is short, need not specific apparatus, detection sensitivity height, specificity is good) of current these two class methods, has not yet to see the method that this method of use detects Chinese papaya curve leaf disease virus.The LAMP method had both guaranteed the susceptibility and the specificity that detect, had simplified operation again simultaneously, provided cost savings, for the investigation of this disease from now on provides a kind of simple but effective method.
Summary of the invention
The present invention has overcome shortcoming of the prior art, for the detection of Chinese papaya curve leaf disease virus provides a kind of quick, easy, sensitive method.
1, design of primers:
According to the Chinese papaya curve leaf disease virus nucleotide sequence design primer of having reported on the NCBI, primer sequence is as follows:
P-F3：5`-CAAACTTCCCTATGCAATCG-3`
P-B3：5`-GATTTGAATTAAATAGGCTTTGAGG-3`
P-FIP：5`GCCGCTTTTGGATTTTGAATTTTGA-ATGGGTCCTTATTTATATGTGAG-3`
P-BIP：5`-TCCGTATAATATTACCGGATGGCC-GTTTTAAATGGAGGACATTTCTTTG-3`
2, total DNA extraction:
Get the sick leaf of 10mg tomato in the 1.5mL centrifuge tube, add 100 μ L 0.5mol/LNaOH, the centrifugal 5min of 5000rpm after the homogenate, supernatant liquor is got 1 μ L and is carried out pcr amplification as template with 100 times of 0.1mol/L Tris-HCl (pH8.0) dilutions.
3, pcr amplification:
Reaction system: totally be 25 μ L, DNA 2 μ L wherein, Bst archaeal dna polymerase (8U/ μ L) 1 μ L, other component final concentration is respectively P-F3 and P-B30.2 μ mol/L; P-FIP and P-BIP 1.6 μ mol/L; Betaine 0.8mol/L; DNTPs 1.4mmol/L, 1 times Thermopolbuffer (containing MgSO42mmol/L); MgCl
28mmol/L.
Reaction conditions: 65 ℃ of thermostat water bath 60min; 80 ℃ of 5min.
4, detect:
Electrophoresis detection: get 10 μ L products through 1% sepharose electrophoresis 40min under 0.5 * tbe buffer liquid and 120V voltage conditions, dyeing 10min in the ethidium bromide (EB) of 0.5 μ g/mL, the dyeing back is observed in gel imaging system, can observe the bright nucleic acid swimming band that is the disperse shape in the sample of infection of Chinese papaya curve leaf disease virus.
After fluorescence dye: LAMP reaction finishes, add SYBR Greenl (1: 1000) 2 μ l in its product, examine the result behind the 1-5min, the sample of infection of Chinese papaya curve leaf disease virus becomes green, and does not infect the orange of sample and blank maintenance dyestuff.
Wherein said 65 ℃ temperature of reaction also can be 63-65 ℃
The invention has the beneficial effects as follows primer according to Chinese papaya curve leaf disease virus nucleotide sequence design LAMP, by grope optimal conditions realized should virus rapid detection, for the investigation of this disease provides a kind of simple and effective method.
Description of drawings
Embodiment
Example one:
Tomato sample with infection of Chinese papaya curve leaf disease virus is a detected object, with the negative contrast of healthy tomato sample.
1, design of primers:
According to the Chinese papaya curve leaf disease virus nucleotide sequence design primer of having reported on the NCBI, primer sequence is as follows:
P-F3：5`-CAAACTTCCCTATGCAATCG-3`
P-B3：5`-GATTTGAATTAAATAGGCTTTGAGG-3`
P-FIP：5`-GCCGCTTTTGGATTTTGAATTTTGA-ATGGGTCCTTATTTATATGTGAG-3`
P-BIP：5`TCCGTATAATATTACCGGATGGCC-GTTTTAAATGGAGGACATTTCTTTG-3`
2, total DNA extraction:
Get the sick leaf of 10mg tomato in the 1.5mL centrifuge tube, add 100 μ L 0.5mol/LNaOH, the centrifugal 5min of 5000rpm after the homogenate, supernatant liquor is got 1 μ L and is carried out pcr amplification as template with 100 times of 0.1mol/LTris-HCl (pH8.0) dilutions.
3, pcr amplification:
Reaction system: totally be 25 μ L, DNA 2 μ L wherein, Bst archaeal dna polymerase (8U/ μ L) 1 μ L, other component final concentration is respectively P-F3 and P-B30.2 μ mol/L; P-FIP and P-BIP 1.6 μ mol/L; Betaine 0.8mol/L; DNTPs 1.4mmol/L, 1 times Thermopolbuffer (containing MgSO42mmol/L); MgCl
28mmol/L.
Reaction conditions: 65 ℃ of thermostat water bath 60min; 80 ℃ of 5min.
4, detect:
Electrophoresis detection: get 10 μ L products through 1% sepharose electrophoresis 40min under 0.5 * tbe buffer liquid and 120V voltage conditions, dyeing 10min in the ethidium bromide (EB) of 0.5 μ g/mL, the dyeing back is observed in gel imaging system.
After fluorescence dye: LAMP reaction finishes, in its product, add SYBR Greenl (1: 1000) 2 μ l, examine the result behind the 1-5min.
5, interpretation of result:
Electrophoresis detection: in the sample of infection of Chinese papaya curve leaf disease virus, can observe the bright nucleic acid swimming band that is the disperse shape;
Fluorescent dye: the sample of infection of Chinese papaya curve leaf disease virus becomes green, and does not infect the orange of sample and blank maintenance dyestuff.
Claims (3)
1. method of utilizing LAMP rapid detection China papaya curve leaf disease virus may further comprise the steps:
(1) design of primers:
According to the Chinese papaya curve leaf disease virus nucleotide sequence design primer of having reported on the NCBI, primer sequence is as follows:
P-F3：5`-CAAACTTCCCTATGCAATCG-3`
P-B3：5`-GATTTGAATTAAATAGGCTTTGAGG-3`
P-FI?P：5`-GCCGCTTTTGGATTTTGAATTTTGA-ATGGGTCCTTATTTATATGTGAG-3`
P-BI?P：5`-TCCGTATAATATTACCGGATGGCC-GTTTTAAATGGAGGACATTTCTTTG-3`
(2) total DNA extraction:
Get the sick leaf of 10mg tomato in the 1.5mL centrifuge tube, add 100 μ L 0.5mol/L NaOH, the centrifugal 5min of 5000rpm after the homogenate, supernatant liquor is got 1 μ L and is carried out pcr amplification as template with 100 times of 0.1mol/L Tris-HCl (pH8.0) dilutions.
(3) LAMP reaction:
Reaction system: totally be 25 μ L, DNA 2 μ L wherein, Bst archaeal dna polymerase (8U/ μ L) 1 μ L, other component final concentration is respectively P-F3 and P-B30.2 μ mol/L; P-FIP and P-BIP 1.6 μ mol/L; Betaine 0.8mol/L; DNTPs 1.4mmol/L, 1 times Thermopolbuffer (containing MgSO42mmol/L); MgCl
28mmol/L.
Reaction conditions: 65 ℃ of thermostat water bath 60min; 80 ℃ of 5min.
(4) detect:
Electrophoresis detection: get 10 μ L products through 1% sepharose electrophoresis 40min under 0.5 * tbe buffer liquid and 120V voltage conditions, dyeing 10min in the ethidium bromide (EB) of 0.5 μ g/mL, the dyeing back is observed in gel imaging system, can observe the bright nucleic acid swimming band that is the disperse shape in the sample of infection of Chinese papaya curve leaf disease virus.
After fluorescence dye: LAMP reaction finishes, add SYBR GreenI (1: 1000) 2 μ l in its product, examine the result behind the 1-5min, the sample of infection of Chinese papaya curve leaf disease virus becomes green, and does not infect the orange of sample and blank maintenance dyestuff.
2. the method for utilizing LAMP rapid detection China papaya curve leaf disease virus according to claim 1 is characterized in that the temperature of reaction in the LAMP amplification is 62-65 ℃.
3. the method for utilizing LAMP rapid detection China papaya curve leaf disease virus according to claim 1, the final concentration that it is characterized in that magnesium ion is 10mmol/L.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925589A (en) * | 2012-11-13 | 2013-02-13 | 新疆农业大学 | Establishment of reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection method for Prunus necrotic ringspot virus (PNRSV) |
| CN103215375A (en) * | 2013-05-20 | 2013-07-24 | 新疆农业大学 | Loop-mediated isothermal amplification rapid detection method of chickpea ascochyta leaf blight |
| CN103436532A (en) * | 2013-08-21 | 2013-12-11 | 中国检验检疫科学研究院 | Primer combination for assisting in identifying cotton leaf curl viruses and application of primer combination |
| CN105525042A (en) * | 2016-02-19 | 2016-04-27 | 彭冬青 | LAMP (loop-mediated isothermal amplification) detection kit and detection method for TYLCV (Tomato yellow leaf curl virus) |
| CN109825647A (en) * | 2019-03-26 | 2019-05-31 | 安徽省农业科学院植物保护与农产品质量安全研究所 | A kind of quick detection kit of Chinese milk vetch dwarf virus |
| CN111254214A (en) * | 2020-04-01 | 2020-06-09 | 江西省农业科学院作物研究所 | Amplification method of sesame SSR molecular marker and application thereof |
| 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 |
|---|---|---|---|---|
| CN101845514A (en) * | 2009-11-10 | 2010-09-29 | 江苏省农业科学院 | Method for rapidly identifying tomato yellow leaf curl virus and papaya leaf curl China virus infecting tomato |
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2011
- 2011-05-10 CN CN2011101196580A patent/CN102154526B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845514A (en) * | 2009-11-10 | 2010-09-29 | 江苏省农业科学院 | Method for rapidly identifying tomato yellow leaf curl virus and papaya leaf curl China virus infecting tomato |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925589A (en) * | 2012-11-13 | 2013-02-13 | 新疆农业大学 | Establishment of reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection method for Prunus necrotic ringspot virus (PNRSV) |
| CN103215375A (en) * | 2013-05-20 | 2013-07-24 | 新疆农业大学 | Loop-mediated isothermal amplification rapid detection method of chickpea ascochyta leaf blight |
| CN103215375B (en) * | 2013-05-20 | 2015-04-08 | 新疆农业大学 | Loop-mediated isothermal amplification rapid detection method of chickpea ascochyta leaf blight |
| CN103436532A (en) * | 2013-08-21 | 2013-12-11 | 中国检验检疫科学研究院 | Primer combination for assisting in identifying cotton leaf curl viruses and application of primer combination |
| CN103436532B (en) * | 2013-08-21 | 2015-04-29 | 中国检验检疫科学研究院 | Primer combination for assisting in identifying cotton leaf curl viruses and application of primer combination |
| CN105525042A (en) * | 2016-02-19 | 2016-04-27 | 彭冬青 | LAMP (loop-mediated isothermal amplification) detection kit and detection method for TYLCV (Tomato yellow leaf curl virus) |
| CN109825647A (en) * | 2019-03-26 | 2019-05-31 | 安徽省农业科学院植物保护与农产品质量安全研究所 | A kind of quick detection kit of Chinese milk vetch dwarf virus |
| CN111254214A (en) * | 2020-04-01 | 2020-06-09 | 江西省农业科学院作物研究所 | Amplification method of sesame SSR molecular marker and application thereof |
| 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 |
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